Showing papers on "Ribosomal DNA published in 2018"

Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression

Abstract: The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome’s molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.

Evolutionary trends in animal ribosomal DNA loci: introduction to a new online database.

Abstract: Ribosomal DNA (rDNA) loci encoding 5S and 45S (18S-5.8S-28S) rRNAs are important components of eukaryotic chromosomes. Here, we set up the animal rDNA database containing cytogenetic information about these loci in 1343 animal species (264 families) collected from 542 publications. The data are based on in situ hybridisation studies (both radioactive and fluorescent) carried out in major groups of vertebrates (fish, reptiles, amphibians, birds, and mammals) and invertebrates (mostly insects and mollusks). The database is accessible online at www.animalrdnadatabase.com . The median number of 45S and 5S sites was close to two per diploid chromosome set for both rDNAs despite large variation (1–74 for 5S and 1–54 for 45S sites). No significant correlation between the number of 5S and 45S rDNA loci was observed, suggesting that their distribution and amplification across the chromosomes follow independent evolutionary trajectories. Each group, irrespective of taxonomic classification, contained rDNA sites at any chromosome location. However, the distal and pericentromeric positions were the most prevalent (> 75% karyotypes) for 45S loci, while the position of 5S loci was more variable. We also examined potential relationships between molecular attributes of rDNA (homogenisation and expression) and cytogenetic parameters such as rDNA positions, chromosome number, and morphology.

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers.

Abstract: ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Malassezia vespertilionis sp. nov.: a new cold-tolerant species of yeast isolated from bats

Abstract: Malassezia is a genus of medically-important, lipid-dependent yeasts that live on the skin of warmblooded animals. The 17 described species have been documented primarily on humans and domestic animals, but few studies have examined Malassezia species associated with more diverse host groups such as wildlife. While investigating the skin mycobiota of healthy bats, we isolated a Malassezia sp. that exhibited only up to 92 % identity with other known species in the genus for the portion of the DNA sequence of the internal transcribed spacer region that could be confidently aligned. The Malassezia sp. was cultured from the skin of nine species of bats in the subfamily Myotinae; isolates originated from bats sampled in both the eastern and western United States. Physiological features and molecular characterisation at seven additional loci (D1/D2 region of 26S rDNA, 18S rDNA, chitin synthase, second largest subunit of RNA polymerase II, β-tubulin, translation elongation factor EF-1α, and minichromosome maintenance complex component 7) indicated that all of the bat Malassezia isolates likely represented a single species distinct from other named taxa. Of particular note was the ability of the Malassezia sp. to grow over a broad range of temperatures (7–40 °C), with optimal growth occurring at 24 °C. These thermal growth ranges, unique among the described Malassezia, may be an adaptation by the fungus to survive on bats during both the host’s hibernation and active seasons. The combination of genetic and physiological differences provided compelling evidence that this lipid-dependent yeast represents a novel species described herein as Malassezia vespertilionis sp. nov. Whole genome sequencing placed the new species as a basal member of the clade containing the species M. furfur, M. japonica, M. obtusa, and M. yamatoensis. The genetic and physiological uniqueness of Malassezia vespertilionis among its closest relatives may make it important in future research to better understand the evolution, life history, and pathogenicity of the Malassezia yeasts.

CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy.

Abstract: Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear–vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA–nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.

Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing

Abstract: Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ∼0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

Nucleolus and chromatin.

Abstract: The nucleolus as site of ribosome biogenesis holds a pivotal role in cell metabolism. It is composed of ribosomal DNA (rDNA), which is present as tandem arrays located in nucleolus organizer regions (NORs). In interphase cells, rDNA can be found inside and adjacent to nucleoli and the location is indicative for transcriptional activity of ribosomal genes—inactive rDNA (outside) versus active one (inside). Moreover, the nucleolus itself acts as a spatial organizer of non-nucleolar chromatin. Microscopy-based approaches offer the possibility to explore the spatially distinct localization of the different DNA populations in relation to the nucleolar structure. Recent technical developments in microscopy and preparatory methods may further our understanding of the functional architecture of nucleoli. This review will attempt to summarize the current understanding of mammalian nucleolar chromatin organization as seen from a microscopist’s perspective.

Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells.

Abstract: rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

Copy Number of Human Ribosomal Genes With Aging: Unchanged Mean, but Narrowed Range and Decreased Variance in Elderly Group.

Abstract: Introduction: The multi-copied genes coding for the human 18, 5.8, and 28S ribosomal RNA (rRNA) are located in five pairs of acrocentric chromosomes forming so-called rDNA. Human genome contains unmethylated, slightly methylated, and hypermethylated copies of rDNA. The major research question: What is the rDNA copy number (rDNA CN) and the content of hypermethylated rDNA as a function of age? Materials and Methods: We determined the rDNA CN in the blood leukocyte genomes of 651 subjects aged 17 to 91 years. The subjects were divided into two subgroups: "elderly" group (E-group, N = 126) - individuals over 72 years of age (the age of the population's mean lifetime for Russia) and "non-elderly" group (NE-group, N = 525). The hypermethylated rDNA content was determined in the 40 DNA samples from the each group. The change in rDNA during replicative cell senescence was studied for the cultured skin fibroblast lines of five subjects from NE-group. Non-radioactive quantitative dot- and blot-hybridization techniques (NQH) were applied. Results: In the subjects from the E-group the mean rDNA CN was the same, but the range of variation was narrower compared to the NE-group: a range of 272 to 541 copies in E-group vs. 200 to 711 copies in NE-group. Unlike NE-group, the E-group genomes contained almost no hypermethylated rDNA copies. A case study of cultured skin fibroblasts from five subjects has shown that during the replicative senescence the genome lost hypermethylated rDNA copies only. Conclusion: In the elderly group, the mean rDNA CN is the same, but the range of variation is narrower compared with the younger subjects. During replicative senescence, the human fibroblast genome loses hypermethylated copies of rDNA. Two hypotheses were put forward: (1) individuals with either very low or very high rDNA content in their genomes do not survive till the age of the population's mean lifetime; and/or (2) during the aging, the human genome eliminates hypermethylated copies of rDNA.

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis.

Abstract: Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate successful adaptation to various environmental conditions, increasing host compatibility of these symbiotic fungi. Taro Maeda et al. report a high degree of sequence polymorphism and lack of tandem repeat structure in the ribosomal DNA of arbuscular mycorrhizal fungus Rhizophagus irregularis. This study puts forward an adaptation strategy that uses diverse translation systems to generate phenotypic plasticity.

The long-range interaction map of ribosomal DNA arrays.

Abstract: The repeated rDNA array gives rise to the nucleolus, an organelle that is central to cellular processes as varied as stress response, cell cycle regulation, RNA modification, cell metabolism, and genome stability. The rDNA array is also responsible for the production of more than 70% of all cellular RNAs (the ribosomal RNAs). The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1 while the 45S rDNA arrays reside on the short arm of five human acrocentric chromosomes. These critical genome elements have remained unassembled and have been excluded from all Hi-C analyses to date. Here we built the first high resolution map of 5S and 45S rDNA array contacts with the rest of the genome combining over 15 billion Hi-C reads from several experiments. The data enabled sufficiently high coverage to map rDNA-genome interactions with 1MB resolution and identify rDNA-gene contacts. The map showed that the 5S and 45S arrays display preferential contact at common sites along the genome but are not themselves sufficiently close to yield 5S-45S Hi-C contacts. Ribosomal DNA contacts are enriched in segments of closed, repressed, and late replicating chromatin, as well as CTCF binding sites. Finally, we identified functional categories whose dispersed genes coalesced in proximity to the rDNA arrays or instead avoided proximity with the rDNA arrays. The observations further our understanding of the spatial localization of rDNA arrays and their contribution to the architecture of the cell nucleus.

Molecular and Cytogenetic Study of East African Highland Banana

Abstract: East African highland bananas (EAHBs) are staple food crop in Uganda, Tanzania, Burundi, and other countries in the African Great Lakes region. Even though several morphologically different types exist, all EAHBs are triploid and display minimal genetic variation. To provide more insights into the genetic variation within EAHBs, genotyping using simple sequence repeat (SSR) markers, molecular analysis of ITS1-5.8S-ITS2 region of ribosomal DNA locus, and the analysis of chromosomal distribution of ribosomal DNA sequences were done. A total of 38 triploid EAHB accessions available in the Musa germplasm collection (International Transit Centre, Leuven, Belgium) were characterized. Six diploid accessions of Musa acuminata ssp. zebrina, ssp. banksii, and ssp. malaccensis representing putative parents of EAHBs were included in the study. Flow cytometric estimation of 2C nuclear DNA content revealed small differences (max ~6.5%) in genome size among the EAHB clones. While no differences in the number of 45S and 5S rDNA loci were found, genotyping using 19 SSR markers resulted in grouping the EAHB accessions into four clusters. The DNA sequence analysis of the internal transcribed spacer region indicated a relation of EAHB clones with M. acuminata and, surprisingly, also with M. schizocarpa. The results suggest that EAHB cultivars originated from a single hybrid clone with M. acuminata ssp. zebrina and ssp. banksii being its most probable parents. However, M. schizocarpa seems to have contributed to the formation of this group of banana.

Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites

Abstract: Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

Diversity and evolution of the repetitive genomic content in Cannabis sativa.

Abstract: The repetitive content of the genome, once considered to be “junk DNA”, is in fact an essential component of genomic architecture and evolution. In this study, we used the genomes of three varieties of Cannabis sativa, three varieties of Humulus lupulus and one genotype of Morus notabilis to explore their repetitive content using a graph-based clustering method, designed to explore and compare repeat content in genomes that have not been fully assembled. The repetitive content in the C. sativa genome is mainly composed of the retrotransposons LTR/Copia and LTR/Gypsy (14% and 14.8%, respectively), ribosomal DNA (2%), and low-complexity sequences (29%). We observed a recent copy number expansion in some transposable element families. Simple repeats and low complexity regions of the genome show higher intra and inter species variation. As with other sequenced genomes, the repetitive content of C. sativa’s genome exhibits a wide range of evolutionary patterns. Some repeat types have patterns of diversity consistent with expansions followed by losses in copy number, while others may have expanded more slowly and reached a steady state. Still, other repetitive sequences, particularly ribosomal DNA (rDNA), show signs of concerted evolution playing a major role in homogenizing sequence variation.

Structural basis of RNA polymerase I stalling at UV light-induced DNA damage.

Abstract: RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 A. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species.

Abstract: The genus Phytophthora is agriculturally and ecologically important. As the number of described Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (β-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and β-tub. Resolution of these four markers also varied with (sub)clade. β-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99 % PCR success rate) and β-tub (96 %) were easier to amplify than cox1 (75 %) and tigA (71 %). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade.

Morphologically indistinguishable hybrid Carassius female with 156 chromosomes: A threat for the threatened crucian carp, C. carassius, L.

Abstract: The crucian carp Carassius carassius (Linnaeus, 1758), is native to many European freshwaters. Despite its wide distribution, the crucian carp is declining in both the number and sizes of populations across much of its range. Here we studied 30 individuals of a putative pure population from Helsinki, Finland. Despite clear external morphological features of C. carassius, an individual was of a higher ploidy level than the others. We therefore applied a set of molecular genetic (S7 nuclear and cytochrome b mitochondrial genes) and cytogenetic tools (sequential fluorescent 4’, 6-diamidino-2-phenylindole [DAPI], Chromomycin A3 [CMA3], C-banding and in situ hybridization [FISH] with both 5S and 28S ribosomal DNA probes) to determine its origin. While all examined characteristics of a diploid representative male (CCAHe2Fi) clearly corresponded to those of C. carassius, a triploid individual (CCAHe1Fi) was more complex. Phylogenetic analysis revealed that the nuclear genome of CCAHe1Fi contained three haploid sets: two C. gibelio and one C. carassius. However the mitochondrial DNA was that of C. gibelio, demonstrating its hybrid origin. The FISH revealed three strong (more intensive) 5S rDNA loci, confirming the triploid status, and an additional 24 weak (less intensive) signals were observed in the chromosome complement of CCAHe1Fi. On the other hand, only two strong and 16 weak 5S rDNA signals were visible on the chromosomes of the CCAHe2Fi male. 28S rDNA FISH revealed four strong signals in both CCAHe1Fi and CCAHe2Fi individuals. CMA3 staining revealed four to six CMA3-positive bands of CCAHe1Fi, while that of diploids contained only two to four. The fact that a polyploid hybrid Carassius female with a strong invasive potential may share morphological characters typical for endangered C. carassius highlights a need to combine genetic investigations of Carassius cryptic diversity with conservation measures of C. carassius in Europe.

Three new species of soil-inhabiting Trichoderma from southwest China

Abstract: Fungi in the genus Trichoderma are widely distributed in China, including in Yunnan province. In this study, we report three new soil-inhabiting species in Trichoderma, named as T.kunmingense, T.speciosum and T.zeloharzianum. Their colony and mycelial morphology, including features of asexual states, were described. For each species, their DNA sequences were obtained from three loci, the internal transcribed spacer (ITS) regions of the ribosomal DNA, the translation elongation factor 1-α encoding gene (tef1) and the gene encoding the second largest nuclear RNA polymerase subunit (rpb2). Our analyses indicated that the three new species showed consistent divergence amongst each other and from other known and closely related species. Amongst the three, T.speciosum and T.kunmingense belong to the Viride Clade. Specifically, T.speciosum is related to three species - T.hispanicum, T.samuelsii and T.junci and is characterised by tree-like conidiophores, generally paired branches, curved terminal branches, spindly to fusiform phialides and subglobose to globose conidia. In contrast, T.kunmingense morphologically resembles T.asperellum and T.yunnanense and is distinguished by its pyramidal conidiophores, ampulliform to tapered phialides, discrete branches and ovoidal, occasionally ellipsoid, smooth-walled conidia. The third new species, T.zeloharzianum, is a new member of the Harzianum Clade and is closely associated with T.harzianum, T.lixii and T.simmonsii but distinguished from them by having smaller, subglobose to globose, thin-walled conidia.

RNA polymerase I activation and hibernation: unique mechanisms for unique genes.

Abstract: In yeast, transcription of ribosomal DNA (rDNA) by RNA polymerase I (Pol I) is regulated by unique mechanisms acting at the level of the enzyme. Under stress situations such as starvation, Pol I hibernates through dimerization. When growth conditions are restored, dimer disassembly and Rrn3 binding drive enzyme activation and subsequent recruitment to rDNA.

Sex Chromosomes of the Iconic Moth Abraxas grossulariata (Lepidoptera, Geometridae) and Its Congener A. sylvata.

Abstract: The magpie moth, Abraxas grossulariata, is an iconic species in which female heterogamety was discovered at the beginning of the 20th century. However, the sex chromosomes of this species have not yet been cytologically identified. We describe the sex chromosomes of A. grossulariata and its congener, A. sylvata. Although these species split only around 9.5 million years ago, and both species have the expected WZ/ZZ chromosomal system of sex determination and their sex chromosomes share the major ribosomal DNA (rDNA) representing the nucleolar organizer region (NOR), we found major differences between their karyotypes, including between their sex chromosomes. The species differ in chromosome number, which is 2n = 56 in A. grossularita and 2n = 58 in A. sylvata. In addition, A. grossularita autosomes exhibit massive autosomal blocks of heterochromatin, which is a very rare phenomenon in Lepidoptera, whereas the autosomes of A. sylvata are completely devoid of distinct heterochromatin. Their W chromosomes differ greatly. Although they are largely composed of female-specific DNA sequences, as shown by comparative genomic hybridization, cross-species W-chromosome painting revealed considerable sequence differences between them. The results suggest a relatively rapid molecular divergence of Abraxas W chromosomes by the independent spreading of female-specific repetitive sequences.

Complete mitochondrial and rDNA complex sequences of important vector species of Biomphalaria, obligatory hosts of the human-infecting blood fluke, Schistosoma mansoni.

Abstract: Using high throughput Illumina sequencing technology, we determined complete sequences for the mitochondrial genome (mitogenome) and nuclear ribosomal DNA (rDNA) complex for three African freshwater snail taxa within the genus Biomphalaria, B. pfeifferi, B. sudanica and B. choanomphala, and for two laboratory strains of B. glabrata originating from the Neotropics. Biomphalaria snails are obligate vectors of the blood fluke Schistosoma mansoni, a major etiologic agent of human intestinal schistosomiasis. Our data show that mitogenomes from African and Neotropical Biomphalaria are highly conserved. With respect to rDNA, the two internal transcribed spacers (ITS1 and 2) were found to be highly variable whereas the three ribosomal RNA genes (28S, 5.8S and 18S rRNA) exhibited no or very limited variation. Our analyses reveal that the two taxa inhabiting Lake Victoria, B. sudanica and B. choanomphala, are very similar to one another relative to the similarity either shows to B. pfeifferi or B. glabrata. This new sequence information may prove useful for developing new markers for snail identification, environmental detection/monitoring purposes or for tracking epidemiology and snail dependencies of S. mansoni in endemic areas. It also provides new information pertinent to still unresolved questions in Biomphalaria systematics and nomenclature.

NGS barcode sequencing in taxonomy and diagnostics, an application in “ Candida ” pathogenic yeasts with a metagenomic perspective

Abstract: Species identification of yeasts and other Fungi is currently carried out with Sanger sequences of selected molecular markers, mainly from the ribosomal DNA operon, characterized by hundreds of tandem repeats of the 18S, ITS1, 5.8S, ITS2 and LSU loci. The ITS region has been recently proposed as a primary barcode marker making this region the most used one in taxonomy, phylogeny and diagnostics. The introduction of NGS is providing tools of high efficacy and relatively low cost to amplify two or more markers simultaneously with great sequencing depth. However, the presence of intra-genomic variability between the repeats requires specific analytical procedures and pipelines. In this study, 286 strains belonging to 11 pathogenic yeasts species were analysed with NGS of the region spanning from ITS1 to the D1/D2 domain of the LSU encoding ribosomal DNA. Results showed that relatively high heterogeneity can hamper the use of these sequences for the identification of single strains and even more of complex microbial mixtures. These observations point out that the metagenomics studies could be affected by species inflection at levels higher than currently expected.

Hidden mycota of pine needles: Molecular signatures from PCR-DGGE and Ribosomal DNA phylogenetic characterization of novel phylotypes

Abstract: Previous studies for enumerating fungal communities on pine needles relied entirely on phenotypic diversity (microscopy) or identification based on DNA sequence data from those taxa recovered via cultural studies. To bypass limitations of the culturing methods and provide a more realistic diversity estimate, we employed and assessed a PCR-DGGE based method coupled with rDNA phylogenetic sequence analyses to characterize fungal taxa associated with pine needles. Fresh (living) and decayed needles from three hosts of the Pinaceae (Keteleeria fortunei, Pinus elliottii and P. massoniana) were examined. Morphological studies reveal that the most abundant species associated with decayed needles were Cladosporium cladosporioides and an unidentified Trichoderma species followed by Gliocephalotrichum sp., Gliocladium sp., Lophodermium pinastri, Paecilomyces varioti, Phaeostalagmus cyclosporus and a Phoma sp, which are commonly occurring fungi. Community genomic data from freshly collected and decayed pine needles recovered 40 operational taxonomic units, which appear to be mostly undetected members of the natural fungal consortium. Sequence analyses revealed a number of phylotypes or "species" that were not recovered using traditional morphological and cultural approaches previously used. Phylogenetic data from partial 18S rDNA sequence data reveal that most phylotypes represent potential novel phylogenetic fungal lineages with affinities to the Dothideomycetes, Leotiomycetes, Lecanoromycetes and Sordariomycetes and were not identical to previously known endophytes or saprobes. Although the major ecological roles of these phylotypes in pine needles are still enigmatic, this study provides new insights in hidden fungal diversity that mycologists are possibly ignoring given the discrepancies associated with available methods. To what extent do previously recovered identified species (either as saprobes or endophytes) from morphological or culturing studies act as pioneer decomposers or constitute an integral part of endophytic community warrants further investigation.

Arabidopsis RIBOSOMAL RNA PROCESSING7 Is Required for 18S rRNA Maturation.

Abstract: Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. In a screen for MAS2 interactors, we identified RIBOSOMAL RNA PROCESSING 7 (RRP7), an ortholog of yeast rRNA processing protein 7 (Rrp7), which is required for 18S ribosomal RNA (rRNA) maturation. Arabidopsis rrp7 mutants exhibit a pleiotropic phenotype including slow growth, altered shoot phyllotaxy, aberrant venation in lateral organs, partial infertility, and abscisic acid hypersensitivity in seedlings. In Arabidopsis, RRP7 localizes mainly to the nucleolus, the site of the 45S rDNA transcription that produces a 45S pre-rRNA primary transcript, precursor of the 25S, 18S and 5.8S rRNAs. Lack of RRP7 function perturbs 18S rRNA maturation, causes nucleolar hypertrophy, and results in an increased 25S/18S rRNA ratio. Arabidopsis contains hundreds of 45S rDNA genes whose expression is epigenetically regulated, and deregulated, in rrp7 mutants. Double mutant analysis revealed synergistic interactions between RRP7 alleles and alleles of MAS2, NUCLEOLIN1 (NUC1), and HISTONE DEACETYLASE 6 (HDA6), which encode epigenetic regulators of 45S rDNA transcription. Our results reveal the evolutionarily conserved but divergent roles of RRP7 as a ribosome biogenesis factor.

Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa).

Abstract: Myxozoa are extremely diverse microscopic parasites belonging to the Cnidaria. Their life-cycles alternate between vertebrate and invertebrate hosts, predominantly in aquatic habitats. Members of the phylogenetically well-defined Sphaerospora (sensu stricto) clade predominantly infect the urinary system of marine and freshwater fishes and amphibians. Sphaerosporids are extraordinary due to their extremely long and unique insertions in the variable regions of their 18S and 28S rDNA genes and due to the formation of motile proliferative stages in the hosts' blood. To date, DNA sequences of only 19 species have been obtained and information on the patterns responsible for their phylogenetic clustering is limited. We screened 549 fish kidney samples from fish of various geographical locations, mainly in central Europe, to investigate sphaerosporid biodiversity microscopically and by 18S rDNA sequences. We performed multiple phylogenetic analyses to explore phylogenetic relationships and evolutionary trends within the Sphaerospora (s.s.) clade, by matching host and habitat features to the resultant 18S rDNA trees. The apparent co-clustering of species from related fish hosts inspired us to further investigate host-parasite co-diversification, using tree-based (CoRE-PA) and distance-based (ParaFit) methods. Our study considerably increased the number of 18S rDNA sequence data for Sphaerospora (s.s.) by sequencing 17 new taxa. Eight new species are described and one species (Sphaerospora diminuta Li & Desser, 1985) is redescribed, accompanied by sufficient morphological data. Phylogenetic analyses showed that sphaerosporids cluster according to their vertebrate host order and habitat, but not according to geography. Cophylogenetic analyses revealed a significant congruence between the phylogenetic trees of sphaerosporids and of their vertebrate hosts and identified Cypriniformes as a host group of multiple parasite lineages and with high parasite diversity. This study significantly contributed to our knowledge of the biodiversity and evolutionary history of the members of the Sphaerospora (s.s.) clade. The presence of two separate phylogenetic lineages likely indicates independent historical host entries, and the remarkable overlap of the larger clade with vertebrate phylogeny suggests important coevolutionary adaptations. Hyperdiversification of sphaerosporids in cypriniform hosts, which have undergone considerable radiations themselves, points to host-driven diversification.

A multi-gene phylogeny of Chlorophyllum (Agaricaceae, Basidiomycota): new species, new combination and infrageneric classification.

Abstract: Taxonomic and phylogenetic studies of Chlorophyllum were carried out on the basis of morphological differences and molecular phylogenetic analyses. Based on the phylogeny inferred from the internal transcribed spacer (ITS), the partial large subunit nuclear ribosomal DNA (nrLSU), the second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-α (tef1) sequences, six well-supported clades and 17 phylogenetic species are recognised. Within this phylogenetic framework and considering the diagnostic morphological characters, two new species, C. africanum and C. palaeotropicum, are described. In addition, a new infrageneric classification of Chlorophyllum is proposed, in which the genus is divided into six sections. One new combination is also made. This study provides a robust basis for a more detailed investigation of diversity and biogeography of Chlorophyllum.