Molecular phylogeny of Candidula (Geomitridae) land snails inferred from mitochondrial and nuclear markers reveals the polyphyly of the genus.

The genus Candidula (Geomitridae), consisting of 28 species in Western Europe as currently described, has a disjunct distribution in the Iberian Peninsula, Italy, the Balkans, the Aegean Islands, and one species on the Canary Islands. Although the genus is seemingly well defined by characters of the reproductive system, the relationships within the genus are still unclear and some authors have indicated a possible subgeneric division based on the internal morphology of the dart sac. Despite substantial phylogenetic incongruence, we present a well-resolved molecular phylogeny of Candidula based on two mitochondrial genes (COI and 16S rRNA), the nuclear rDNA region (5.8S rNRA + ITS2 + 28S rRNA) and seven additional nuclear DNA regions developed specifically for this genus (60SL13, 60SL17, 60SL7, RPL14, 40SS6, 60SL9, 60SL13a), in total 5595 bp. Six reciprocally monophyletic entities including Candidula species were recovered, grouping into two major clades. The incorporation of additional geomitrid genera allowed us to unequivocally demonstrate the polyphyly of the genus Candidula. One major clade grouped species from southern France and Italy with the widely distributed species C. unifasciata. The second major clade grouped all the species from the Iberian Peninsula, including C. intersecta and C. gigaxii. Candidula ultima from the Canary Islands was recovered as separated lineage within the latter clade and related to African taxa. The six monophyla were defined as six new genera belonging to different tribes within the Helicellinae. Thus, we could show that similar structures of the stimulatory apparatus of the genital system in different taxa do not necessarily indicate a close phylogenetic relationship in the Geomitridae. More genera of the family are needed to clarify their evolutionary relationships, and to fully understand the evolution of the stimulatory apparatus of the genital system within the Geomitridae.

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits.

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.

Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6.

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.

Deamination of adenosines in extracellular RNA spontaneously internalized by human cells.

Ribonucleic acids circulating in mammalian extracellular fluids as well as RNAs accumulating in culture medium condensed by mammalian cells are internalized by acceptor cells and distributed among cellular compartments. The internalized RNA can be involved in the induction and regulation of cellular processes as guide or signaling molecules. The internalization of RNA may be accompanied by covalent modifications influencing the stability and functionality of this RNA. To analyze nucleotide modifications introduced by human cells in internalized extracellular RNA, 5.8S rRNA of S. cerevisiae was used. It was shown that 5.8S rRNA of S. cerevisiae is captured by human adenocarcinoma MCF-7 cells from culture medium and delivered to cytoplasm and nuclei. Most of the internalized RNA was hydrolyzed to mono- and oligonucleotides. Full-length RNA uptake was detected by RT-PCR in the cytoplasm and in nuclei after the pulse addition of RNA to the culture medium. 5'-Inosine was the only detectable modified nucleotide in the hydrolysate of cell-internalized RNAs. Consequently, extracellular RNAs entering human cells were subjected to partial adenosine deamination. Sequencing of cDNA confirmed that full-length extracellular RNA that accumulated in the nucleus, but not in the cytoplasm, was partially edited by adenosine deaminases. The deamination revealed in nuclear-stored, full-length RNA was site-specific. Adenosines edited in S. cerevisiae 5.8S rRNA are stack-closing A-U pairs A53 and A96, as well as unpaired A44 and A65. Our data emphasize the participation of adenosine deaminases in capturing and intracellular trafficking of internalized RNAs.

Secondary structure of the rRNA ITS2 region reveals key evolutionary patterns in acroporid corals.

This study investigates the ribosomal RNA transcript secondary structure in corals as confirmed by compensatory base changes in Isopora/Acropora species. These species are unique versus all other corals in the absence of a eukaryote-wide conserved structural component, the helix III in internal transcriber spacer (ITS) 2, and their variability in the 5.8S-LSU helix basal to ITS2, a helix with pairings identical among all other scleractinian corals. Furthermore, Isopora/Acropora individuals display at least two, and as many as three, ITS sequence isotypes in their genome which appear to be capable of function. From consideration of the conserved elements in ITS2 and flanking regions, it appears that there are three major groups within the IsoporaAcropora lineage: the Isopora + Acropora "longi" group, the large group including Caribbean Acropora + the Acropora "carib" types plus the bulk of the Indo-Pacific Acropora species, and the remaining enigmatic "pseudo" group found in the Pacific. Interbreeding is possible among Caribbean A. palmata and A. cervicornis and among some species of Indo-Pacific Acropora. Recombinant ITS sequences are obvious among these latter, such that morphology (as represented by species name) does not correlate with common ITS sequence. The combination of characters revealed by RNA secondary structure analyses suggests a recent past/current history of interbreeding among the Indo-Pacific Acropora species and a shared ancestry of some of these with the Caribbean Acropora. The unusual absence of helix III of ITS2 of Isopora/Acropora species may have some causative role in the equally unusual instability in the 5.8S-LSU helix basal to ITS2 of this species complex.

A polyphasic approach to the identification of ochratoxin A-producing black Aspergillus isolates from vineyards in Sicily.

Aspergillus strains belonging to section Nigri isolated during a two year survey in eight Sicilian vineyards located on the slopes of Mount Etna (Sicily, Italy) were analysed analyzed in order to characterize species responsible for ochratoxin A (OTA) contamination of grapes. The polyphasic approach permitted analysis of biodiversity of Aspergillus isolates in relation to their morphology, ochratoxigenicity and genetic variability. We assessed OTA production by A. carbonarius, A. niger, A. tubingensis and A. japonicus using an enzyme-linked immunosorbent assay. A. carbonarius isolates were the strongest OTA producers. A subset of 66 representative strains was selected for further DNA-based characterization. PCR assays using species-specific primers discriminated between A. niger, A. carbonarius and A. japonicus on the basis of the target sequences for each species. The PCR-based methods matched morphological characterization in identifying all the black aspergilli (BA) isolates tested, whereas RFLP analysis with RsaI of isolates positive to PCRs with A. niger specific primers identified three A. tubingensis isolates. The identification of thirteen isolates was further confirmed by ITS analysis. By this method, each of the isolates was identified and assigned to an Aspergillus species. The fAFLP analysis of 40 isolates highlighted the power of this technique to discriminate different species and single strains, to verify the presence of mixed populations in the same vineyard, through homogeneous species clusters. No correlation was observed between the clusters and OTA production level or origin.

Joergensenia, a new genus to accommodate Psoroma cephalodinum (lichenized Ascomycota).

The taxonomy of the genus Psoroma (lichenized Ascomycota) is currently under revision, as it has long been recognized as heterogeneous. The aim of the present study was to reconstruct the phylogeny of Psoroma and related genera. The ITS region of nu-rDNA and the mtSSU rDNA from 22 collections of Psoroma and Pannaria were amplified. Sequences from GenBank were also used. For the phylogenetic analysis, direct optimization was implemented, using the program POY, and standard MP using PAUP. Both analyses resulted in similar trees. Two main clades were obtained, one including Santesoniella and most Psoroma species and the other including Pannaria and two Psoroma species, indicating that Psoroma is polyphyletic. Based on its phylogenetic position and ascus type, P. implexum is transferred to the genus Pannaria, and the new combination P. implexa is proposed. Psoroma cephalodinum clusters with Parmeliella and Degelia as a basal group within the Pannariaceae. The ascus apex of P. cephalodinum has a strong amyloid reaction revealing a compact cap-shaped plug. This differs from most of the ascus apical structures observed in the family. Both ascus internal structure and phylogenetic position suggest that P. cephalodinum is distantly related to Psoroma or Pannaria, and thus a new genus Joergensenia is proposed.

Debaryomyces singareniensis sp. nov., a novel yeast species isolated from a coal mine soil in India.

Three strains (AP19, AP19-4 and AP19-6) of a novel yeast species were isolated from soil from the Singareni coal mines, Andhra Pradesh, India. They were morphologically, physiologically and phylogenetically identical and produced one to four spherical ascospores per ascus. Phylogenetic analysis using the D1/D2 variable domain of the large-subunit rRNA gene indicated that the closest relative of these strains is Debaryomyces etchellsii (2.6% divergence). Other species related to these strains are D. mycophilus (5.1% divergence) and species of the D. hansenii cluster (4.9-5.6% divergence). The novel species differs by 20 and 15 physiological tests from D. etchellsii and D. mycophilus, respectively. Phylogenetic analysis of the internal transcribed spacer (ITS) region also indicated that strains of the new species are related to D. etchellsii (7.7% divergence), followed by species of the D. hansenii cluster (9-10% divergence). In the small-subunit rRNA gene sequences, they differed from D. etchellsii by seven substitutions and one insertion or deletion of a base in a sequence (indel) and from D. mycophilus by 17 substitutions and 1 indel. The physiological, biochemical and molecular data suggest that these strains belong to a novel species, for which we propose the name Debaryomyces singareniensis sp. nov. The type strain of AP19(T) (=MTCC 7061(T)=CBS 10405(T)). The Mycobank number of the new species is MB510046.

Molecular phylogeny of Hyphoderma and the reinstatement of Peniophorella.

Hyphoderma is a large genus of corticioid homobasidiomycetes. In earlier homobasidiomycete-wide molecular phylogenetic studies the genus has appeared as polyphyletic. This paper describes the results from phylogenetic analyses of 22 species of Hyphoderma using nuclear 5.8 S and 28 S rDNA. Species with echinocysts and stephanocysts form a distinct clade well separated from Hyphoderma s. str. For this group the old genus name Peniophorella is available with P. pubera as the type species. Nineteen new combinations in Peniophorella are made and a key to the species is given. The clade representing Hyphoderma in its restricted sense receives only indicative support and a further subdivision of the genus may become necessary. H. capitatum, H. orphanellum, and H. sibiricum belong neither to Peniophorella nor to Hyphoderma s. str. Hypochnicium is a sister group to Hyphoderma. The phylogenetic analyses support the segregation of Hypochnicium analogum and H. vellereum as Gloeohypochnicium analogum and Granulobasidium vellereum, respectively, and the inclusion of H. detriticum in Hyphodontia. Hyphodermopsis and Bulbillomyces are best regarded as synonyms of Hypochnicium.

Molecular systematics of Zopfiella and allied genera: evidence from multi-gene sequence analyses.

This study aims to reveal the phylogenetic relationships of Zopfiella and allied genera in the Sordariales. Multiple gene sequences (partial 28S rDNA, ITS/5.8S rDNA and partial beta-tubulin) were analysed using MP and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to infer phylogenies. Phylogenetic analyses show that currently recognised Zopfiella species are polyphyletic. Based on sequence analyses and morphology, it appears that Zopfiella should be restricted to species having ascospores with a septum in the dark cell. Our molecular analysis also shows that Zopfiella should be placed in Lasiosphaeriaceae rather than Chaetomiaceae. Cercophora and Podospora are also polyphyletic, which is in agreement with previous studies. Our analyses show that species possessing a Cladorrhinum anamorph are phylogenetically closely related. In addition, there are several strongly supported clades, characterised by species possessing divergent morphological characters. It is difficult to predict which characters are phylogenetically informative for delimiting these clades.

Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits.

Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.

Population genetic structure of the polypore Datronia caperata in fragmented mangrove forests.

Datronia caperata, a basidiomycete fungus, is one of the dominant polypore species found in neotropical mangrove forest fragments, where it is locally specialized on Laguncularia racemosa. We examined the genetic structure of D. caperata populations from four Panamanian mangrove forests using AFLP markers. Using five primer pair combinations, 145 loci were detected, 98.6% of which were polymorphic. Each of the populations showed a high degree of genetic diversity (Nei's h ranging from 0.146 to 0.223). Results from minimum spanning trees and Mantel tests showed little evidence for small-scale spatial structure within sites. A significant amount of total genetic variation was partitioned among populations (phi(ST) = 0.21) separated by 10s to 100s of km, a considerably greater amount than has been detected in other mushroom and wood-decaying fungi sampled at equal or greater geographic distances. These results suggest that despite production of copious basidiospores capable of long distance dispersal, some homobasidiomycete fungi may be susceptible to genetic isolation due to habitat fragmentation.

PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia.

OBJECTIVE: To assess the usefulness of polymerase chain reaction (PCR) assays in the diagnosis of fungal infections in immunocompromised patients. METHODS: A rapid and sensitive PCR-based assay for the detection and identification of fungal pathogens was designed and applicability of this method was investigated in a group of children with cancer and febrile neutropenia (FN). RESULTS: The ITS2 sequences and adjacent regions of 40 fungal pathogens were analyzed and primers for detection of all analyzed fungal species were designed. Amplification product length polymorphism (APLP) and restriction fragment length polymorphism (RFLP) generated genus- or species-specific patterns. The sensitivity of the method was approximately three cells of Candida albicans per 1 mL of blood. The results were available within 8 h after sample collection. The method was tested on 53 blood samples and one lung biopsy sample from 24 children with cancer and febrile neutropenia (FN). The PCR assay detected fungal DNA in 25 clinical samples from ten patients. Blood cultures were positive in only five samples, while another two blood-culture negative patients had positive cultures from throat swabs. The remaining 14 patients were both culture- and PCR-negative. Culture-isolated strains matched completely those obtained by PCR-APLP-RFLP identification. The identity of fungal species was confirmed by direct sequencing of amplified products. CONCLUSION: Our results suggest that PCR-APLP-RFLP assays can be useful in the diagnosis of fungal infections in immunocompromised patients.

Molecular phylogenetic analysis of the genus Gyrodactylus (Platyhelminthes: Monogenea) inferred from rDNA ITS region: subgenera versus species groups.

Analyses of small subunit ribosomal RNA gene sequences of representatives of major taxa of Monopisthocotylea were performed to identify the sister group of Gyrodactylus. Nuclear ribosomal DNA sequences from the complete internal transcribed spacer (ITS) region were used to infer phylogeny of 37 Gyrodactylus species and Gyrodactyloides bychowskii, Macrogyrodactylus polypteri and Gyrdicotylus gallieni, using maximum likelihood, parsimony and Bayesian inference. The genus Gyrodactylus appeared to be a monophyletic group in all analyses, based on the present data set. Within the genus, there were 3 major groups recognized by high bootstrap values and posterior probabilities. None of the 6 subgenera appeared to be monophyletic, and the most basal subgenus G. (Gyrodactylus) was paraphyletic. Characteristics of the excretory system of Gyrodactylus do not seem to be conservative enough to reveal subgenera within Gyrodactylus and we suggest abandoning existing subgenera as indicators of phylogeny. The grouping of species based on the morphology of the ventral bar and marginal hooks seems to have sufficient power to infer relationships between the Gyrodactylus species.

Characterization of RNA with unusual electrophoretic mobility from tissues of patients with Crohn's disease.

RNAs from tissues of patients with Crohn's disease that migrate off the diagonal in a two-dimensional gel electrophoresis system were partially characterized. One of the RNA species was a discrete cleavage product of region V2-9 of 28S rRNA; another is a conformer or variant of 5.8S rRNA; and a third is a mixture of unidentified fragments with mobility similar to that of 7S RNA. The yield of these species from resected tissue and their visualization by silver staining was very sensitive to the details of the preparative procedure. No evidence of viroid-like RNA was found within the range of molecular sizes (< 7S) that we examined.

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.

Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ.

The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.