Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species.

The use and implications of ribosomal DNA sequencing for the discrimination of digenean species

In just over a decade, the use of molecular approaches for the recognition of parasites has become commonplace. For trematodes, the internal transcribed spacer region of ribosomal DNA (ITS rDNA) has become the default region of choice. Here, we review the findings of 63 studies that report ITS rDNA sequence data for about 155 digenean species from 19 families, and then review the levels of variation that have been reported and how the variation has been interpreted. Overall, complete ITS sequences (or ITS1 or ITS2 regions alone) usually distinguish trematode species clearly, including combinations for which morphology gives ambiguous results. Closely related species may have few base differences and in at least one convincing case the ITS2 sequences of two good species are identical. In some cases, the ITS1 region gives greater resolution than the ITS2 because of the presence of variable repeat units that are generally lacking in the ITS2. Intraspecific variation is usually low and frequently apparently absent. Information on geographical variation of digeneans is limited but at least some of the reported variation probably reflects the presence of multiple species. Despite the accepted dogma that concerted evolution makes the individual representative of the entire species, a significant number of studies have reported at least some intraspecific variation. The significance of such variation is difficult to assess a posteriori, but it seems likely that identification and sequencing errors account for some of it and failure to recognise separate species may also be significant. Some reported variation clearly requires further analysis. The use of a yardstick to determine when separate species should be recognised is flawed. Instead, we argue that consistent genetic differences that are associated with consistent morphological or biological traits should be considered the marker for separate species. We propose a generalised approach to the use of rDNA to distinguish trematode species.

Nucleolar localization of aprataxin is dependent on interaction with nucleolin and on active ribosomal DNA transcription

The APTX gene, mutated in patients with the neurological disorder ataxia with oculomotor apraxia type 1 (AOA1), encodes a novel protein aprataxin. We describe here, the interaction and interdependence between aprataxin and several nucleolar proteins, including nucleolin, nucleophosmin and upstream binding factor-1 (UBF-1), involved in ribosomal RNA (rRNA) synthesis and cellular stress signalling. Interaction between aprataxin and nucleolin occurred through their respective N-terminal regions. In AOA1 cells lacking aprataxin, the stability of nucleolin was significantly reduced. On the other hand, down-regulation of nucleolin by RNA interference did not affect aprataxin protein levels but abolished its nucleolar localization suggesting that the interaction with nucleolin is involved in its nucleolar targeting. GFP-aprataxin fusion protein co-localized with nucleolin, nucleophosmin and UBF-1 in nucleoli and inhibition of ribosomal DNA transcription altered the distribution of aprataxin in the nucleolus, suggesting that the nature of the nucleolar localization of aprataxin is also dependent on ongoing rRNA synthesis. In vivo rRNA synthesis analysis showed only a minor decrease in AOA1 cells when compared with controls cells. These results demonstrate a cross-dependence between aprataxin and nucleolin in the nucleolus and while aprataxin does not appear to be directly involved in rRNA synthesis its nucleolar localization is dependent on this synthesis.

Origin and evolution of a myxozoan worm

Buddenbrockia pluinatellae is an active, muscular, worm-shaped parasite of freshwater bryozoans. This rare and enigmatic animal has been assigned to the Myxozoa on the basis of 18S ribosomal DNA sequences and the presence of malacosporean spores. Here we report cloning of four homologous protein-coding genes from Buddenbrockia worms, the putatively conspecific sac-shaped parasite originally described as Tetracapsula bryozoides and the related sac-shaped parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Analyses are consistent with the hypothesis that Buddenbrockia is indeed a malacosporean myxozoan, but do not provide support for conspecificity with either T. bryozoides or T. bryosalmonae. Implications for the evolution of worm-like body plans in the Myxozoa are discussed.

Three sympatric karyomorphs in the fish Astyanax fasciatus (Teleostei, Characidae) do not seem to hybridize in natural populations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chromosomal diversification of diploid number, heterochromatin and rDNAs in two species of Phanaeus beetles (Scarabaeidae, Scarabaeinae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cytogenetic analysis in three Bryconamericus species (Characiformes, Characidae): first description of the 5S rDNA-bearing chromosome pairs in the genus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Immunocytochemistry and metamorphic fate of the larval nervous system of Triphyllozoon mucronatum (Ectoprocta : Gymnolaemata : Cheilostomata)

The development of gymnolaemate Ectoprocta includes a larval stage of either the coronate or the cyphonautes type. Herein, we provide the first description of the larval neural anatomy of a coronate larva using immunocytochemical methods. We used antibodies against the neurotransmitters serotonin and FMRFamide and followed the fate of immunoreactive cells through metamorphosis. The larval serotonergic nervous system of Triphyllozoon mucronatum consists of an apical commissure, one pair of lateral axons, a coronate nerve net, an internal nerve mesh, and one pair of axons innervating the frontal organ. FMRFamide is only found in the larval commissure and in the lateral axons. The entire serotonergic and FMRFamidergic nervous system is lost during metamorphosis and the adult neural structures form independent of the larval ones. In the postlarval zooid, both neurotransmitters are detected in the cerebral commissure, in cell bodies located at the base of the lophophore, and in neurites connecting these somata to the cerebral commissure. These findings differ significantly from that observed in other lophotrochozoans, where certain larval neural features are either incorporated in the adult nervous system and/or have inductive functions during its ontogeny. The occurrence of a larval commissure and the lack of a serotonergic or FMRFamidergic apical organ in T. mucronatum are unique among lophotrochozoan larvae, which usually have a distinct apical organ containing serotonergic cells. Our data show that the larval neuroanatomy and the processes that underlie the reorganization of larval organ systems during metamorphosis may vary much more among lophotrochozoan taxa than previously thought.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i.e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E, galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E, hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of total genomic DNA from 15 red algal species. It resulted in 0.1 mug high quality DNA from 1 mg fresh algal material, with an A(260)/A(280) ratio of 1.68 - 1.90. Using this rapidly isolated DNA, the 18S ribosomal RNA genes ( rDNA) and the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. The tested DNA was suitable for restriction endonuclease digestion, genetic marker analysis and polymerase chain reaction (PCR) amplification, and may be valid for other genetic manipulation.

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences.Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae.Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya.Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.

Gene Mapping of 18S and 5S rDNA Genes in the Karyotype of the Three-Spot Gourami Trichogaster trichopterus (Perciformes, Osphronemidae)

Ornamental fish culture is important as an economic activity and for biodiversity conservation as well. The species of the genus Trichogaster (Perciformes, Osphronemidae), popularly known as three-spot gourami, are among the several commercial species raised around the world. In the present work, eight specimens of Thrichogaster trichopterus from aquarium trade facilities were analyzed. The karyotype was composed of 23 pairs of subtelo/acrocentric chromosomes. Fluorescent in situ hybridization allowed identifying the 18S ribosomal gene at telomeric region on long arms of the largest acrocentric pair. On the other hand, the 5S rRNA gene is located at a proximal region on a pair of medium-sized chromosomes. Such information is extremely useful in face of the risks of introduction and the development of ornamental fish trade, once many fish species can be identified only by genetic studies.

Chromosomal mapping of repetitive DNAs in the beetle Dichotomius geminatus provides the first evidence for an association of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity between the B chromosome and A complement

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)