1,720,979 research outputs found
Diatom-based genetic engineering system methodology for the eco-sustainable production of ovothiols
No full textThe present invention relates to the field of biotechnology, in particular it relates to the set-up of a protocol
of enzymatic engineering of the diatom species Phaeodactylum tricornutum to overexpress the biosynthetic
enzyme leading to ovothiol production. This protocol is eco-sustainable because it uses cells and nutrients
for biosynthesis and does not produce toxic side-compounds.
Microalgal biomass moreover can be exploited for additional uses after ovothiol extraction, making the
production process more cost-effective. The production of ovothiols is relevant for the pharmaceutical, nutraceutical, and cosmeceutical sectors
Selection and validation of reference genes for qPCR analysis in the pennate diatoms Pseudo-nitzschia multistriata and P. arenysensis
No full textDiatoms are eukaryotic microalgae broadly present in freshwater and marine ecosystems. The great ecological interest about diatoms has recently led to increased efforts towards the exploration of the molecular properties of these organisms and the development of molecular tools to study the function of genes. We are using as model system two pennate diatoms, Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis. In order to enable molecular investigations in these two species, we have made use of sequence information from transcriptomic data to identify genes that can be used as reference genes in quantitative PCR (qPCR). We have analyzed the expression of a set of genes commonly used as reference genes: histone H4, TBP (TATA binding protein), RPS (30. S ribosomal protein), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), TUB A and TUB B (tubulin α and tubulin β), ACT (actin), CDK A (cyclin dependent kinase A) and COPA (coatomer protein complex, subunit α). The suitability of these genes as references in qPCR has been tested in different conditions and among different strains. We have found that only TUB A, TUB B and CDK A are stable in all the conditions analyzed. These three genes, in addition to ACT and COPA, are good reference genes for P. multistriata, while GAPDH appears to be differentially expressed in the tested conditions in this species. In P. arenysensis, instead, TUB A, TUB B, CDK A, GAPDH, H4 and RPS show the highest levels of stability and can be considered reliable reference genes. © 2013 Elsevier B.V
Establishment of Genetic Transformation in the Sexually Reproducing Diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and Inheritance of the Transgene
No full textWe report the genetic transformation of the planktonic diatoms Pseudo-nitzschia arenysensis and Pseudo-nitzschia multistriata, members of the widely distributed and ecologically important genus Pseudo-nitzschia. P. arenysensis and P. multistriata present the classical size reduction/restitution life cycle and can reproduce sexually. Genetic transformation was achieved with the biolistic method, using the H4 gene promoter from P. multistriata to drive expression of exogenous genes. The transformation was first optimized introducing the Sh ble gene to confer resistance to the antibiotic zeocin. Integration of the transgene was confirmed by PCR and Southern blot analyses. Subsequently, we simultaneously transformed in P. arenysensis two plasmids, one encoding the beta-glucuronidase (GUS) gene together with the plasmid carrying the Sh ble resistance gene, demonstrating the possibility of co-transformation. By transforming a gene encoding a fusion between the histone H4 and the green fluorescent protein (GFP), we demonstrated that fluorescent tagging is possible and that studies for protein localization are feasible. Importantly, we crossed P. arenysensis- and P. multistriata-transformed strains with a wild-type strain of opposite mating type and demonstrated that the transgene can be inherited in the F1 generation. The possibility to transform two diatom species for which genetic crosses are possible opens the way to a number of new approaches, including classical loss of function screens and the possibility to obtain different combinations of double transformants
Selection and validation of reference genes for qPCR analysis in the pennate diatoms Pseudo-nitzschia multistriata and P. arenysensis
No full textDiatoms are eukaryotic microalgae broadly present in freshwater and marine ecosystems. The great ecological interest about diatoms has recently led to increased efforts towards the exploration of the molecular properties of these organisms and the development of molecular tools to study the function of genes. We are using as model system two pennate diatoms, Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis. In order to enable molecular investigations in these two species, we have made use of sequence information from transcriptomic data to identify genes that can be used as reference genes in quantitative PCR (qPCR). We have analyzed the expression of a set of genes commonly used as reference genes: histone H4, TBP (TATA binding protein), RPS (30 S ribosomal protein), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), TUB A and TUB B (tubulin alpha and tubulin beta), ACT (actin), CDK A (cyclin dependent kinase A) and COPA (coatomer protein complex, subunit alpha). The suitability of these genes as references in qPCR has been tested in different conditions and among different strains. We have found that only TUB A, TUB B and CDK A are stable in all the conditions analyzed. These three genes, in addition to ACT and COPA, are good reference genes for P. multistriata, while GAPDH appears to be differentially expressed in the tested conditions in this species. In P. arenysensis, instead, TUB A, TUB B, CDK A, GAPDH, H4 and RPS show the highest levels of stability and can be considered reliable reference genes. (C) 2013 Elsevier B.V. All rights reserved
Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of Nitric Oxide Synthase genes in diatoms
Full text linkDiatoms are among the most diverse eukaryotic microorganisms on Earth, they are responsible for a large fraction of primary production in the oceans and can be found in different habitats. Pseudo-nitzschia are marine planktonic diatoms responsible for blooms in coastal and oceanic waters. We analyzed the transcriptome of three species, Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata, with different levels of genetic relatedness. These species have a worldwide distribution and the last one produces the neurotoxin domoic acid. We were able to annotate about 80% of the sequences in each transcriptome and the analysis of the relative functional annotations allowed comparison of the main metabolic pathways, pathways involved in the biosynthesis of isoprenoids (MAV and MEP pathways), and pathways putatively involved in domoic acid synthesis. The search for homologous transcripts among the target species and other congeneric species resulted in the discovery of a sequence annotated as Nitric Oxide Synthase (NOS), found uniquely in Pseudo-nitzschia multistriata. The predicted protein product contained all the domains of the canonical metazoan sequence. Putative NOS sequences were found in other available diatom datasets, supporting a role for nitric oxide as signaling molecule in this group of microalgae
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
lipoxygenase transcript leads to reduced oxylipin production and impaired growth
No full textBecause of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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