Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns

Background: Research using the Pacific oyster Crassostrea gigas as a model organism has experienced rapid growth in recent years due to the development of high-throughput molecular technologies. As many as 56,268 EST sequences have been sequenced to date, representing a genome-wide resource that can be used for transcriptomic investigations. Results: In this paper, we developed a Pacific oyster microarray containing oligonucleotides representing 31,918 transcribed sequences selected from the publicly accessible GigasDatabase. This newly designed microarray was used to study the transcriptome of male and female gonads, mantle, gills, posterior adductor muscle, visceral ganglia, hemocytes, labial palps and digestive gland. Statistical analyses identified genes differentially expressed among tissues and clusters of tissue-enriched genes. These genes reflect major tissue-specific functions at the molecular level, such as tissue formation in the mantle, filtering in the gills and labial palps, and reproduction in the gonads. Hierarchical clustering predicted the involvement of unannotated genes in specific functional pathways such as the insulin/NPY pathway, an important pathway under study in our model species. Microarray data also accurately identified reference genes whose mRNA level appeared stable across all the analyzed tissues. Adp-ribosylation factor 1 9arf1) appeared to be the most robust reference for normalizing gene expression data across different tissues and is therefore proposed as a relevant reference gene for further gene expression analysis in the Pacific oyster. Conclusions: This study provides a new transcriptomic tool for studies of oyster biology, which will help in the annotation of its genome and which identifies candidate reference genes for gene expression analysis

From qualitative data to quantitative models: analysis of the phage shock protein stress response in Escherichia coli

Background Bacteria have evolved a rich set of mechanisms for sensing and adapting to adverse conditions in their environment. These are crucial for their survival, which requires them to react to extracellular stresses such as heat shock, ethanol treatment or phage infection. Here we focus on studying the phage shock protein (Psp) stress response in Escherichia coli induced by a phage infection or other damage to the bacterial membrane. This system has not yet been theoretically modelled or analysed in silico. Results We develop a model of the Psp response system, and illustrate how such models can be constructed and analyzed in light of available sparse and qualitative information in order to generate novel biological hypotheses about their dynamical behaviour. We analyze this model using tools from Petri-net theory and study its dynamical range that is consistent with currently available knowledge by conditioning model parameters on the available data in an approximate Bayesian computation (ABC) framework. Within this ABC approach we analyze stochastic and deterministic dynamics. This analysis allows us to identify different types of behaviour and these mechanistic insights can in turn be used to design new, more detailed and time-resolved experiments. Conclusions We have developed the first mechanistic model of the Psp response in E. coli. This model allows us to predict the possible qualitative stochastic and deterministic dynamic behaviours of key molecular players in the stress response. Our inferential approach can be applied to stress response and signalling systems more generally: in the ABC framework we can condition mathematical models on qualitative data in order to delimit e.g. parameter ranges or the qualitative system dynamics in light of available end-point or qualitative information.Medical Research Council (Great Britain)Biotechnology and Biological Sciences Research Council (Great Britain)Wellcome Trust (London, England)Royal Society (Great Britain) (Wolfson Research Merit Award

Étude fonctionnelle de l'AMP-activated protein kinase chez l'huître creuse Crassostrea gigas

L objectif de cette thèse était de caractériser les éléments appartenant à la voie de signalisation énergétique AMP-activated protein kinase chez l huître creuse Crassostrea gigas afin de comprendre son implication dans la gestion de l énergie, en particulier en réponse à des conditions physiologiques qui sollicitent de l énergie telles que la reproduction, ou à des stress environnementaux comme l hypoxie ou le jeûne. Au niveau génomique, les trois sous-unités constitutives du trimère AMPK ainsi que plusieurs éléments impliqués dans cette voie de signalisation et dans les métabolismes glucidiques et lipidiques, potentiellement cibles de l AMPK, ont été décrits. Au niveau protéique, plusieurs anticorps hétérologues ciblant les isoformes de la sous-unité a et la phosphorylation du résidu thréonine 172 de la sous-unité a, témoin indirect de l activité AMPK, ont été utilisés. Deux sous-unités a tronquées dans le domaine kinase ont été caractérisées principalement dans les tissus musculaires suggérant leurs implications dans la fonction musculaire. Au cours d un stress hypoxique, une augmentation significative des quantités de sous-unités a tronquées a été observée dans le muscle lisse. Ce résultat suggère que pendant une durée d au moins 6 h, ces protéines tronquées sont nécessaires au maintien du métabolisme aérobie dans le muscle lisse, lui permettant ainsi de remplir son rôle de fermeture statique des valves. Nous avons suggéré une hypothèse indiquant que l accumulation in vivo de ces sous-unitésa tronquées pourrait exercer un rôle de modulation ou de transdomination négative de l activité de la sous-unité a entière. Dans la gonade, nous avons observé une activation de l AMPK tout au long du processus de gamétogénèse afin de supporter les processus cataboliques de création de gamètes. Une diminution de cette activation a été observée lors du stade anabolique de mise en réserve des ovocytes. Enfin, lors d un conditionnement en milieu contrôlé, une approche physiologique par privation de nourriture et une approche pharmacologique par injection d AICAR ont été réalisées pour provoquer une modulation de l AMPK. Les analyses ont montré que ni le jeûne ni l AICAR n ont induit une augmentation de la phosphorylation de la sous-unité a. Cependant, plusieurs changements liés à l injection de l AICAR ont été observés sur la physiologie de l huître : la modification du rapport AMP:ATP chez les huîtres nourries en comparaison aux huîtres à jeun, et une mortalité dépendante de la dose injectée d AICAR chez les huîtres mises à jeun. La caractérisation de l AMPK chez C. gigas ouvre de nombreuses perspectives exigeant des études fonctionnelles poussées afin de démontrer le rôle pivot de cette kinase dans la gestion de l énergie, comme démontré chez de nombreuses espèces de vertébrés, et ainsi décrypter le métabolisme énergétique de l huître.The objective of this thesis was to characterize elements implicated in the energypathway of the AMP-activated protein kinase of the Pacific oyster Crassostrea gigas. Thecharacterization of the elements was performed in the scope of understanding their involvementin energy management, particularly in response to physiological conditions requiring energy, asreproduction or environmental stress, such as hypoxia or fasting.At genomic level, the three subunits of AMPK trimer and several elements involved inAMPK signaling pathway and in carbohydrate and lipid metabolism, supposedly under AMPKcontrol, were described. Additionally, at proteomic level, several heterologous antibodiestargeting AMPKa subunit isoforms and threonine 172 phosphorylation site of AMPKa subunit,indirect witness of AMPK activity, were assayed. Two truncated a subunits in the kinase domainwere characterized essentially in muscles, suggesting their involvement in muscle function.During a hypoxic stress, a significant increase of truncated a subunits protein amount wasobserved in smooth muscle. These results suggest that, for a period of at least 6 h, thesetruncated subunits are necessary for the maintenance of aerobic metabolism in smooth muscle ofC. gigas, allowing it to fulfill its static closing valves. We suggested that in vivo accumulation oftruncated AMPKa could serve as modulator or as transdominant negative regulator of the fulllengthAMPKa activity. In the gonad, AMPK appeared to be activated through the process ofgametogenesis, in order to support the catabolic processes of gametes creation. During theanabolic phase, when oocyte reserves were created, a signal disruption was observed. Finally,during controlled experiment, a physiological approach by food deprivation and apharmacological approach using AICAR injections were performed to modulate AMPK signal.This analysis showed that neither fasting nor AICAR induced an increase of AMPKphosphorylation, as expected. Although, several changes related to AICAR injection wereobserved in oysters physiology, such as the change of the AMP:ATP ratio in fed oysters and aAICAR dose-related mortality in fasting oysters. AMPK characterization in C. gigas opens newperspectives demanding extensive functional studies to establish the key role of AMPK in energymanagement, as demonstrated in vertebrates species, in order to understand the oyster s energymetabolism.BREST-SCD-Bib. electronique (290199901) / SudocSudocFranceF

Retroviruses integrate into a shared, non-palindromic DNA motif.

Many DNA-binding factors, such as transcription factors, form oligomeric complexes with structural symmetry that bind to palindromic DNA sequences1. Palindromic consensus nucleotide sequences are also found at the genomic integration sites of retroviruses2-6 and other transposable elements7-9, and it has been suggested that this palindromic consensus arises as a consequence of the structural symmetry in the integrase complex2,3. However, we show here that the palindromic consensus sequence is not present in individual integration sites of human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1), but arises in the population average as a consequence of the existence of a non-palindromic nucleotide motif that occurs in approximately equal proportions on the plus strand and the minus strand of the host genome. We develop a generally applicable algorithm to sort the individual integration site sequences into plus-strand and minus-strand subpopulations, and use this to identify the integration site nucleotide motifs of five retroviruses of different genera: HTLV-1, HIV-1, murine leukaemia virus (MLV), avian sarcoma leucosis virus (ASLV) and prototype foamy virus (PFV). The results reveal a non-palindromic motif that is shared between these retroviruses

"Bons baisers de..." : tourisme et touristes dans la saga James Bond

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P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish

The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used

Comparative study of shell shape and muscle scar pigmentation in the closely related cupped oysters Crassostrea angulata, C-gigas and their reciprocal hybrids

The taxonomic status of the cupped oysters Crassostrea angulata and C. gigas has received considerable attention in the last decades. Based on larval shell morphology, experimental hybridization, allozymes and nuclear DNA studies several authors have considered these two taxa as being synonymous. However, mitochondrial data showed clear genetic differences between the two taxa. In addition, microsatellite- based studies and cytogenetic studies have also provided evidence that supports their differentiation. Considerable differences have also been observed at the phenotypic level in terms of growth rate and ecophysiological parameters. In the present study, C. angulata from Sado estuary ( Portugal) and C. gigas from Seudre estuary ( France) were collected and factorial crosses were performed. Juveniles of the different progenies were reared in Ria Formosa ( Portugal) under common conditions to determine if they exhibited differences in shell shape and in pigmentation of the adductor muscle scar. Significant morphometric differences between C. angulata and C. gigas progenies were indicated by univariate and multivariate analyses. Univariate analysis of size- adjusted shell measurements revealed significant differences between the two taxa for shell depth, muscle scar height, and length of ligamental area. Both reciprocal hybrids showed intermediate morphometric characters between parental lines. In addition, significant differences were also observed between C. angulata and C. gigas progenies in terms of pigmentation of adductor muscle scar. C. angulata and both reciprocal hybrid progenies showed highly pigmented adductor muscle scars whereas in C. gigas progeny the pigmentation was lighter. The differences in shell shape and muscle scar pigmentation observed in the present study support the distinction of the two taxa.info:eu-repo/semantics/publishedVersio

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

Background: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description: In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.htm l. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion: A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism