269 research outputs found
Impact des éléments mobiles de l'ADN sur l'adaptabilité et le transfert horizontal chez Aeromonas salmonicida ssp.salmonicida
Les Ă©lĂ©ments mobiles de lâADN, des sĂ©quences capables de transfert intracellulaire ou extracellulaire, existent dans la plupart des gĂ©nomes (eucaryotes ou procaryotes). Ce sont des vecteurs importants de variation gĂ©nĂ©tique. Certains portent des gĂšnes accessoires qui procurent un avantage sĂ©lectif Ă leur organisme-hĂŽte. Les sĂ©quences dâinsertion (IS) sont un cas spĂ©cial dâĂ©lĂ©ments mobiles bactĂ©riens. Elles sont exemptes de gĂšnes accessoires, mais autonomes pour leur transposition. Elles peuvent donc causer des variations structurales, ce qui peut perturber les fonctions de la bactĂ©rie-hĂŽte. NĂ©anmoins, la plupart des gĂ©nomes bactĂ©riens portent plusieurs copies de diffĂ©rentes familles dâIS. Plusieurs hypothĂšses, allant du parasitisme des IS Ă une forme de commensalisme IS-bactĂ©rie, ont Ă©tĂ© formulĂ©es pour expliquer la persistance de ces Ă©lĂ©ments mobiles dans les gĂ©nomes. Aeromonas salmonicida ssp. salmonicida, lâagent Ă©tiologique de la furonculose chez les salmonidĂ©s, est un modĂšle intĂ©ressant pour Ă©tudier les IS. Son gĂ©nome possĂšde plusieurs familles dâIS prĂ©sentes en diffĂ©rents nombres de copies. De plus, des travaux rĂ©alisĂ©s prĂ©cĂ©demment ont montrĂ© que ces Ă©lĂ©ments mobiles Ă©taient responsables de variations structurales dans les plasmides de la bactĂ©rie et de lâinactivation de certains de ses facteurs de virulence. Bien que les Ă©vĂšnements mentionnĂ©s ci-dessus semblent neutres ou dĂ©savantageux, il nâest pas exclu que certains Ă©vĂšnements impliquant les IS puissent apporter un avantage adaptatif Ă la bactĂ©rie. Lâobjectif de cette thĂšse Ă©tait donc dâexplorer les Ă©lĂ©ments mobiles du gĂ©nome dâA. salmonicida ssp. salmonicida, en portant une attention particuliĂšre aux IS, afin dâidentifier des Ă©vĂšnements Ă rĂ©percussion bĂ©nĂ©fique pour la bactĂ©rie. La richesse dâinformation et lâaccessibilitĂ© grandissante du sĂ©quençage Ă haut dĂ©bit en ont fait une technique de choix pour Ă©tudier les IS et autres transposons en association avec un autre Ă©lĂ©ment mobile essentiel Ă leur transmission intercellulaire, les plasmides. Une premiĂšre Ă©tude a permis de dĂ©couvrir des variants dâun grand plasmide dâA. salmonicida ssp. salmonicida, pAsa4, dont la forme de rĂ©fĂ©rence porte des gĂšnes de rĂ©sistance aux antibiotiques, des IS et un transposon. Les variants identifiĂ©s lors de lâĂ©tude, pAsa4b et pAsa4c, prĂ©sentent des variations structurales par rapport Ă la rĂ©fĂ©rence. Elles ont des consĂ©quences sur les gĂšnes de rĂ©sistance portĂ©s et sur la capacitĂ© de transfert des plasmides. La gĂ©nomique comparative faite entre les pAsa4 suggĂšre que les Ă©lĂ©ments mobiles tiennent un rĂŽle primordial dans la crĂ©ation de certaines variations structurales, tant par leur capacitĂ© de transposition ou dâintĂ©gration que par leur propension Ă la recombinaison homologue. LâĂ©largissement de la famille des pAsa4 Ă trois variants procure dâautres exemples de variabilitĂ© attribuable aux Ă©lĂ©ments mobiles de lâADN. Dans une second temps, nous avons approfondi un Ă©vĂšnement Ă©tudiĂ© prĂ©cĂ©demment : la perte du locus du systĂšme de sĂ©crĂ©tion de type trois (SSTT). Cette rĂ©gion, situĂ©e sur le grand plasmide pAsa5, peut ĂȘtre perdue lorsque A. salmonicida ssp. salmonicida est cultivĂ©e Ă 25°C. Dans certains cas, la recombinaison homologue dâIS flanquant les rĂ©gions touchĂ©es avait Ă©tĂ© mise en cause. Dans le cadre de ce projet, le gĂ©nome de la souche 01-B526, qui avait Ă©tĂ© utilisĂ©e pour gĂ©nĂ©rer des pAsa5 rĂ©arrangĂ©s, a Ă©tĂ© sĂ©quencĂ© par une combinaison de technologies Ă courtes et Ă longues lectures. Un nouveau plasmide, pAsa9, ainsi quâune nouvelle IS de pAsa5, ISAS5Z, ont Ă©tĂ© dĂ©couverts. Ensemble, ces deux Ă©lĂ©ments ont permis de proposer un nouveau schĂ©ma de recombinaison homologue qui sâest avĂ©rĂ© exact pour tous les rĂ©arrangements de pAsa5 gĂ©nĂ©rĂ©s lors de lâexpĂ©rience prĂ©cĂ©dente et considĂ©rĂ©s comme irrĂ©guliers. Ces nouveaux rĂ©sultats permettent de regrouper tous les scĂ©narios de rĂ©arrangement de pAsa5 sous un seul mĂ©canisme, la recombinaison homologue entre IS, en plus dâajouter une nouvelle entitĂ© au plasmidome dâA. salmonicida ssp. salmonicida. Plusieurs Ă©vĂšnements ont donc Ă©tĂ© ajoutĂ©s au catalogue des variations structurales causĂ©es par les Ă©lĂ©ments mobiles de lâADN dâA. salmonicida ssp. salmonicida. Dans les derniers chapitres de cette thĂšse, les consĂ©quences de ces diffĂ©rents Ă©vĂšnements sont discutĂ©es. Certaines variations sur pAsa4 apportent des gĂšnes accessoires qui sont bĂ©nĂ©fiques dans un environnement donnĂ©. Autrement, lâĂ©tude des Ă©lĂ©ments observĂ©s est limitĂ©e par le manque de connaissance sur les facteurs environnementaux qui causent ces variations et sur leur impact in vivo, chez le poisson. Quoi quâil en soit, les IS du gĂ©nome dâA. salmonicida ssp. salmonicida influencent certainement les relations hĂŽte-pathogĂšne de cette bactĂ©rie et participent intimement Ă son histoire Ă©volutive.Mobile genetic elements are sequences capable of intracellular and extracellular transfer. They exist in the majority of eukaryotic and prokaryotic genomes, and are important vectors of genetic variability. Some mobile genetic elements carry accessory genes that confer selective advantages to their host. Insertion sequences (IS), do not belong to the latter group, as they are exempt of accessory genes, yet they are autonomous in their transposition. ISs are thus important structural variation producers, which may endanger the hostâs functions and its genomic integrity. However, most bacterial genomes carry ISs. Hypotheses ranging from parasitism to commensalism were formulated to explain how ISs persist in genomes. A. salmonicida subsp. salmonicida, the causative agent of furunculosis in salmonids, is a good model to study ISs. Its genome carries many ISs divided in different families. Moreover, previous work showed that these elements cause structural variations in plasmids and virulence factor inactivations. Even though the events observed to date seemed neutral or detrimental for the bacteria, we cannot exclude that ISs may procure an adaptative advantage in certain cases. This thesisâ objective was to explore the A. salmonicida subsp. salmonicida mobile genetic elements, especially ISs, to identify events with a positive outcome for the bacteria. ISs and transposons were studied in association with another mobile element accountable for their intercellular transfer, plasmids. High-throughput sequencing was used as a preferred method because of its growing availability and the richness of information it generates. In a first article, variants of a large plasmid that carries antibiotic resistance genes, pAsa4, were discovered. pAsa4b and pAsa4c display structural variations when compared to the reference plasmid that impact their antibiotic resistance genes and their conjugation capabilities. Comparative genomics between the three variants shows that mobile elements, either by transposition or recombination, have an important role in some variations. Adding two new members to the pAsa4 family of plasmids gave further examples of genetic diversity driven by mobile elements. In a second article, we investigated previous results concerning the type three secretion system (TTSS) loss. This is an essential virulence factor for A. salmonicida subsp. salmonicida, and its genes are mainly encoded in a single locus of the large plasmid pAsa5. The region can be lost when the bacterium is cultivated at 25°C or higher. In some cases, homologous recombinations between IS copies flanking the TTSS locus have been identified as causing the deletion. In this study, the genome of the strain 01-B526, which was used in a previous study to produce TTSS-negative mutants, was sequenced using Illumina and PacBio technologies. A new plasmid, named pAsa9, and a new pAsa5 IS, ISAS5Z, were discovered. Together, those elements permitted to build a new IS-driven homologous recombination model that was tested in the mutant strains. All TTSS-negative strains generated in previous study and that were not explained by other IS recombination patterns fitted with the new ISAS5 homologous recombination model. These results allowed to regroup all pAsa5 rearrangements under a consistent mechanism: recombination using IS copies as a template. In this thesis, more structural variation events involving ISs were uncovered. In the last chapters, their involvement in A. salmonicida subp. salmonicida fitness in vitro and in the fish host is discussed. Some variations in pAsa4 bring new resistance genes, which are beneficial for the bacteria in an aquaculture context. However, for most events, we lack situational information to properly conclude their fitness impact. Regardless, ISs of the A. salmonicida subp. salmonicida genome certainly influence host-pathogen relations and participate in the bacteria evolutive history
Variants of a genomic island in Aeromonas salmonicida subsp. salmonicida link isolates with their geographical origins
Aeromonas salmonicida subsp. salmonicida is a fish pathogen. Analysis of its genomic characteristics is required to determine the worldwide distribution of the various populations of this bacterium. Genomic alignments between the 01-B526 pathogenic strain and the A449 reference strain have revealed a 51-kb chromosomal insertion in 01-B526. This insertion (AsaGEI1a) has been identified as a new genomic island (GEI) bearing prophage genes. PCR assays were used to detect this GEI in a collection of 139 A. salmonicida subsp. salmonicida isolates. Three forms of this GEI (AsaGEI1a, AsaGEI1b, AsaGEI2a) are now known based on this analysis and the sequencing of the genomes of seven additional isolates. A new prophage (prophage 3) associated with AsaGEI2a was also discovered. Each GEI appeared to be strongly associated with a specific geographic region. AsaGEI1a and AsaGEI2a were exclusively found in North American isolates, except for one European isolate bearing AsaGEI2a. The majority of the isolates bearing AsaGEI1b or no GEI were from Europe. Prophage 3 has also a particular geographic distribution and was found only in North American isolates. We demonstrated that A. salmonicida subsp. salmonicida possesses unsuspected elements of genomic heterogeneity that could be used as indicators to determine the geographic origins of isolates of this bacterium.Keywords : Bacteria, Genomics-functional genomics-comparative genomics; Furunculosis; Aeromonas salmonicida; Fish pathogen; Genomic island; Geographical distributio
Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens
We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region's past and offers a new context within which the late Holocene "Earthmovers" of the Llanos de Moxos could have emerged. © 2013 Lombardo et al
An Insertion Sequence-Dependent Plasmid Rearrangement in Aeromonas salmonicida Causes the Loss of the Type Three Secretion System
Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida
Overexpression of the oncostatin-M receptor in cervical squamous cell carcinoma is associated with epithelial-mesenchymal transition and poor overall survival.
BACKGROUND: Copy-number gain of the oncostatin-M receptor (OSMR) occurs frequently in cervical squamous cell carcinoma (SCC) and is associated with adverse clinical outcome. We previously showed that OSMR overexpression renders cervical SCC cells more sensitive to the major ligand oncostatin-M (OSM), which increases migration and invasion in vitro. We hypothesised that a major contribution to this phenotype would come from epithelial-mesenchymal transition (EMT). METHODS: We performed a comprehensive integrated study, involving in vitro cell line studies, in vivo animal models and numerous clinical samples from a variety of anatomical sites. RESULTS: In independent sets of cervical, head/neck and lung SCC tissues, OSMR expression levels correlated with multiple EMT-associated phenotypic markers and transcription factors. OSM treatment of OSMR overexpressing cervical SCC cells produced consistent EMT changes and increased tumour sphere formation in suspension culture. In a mouse model, OSMR overexpressing SCC cells treated with OSM showed significant increases in lung colonisation. The biological effects of exogenous OSM were mirrored by highly significant adverse overall survival in cervical SCCs with OSMR overexpression (N=251). CONCLUSIONS: OSM:OSMR interactions are able to induce EMT, increased cancer stem cell-like properties and enhanced lung colonisation in SCC cells. These changes are likely to contribute to the highly significant adverse outcome associated with OSMR overexpression in cervical SCCs.This work was supported by Cancer Research UK (Programme Grant A13080).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Nature Publishing Group
Novel features of ARS selection in budding yeast Lachancea kluyveri
<p>Abstract</p> <p>Background</p> <p>The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.</p> <p>Results</p> <p>In this study we have isolated and characterized autonomously replicating sequences (ARSs) in <it>Lachancea kluyveri </it>- a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that <it>L. kluyveri </it>ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in <it>Saccharomyces cerevisiae</it>. Moreover, compared with <it>S. cerevisiae </it>and <it>K. lactis</it>, the replication licensing machinery in <it>L. kluyveri </it>seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all <it>S. cerevisiae </it>ARSs tested and most <it>Kluyveromyces lactis </it>ARSs. In contrast, only about half of the <it>L. kluyveri </it>ARSs function in <it>S. cerevisiae </it>and less than 10% function in <it>K. lactis</it>.</p> <p>Conclusions</p> <p>Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.</p
A Genome-Wide Characterization of MicroRNA Genes in Maize
MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCRâRACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with âŒ35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes
- âŠ