Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

My dissertation encompasses five different studies that are linked by a common theme the investigation of transposable element (TE) contributions to eukaryotic gene sequences. A detailed analysis of exonization events of LTR elements in the human genome shows the preference towards the fixation of LTR elements in gene untranslated regions, which supports the existing concept of a major role of LTR elements as a natural source of regulatory sequences. The ability of different classes of sequence similarity search methods to detect TE-derived sequences was evaluated. In general, the different search methods are found to be complementary, and combined search approaches are needed to systematically check any data set for all potential TE-associated coding sequences. On average, TE-derived exon sequences have low protein coding potential. In particular, non-coding TEs, are frequently exonized but unlikely to encode protein sequences. Many of these non-coding exonized TEs may be actually involved in gene regulation via the formation of double stranded RNA complexes with complementary TE-derived exons. The investigation of the relationship between human miRNAs and TEs shows that 55 experimentally verified human miRNA genes (~12%) originated from TEs. Overall, TE-derived miRNA genes are less conserved than non TE-derived miRNAs. The potential regulatory and functional significance of TE-derived miRNAs was explored. An ab initio prediction algorithm I developed was used to discover putative cases of novel TE-derived miRNA genes. A miRNA gene family, hsa-mir-548, was found to be derived from Made1 family of MITEs. The palindromic structure of the Made1 elements, and MITEs in general, points to a specific mechanism by which these sequences can be recognized and processed by the miRNA biogenesis pathway. MITEs may also represent an evolutionary link between siRNAs and miRNAs. An original model for a siRNA-to-miRNA evolutionary transition mediated by DNA-type TEs is proposed. This model is supported by the presence of evolutionary intermediate TE sequences that encode both siRNAs and miRNAs in the Arabidopsis and rice genomes. The siRNA-to-miRNA evolutionary transition is representative of a number of other regulatory mechanisms that evolved to silence TEs and were later co-opted to serve as regulators of host gene expression.

Made available in DSpace on 2014-06-11T19:32:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-25Bitstream added on 2014-06-13T18:43:21Z : No. of bitstreams: 1 lopes_fr_dr_sjrp.pdf: 1411366 bytes, checksum: ca55952aff87e294af96683898e3b1e3 (MD5) / Elementos de transposição (TEs) são entidades genéticas que podem ter profundos impactos, estrutural, funcional, intra e interespecíficos na evolução dos genomas. A contribuição dos TEs para formação de novas seqüências codificadoras de proteínas é de particular interesse porque sua inserção em exons pode alterar a seqüência protéica influenciando diretamente o fenótipo. Além disso, o estudo das condições que provocam a ativação de TEs, e os mecanismos que os regulam, justifica o interesse de se identificar TEs expressos em tecidos ou condições específicas. Este estudo foca tais questões usando um modelo vegetal: C. arabica, única espécie híbrida e poliplóide do seu gênero, derivada de uma hibridização recente e natural entre C. canephora e C. eugenioides. As análises foram realizadas por meio de uma variedade de abordagens: 1) análises computacionais usando uma combinação de RepeatMasker, tBLASTx e diversas bibliotecas de TEs referência estocadas no Repbase; 2) análises de expressão baseadas em macroarranjos de DNA; e 3) avaliação do número de cópias e distribuição cromossomal de TEs ativos por Hibridização in situ fluorescente (FISH). Foram identificados 180 unigenes com fragmentos de TEs nas três espécies de café. Em uma primeira análise, com base em unigenes selecionados, foi possível sugerir 26 putativas proteínas com inserção de cassetes de TEs, demonstrando uma provável contribuição para a variabilidade do repertório protéico hospedeiro. Por outro lado, 327 ESTs similares a TEs expressos foram identificadas com uma possível abundância diferencial para duas famílias de Ty3/Gypsy (dea1 e Retrosat) identificadas em apenas duas bibliotecas de C. canephora (sementes e pericarpo). Análises de expressão mostraram que muitos dos mRNAs quiméricos e TEs ativos apresentam baixa expressão... / Transposable elements (TEs) are genetic entities that can have profound structural, intra, interspecific impacts in evolution of the genomes. The contribution of the TEs in the protein coding region is of particular interest because insertions of TE into exons can alter the protein sequence influencing directly the phenotype. Moreover, the analysis of the conditions that cause the TE activation and the mechanisms that regulate them justify the interest of identifying expressed TEs in tissues or specific conditions. This study focus such subjects using the plant model: C. arabica, unique hybrid species and polyploidy of their genus, derived of a recent and natural hybridization between C. canephora and C. eugenioides. The analyses were performed by a variety of approaches: 1) computational analyses using a combination of RepeatMasker, tBLASTx e several libraries of reference TEs stored in Repbase; 2) expression analysis based in macroarrays; and 3) evaluation of copy number e chromosomal distribution of expressed TEs by Fluorescent in situ hybridization (FISH). 180 unigenes containing TE fragments were identified in the three Coffea species. Based in selected unigenes, it was possible to identify 26 putative proteins harboring TE-cassettes, demonstrating a probable contribution for the host protein repertory variability. In addition, 327 ESTs similar to expressed TEs were identified with a probable differential abundance for two families of Ty3/Gypsy (dea1 and Retrosat) identified only in two cDNA libraries from C. canephora (seeds and pericarp). Our expression analyses showed that most of the chimerical mRNAs and expressed TEs has low or null expression. On the other hand, several transcripts had their expression reestablished in cell culture treated with Cycloheximide, a drug that permit the accumulation of transcripts previously silenced by reverting a mechanisms... (Complete abstract click electronic access below)

Chez les métazoaires, la mobilisation des éléments transposables est régulée par de petits ARN non codants appelés piARN pour "PIWI interacting RNA". Cette répression est très étudiée dans la lignée germinale adulte où elle est particulièrement efficace. Néanmoins, la mobilisation de ces éléments doit être régulée tout au long du développement de la lignée germinale, qui transmet l’information génétique à travers les générations. Durant ma thèse, j’ai utilisé le modèle D. melanogaster pour étudier la répression des éléments transposables au cours du développement de la lignée germinale femelle. J’ai ainsi pu montrer qu’une répression fonctionnelle par les piARN existe dès la fin de l’embryogenèse et que les gènes liés à la régulation chez l'adulte sont également nécessaires pour la répression au cours du développement. L’analyse de données de séquençage haut débit m’a permis de mettre en évidence la production de novo de piARN fonctionnels dans les gonades en formation. De plus, comme dans les ovaires adultes, j'ai pu remarquer une répression incomplète, ressemblant à la variégation, à tous les stades du développement. Des expériences de lignage cellulaire suggèrent fortement qu'une mémoire épigénétique précoce est initiée dans les cellules germinales embryonnaires et maintenue jusqu'au stade adulte. L'implication de l'Heterochromatin Protein 1a (HP1a) dans la production des piARN télomériques montrée par séquençage des piARN pourrait expliquer ce phénomène . Les données présentées ici montrent que piARN et leurs partenaires protéiques sont les composants d'un système de répression épigénétique continu tout au long de la vie des cellules germinales. / In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous investigations in Drosophila have enlightened the mechanism of this repression in the adult germline. However, very little is known about piRNA-mediated repression during germline development. Nevertheless, to maintain the integrity of the genome, repression should occur throughout the lifespan of germ cells. During my PhD, I show that piRNA-mediated repression is active in the female germline, from late embryonic to pupal primordial germ cells, and that genes related to the adult piRNA pathway are required for repression during development. rhino-dependent piRNAs, exhibiting the molecular signature of the piRNA pathway "ping-pong" amplification step, are detected in larval gonads, arguing for de novo biogenesis of functional piRNAs during development. I also show that production of telomeric piRNAs depends on Heterochromatin Protein 1a (HP1a). Furthermore, as in adult ovaries, I observe an incomplete, bimodal and stochastic repression resembling variegation at all developmental stages. Clonal analyzes of this incomplete silencing strongly suggest that a cellular memory of an early repression decision is initiated in embryonic germ cells and further maintained until the adult stage. Taken together, the data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.

Le développement de la seconde génération de séquenceurs haut débit a généralisé l'accès à l'étude du transcriptome via le protocole RNAseq. Celui-ci permet d'obtenir à la fois la séquence et l'abondance des transcrits d'un échantillon. De nombreuses méthodes bioinformatiques ont été et sont encore développées pour permettre l'analyse des données issues du RNAseq et en tirer le maximum d'information. Ce type d'analyse est notamment possible sans utiliser de génome de référence, et donc pour les espèces modèles ou non-modèles, grâce à des méthodes d'assemblage. Durant ma thèse, j'ai principalement travaillé à partir de données RNA-seq issues d'espèces non modèles. Je me suis intéressée dans un premier temps à l'impacte de l'hybridation inter spécifique sur la stabilité des génomes chez les hybrides issus des croisements réciproques de D. mojavensis et D. arizonae. Nos résultats ne montrent pas une dérégulation globale, mais plutôt quelques gènes et éléments transposables qui sont spécifiquement dérégulés. La pipeline d'analyse mis en place ici sera réutilisée pour l'étude des niveaux d'expression des transcrits chez les mâles ainsi que pour les croisements issus d'autres lignées de D. mojavensis avec D. arizonae, conduisant à une fertilité variable chez les hybrides.Dans un second temps, j'ai participé à la validation du logiciel KisSplice pour la détection de SNP dans des données RNA-seq sans génome de référence. Celui-ci permet de trouver différents types de variants (épissage, indels) directement dans le graphe de de Bruijn construit à partir des lectures séquencées. J'ai également participé au développement d'outils de post-traitement permettant de prédire l'impact des SNP sur les protéines / Next-generation high throughput sequencing technologies provide efficient, rapid, and low cost access to sequencing. Its application to transcriptomes, called RNA-seq, enables the study of both the sequence and the expression of the transcripts. Many bio-informatics methods are still developed for RNA-seq data processing, trying to get the maximum out of it. Assembly methods allow us to study non-model species (no reference genome available) as well as model species. The work presented here is mostly related to RNA-seq data on non-model species.In the first study, to understand the initiation of hybrid incompatibility, we performed a genome-wide transcriptomic analysis on ovaries from parental lines and on hybrids from reciprocal crosses of \emph{D. mojavensis} and \emph{D. arizonae}. We didn't see a global deregerulation of genes or transposable element. Instead, we show that reciprocal hybrids presented specific gene categories and few transposable element families misexpressed relative to the parental lines. The analytical workflow developed for this project will be used to analyze transcriptomic data from the testis, but also to study the reciprocal crosses from other lines of D. mojavensis with D. arizonae leading to variable levels of sterility in hybrids. A second project tacked here is the identification and quantification of SNPs from RNA-seq data without a reference genome with KisSplice. Kissplice was developed to identified several type of variants (splicing events, indels) directly from the de Bruijn graph, build from the sequenced reads. We also developed other KisSplice-tools, for downstream analyses of the SNPs, including the prediction of their impact on the protein sequence

No description available.

Les Eléments Transposables (ET) sont des parasites du génome caractérisés par leur capacité à se répliquer plus rapidement que les autres éléments génétiques du génome. La régulation par la voie des piARN joue un rôle essentiel pour limiter l’expansion des ET dans les lignées germinales des animaux.La première question posée est comment le génome répond face à une nouvelle invasion par un ET. Dans ce but, nous avons introduit le transposon de Classe II mariner (sous-famille mos1) chez D. melanogaster, qui ne contient naturellement pas l’élément. Nous avons montré, qu’après son amplification autonome dans le génome, l’élément avait atteint un équilibre en termes de nombre de copies, depuis qu’une régulation de novo par les piARN avait été acquise.Deuxièmement, nous avons étudié la mobilisation de l’élément mariner au cours du processus de colonisation des régions géographiques tempérées. A partir d’un large panel de populations naturelles nous avons trouvé que l’activité moyenne de mariner était remarquablement augmentée dans les populations non-Africaines en comparaison aux populations Africaines. Ces variations peuvent s’expliquer par un fort polymorphisme d’expression (transcriptionnel et traductionnel) des gènes de la voie des piARN.Le troisième chapitre soutient que la forte activité des ET dans la lignée germinale mâle est un phénomène global chez les drosophiles. Par ailleurs, le contenu en ET chez les espèces sœurs (D. melanogaster et D. simulans) a fortement divergé et, cela a affecté la réponse associée à la production des piARN. Chez D. melanogaster, de nombreux « burst » de transposition ont eut lieu récemment. Ces familles d’ET sont activement réprimées par les piARN dans l’ovaire et donc, se retrouvent massivement surexprimés dans les testicules. Chez D. simulans, nous pensons que la réponse par les piARN résulte principalement d’une régulation passée qui semble être la relique d’anciennes invasions d’ET.La voie des piARN est supposé être « garante de l’intégrité du génome » de par son rôle actif dans la défense contre les ET. Cependant, si la sélection naturelle purge les génomes de ces parasites délétères, il semble que les mécanismes de régulation de l’hôte contribuent au maintien de l’homéostasie du génome en limitant leur expansion, et quelque part en favoriser le maintien sur long terme. Ainsi, une autre interprétation pourrait être que la voie des piARN est « garante de la diversification du génome » de par son rôle à faciliter l’accumulation des ET. / Transposable Elements (TEs) are genomic parasites characterized by their ability to replicate faster than any other genetic element in the genomes. The piRNA mediated silencing is of central importance to limit TE expansion in the germline of animal species. The present dissertation explores the relationship between TEs and piRNAs alongside their evolutionary dynamics.The first question raised here was to understand how the genome responds to a new TE invasion. For that purpose, we injected a mariner Class II transposon into D. melanogaster genome that does not naturally contain the element. We found that, after its self-replication into the genome, the element have reached a copy number equilibrium since a de novo piRNA mediated regulation have been acquired.Second, we studied the mariner rewiring activity during the colonization of geographical temperate regions. From a large sampling of D. simulans natural populations, we found the mean activity of mariner to be strikingly higher in non-African populations compared to the African ones. These findings support the idea that selection acting on piRNA effector proteins has been of central importance to explain TE lineages diversification during colonization process.The third chapter provides evidences to propose that, the strong TE activity in testes, is a general phenomenon in Drosophila. We also observed that TE landscape divergence between the two sister species, have affected the genomic response mediated by the piRNAs. As a response of their recent bursts of transposition, TEs overexpressed in testes are preferentially silenced by piRNAs in D. melanogaster ovaries. By contrast, we assumed the D. simulans piRNA response to be the relic of a past regulation that still persists mostly against inactive TEs.The piRNA silencing in the germline, is assumed to be the “vanguard of genome” defense and integrity due to its active role against TEs. However, while natural selection purifies the genome from its deleterious parasites, it seems that the host regulation contributes to genome homeostasis by limiting their expansion, and somehow, favors their longterm maintenance. Thus, another interpretation would have been that piRNA silencing is the “vanguard of genome” diversification due to its active role in facilitating TE accumulation

Transposable elements (TEs) are mobile genetic elements capable of replicating and spreading within, and in some cases, between genomes. I describe a whole-genome analysis of DD34E TEs, which belong to the IS630-Tc1-mariner superfamily of DNA transposable elements, in the African malaria mosquito, Anopheles gambiae. Twenty-six new transposons as well as a new family, gambol, were identified. The gambol family shares the DD34E catalytic motif with Tc1-DD34E transposons, but is distinct from these elements in their phylogenetic relationships. Although gambol appears to be related to a few DD34E transposons from cyanobacteria and fungi, no gambol elements have been reported in any other insects or animals thus far. This discovery expands the already expansive diversity of the IS630-Tc1-mariner TEs, and raises interesting questions as to the origin of gambol elements and their apparent diversity in An. gambiae. Several DD34E transposons discovered in An. gambiae possess characteristics that are associated with recent transposition, such as high sequence identity between copies, and intact terminal-inverted repeats and open reading frames. One such element, AgTango, was also found in a distantly related mosquito species, Aedes aegypti, at high amino acid sequence identity (79.9%). It was discovered that Tango transposons have patchy distribution among twelve mosquito species surveyed using PCR as well as genomic searches, suggesting a possible case for horizontal transfer. Additionally, it was discovered that in some mosquito genomes, there are several Tango transposons. These observations suggest differential evolutionary scenarios and/or TE-host interaction of Tango elements between mosquito species. This strengthened the case that AgTango may be a functional transposase, and I sought to test its potential activity in a cell culture-based inter-plasmid transposition assay using the Herves plasmids as a positive control (Arensburger et al., 2005). AgTango constructs were successfully constructed; however, no transposition events were detected for Tango or Herves. Because the positive control failed to work, no assessment can be made concerning Tango's transposase. Possible causes and solutions for these results, alternative means to detect transposition, as well as future directions with Tango are discussed. / Ph. D.

No description available.