Dissertations / Theses on the topic 'Transposable elements; repeats; evolution'

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.

Cílem práce je seznámení se s problematikou uchovávání informace v DNA, provést rešerši na téma transpozony, bioinformatické nástroje a algoritmy, které jsou používány k jejich detekci v nasekvenovaných genomech a vytvořit tak stručný úvod do obsáhle problematiky, včetně jejího zasazení do kontextu současně probíhajícího výzkumu v dané oblasti. Na základě přehledu stávajících algoritmů a nástrojů pro detekci transpozonů je navržen a implementován nástroj pro hledání tzv. LTR transpozonů.

In this thesis we investigate the mappability of human genome and we look at some reasons that might cause unmappability in a region. We look at transposable elements and genome duplications as the main reasons for unmappability. In this analysis we simulated singled end, paired end and mate paired reads of 6 different lengths and we used BWA to map these simulated reads to the human genome. We assumed that a position in the genome is mappable if there is at least one unique read mapped to that position. We looked at unmappable regions and fraction of transposable elements or genome duplications corresponding to these regions. We also looked at age distribution of transposable elements and genome duplications that are in unmappable regions. Our results shows that regions that are in younger and longer transposable elements are harder to sequence. In order to compare our simulated data with a real sequencing data, we used the output of a sequencing from Illumina to compare coverage of genome in this real data set with our mappability results. We show that 4.1% of genome that is mappable in our simulations result, has low coverage in real sequencing data. We also investigated the reasons behind having low coverage in mappable regions. Our simulation result shows the impact of transposable elements and other repeats on mappability in the human genome and we show that using longer paired end and mate paired reads improves the mappability of the human genome.
Dans cette thèse, nous étudions la "visibilité" du génome humain par des méthodes séquençage modernes et nous regardons quelles sont les raisons qui pourraient causer l'absence de visibilité dans une région donnée. Nous montrons que les éléments transposables et les duplications de génome sont les principaux obstables à la visibilité de régions génomiques. Dans cette analyse, nous avons utilisé des reads simulés, de types individuels ou pairés, de 6 longueurs différentes et nous avons utilisé BWA pour assigner ces reads au génome humain. Nous avons supposé que la position dans le génome est visible s'il y a au moins un read unique assigné à cette position. Nous avons examiné les régions non visibles et la fraction d'éléments transposables ou des duplications de génome correspondant à ces régions. Nous avons également examiné la distribution d'âge des éléments transposables et des duplications de génome qui sont dans les régions non visibles. Nos résultats montrent que les régions qui sont des éléments plus jeunes et plus transposable sont plus difficiles à séquencer. Afin de comparer nos données simulées avec les données réelles de séquençage, nous avons utilisé des données de reséquençage provenant d'un séquençage Illumina pour comparer la couverture observée du génome avec nos résultats provenant de données simulées. Nous montrons que 4,1% du génome qui est visible dans nos simulations a une faible couverture dans les données de séquençage réelles. Nous avons également étudié les raisons pouvant expliquer une faible couverture dans les régions visibles. Les résultats de nos simulations montrent l'impact des éléments transposables et les autres répétitions sur la visibilité dans le génome humain et nous montrent que l'utilisation de long reads pairés améliorent la visibilité du génome humain.

The different genomic environments in which transposable elements reside in the Great Apes and the Drosophila result in substantial differences between the evolution of transposable elements in these two groups of organisms. In the Great Apes, where deletion of transposable elements is relatively rare, elements tend to be retained in the genome to the extent that complete sets of elements belonging to a particular transposable element family can be obtained. In Drosophila, there is a rapid turnover of transposable elements, imposing strong selection pressure on transposable elements to be able to infect new hosts. This study investigates the evolution of transposable elements in these two genomic environments. Complete sets of elements belonging to young Alu subfamilies in humans and closely-related species are used to investigate factors involved in their evolutionary history, such as mutation and gene conversion. The application of the master gene model, and other proposed models of the proliferation of young Alu subfamilies, are considered in light of the results obtained. The evolution of the AluYg, Yh and Yi lineages are investigated using a C++ program to simulate their evolutionary history. The results of the simulations are compared to statistics such as theta and pi, as well as the number of shared mutations and the proliferation time, in order to determine possible, and likely, values for parameters such as the retrotransposition rate and the number of source elements for each subfamily. The results suggest that although the master gene model may apply to some lineages, it is not the best model to explain the evolutionary history of all young Alu subfamilies. The selection pressure on transposable elements in Drosophila results in a high level of horizontal transfer of these elements among species of the Drosophila genus. In this study, the twelve sequenced Drosophila genomes are used to investigate the frequency of horizontal transfer within these twelve species using a large dataset of transposable element sequences from the DNA transposons, as well as LTR, and non-LTR, retrotransposons. Horizontal transfer is inferred where identity between transposable elements of the same family in different species exceeds that between the coding regions of the Adh gene in the relevant species. Cases are further supported by evidence from the distribution of the transposable element family across the Drosophila genus, and phylogenetic incongruence, which in many cases elucidates likely directions of transfer. The results suggest that horizontal transfer may be even more common than previously thought, and appears to be most common for the LTR retrotransposons. The possibility that possession of the env gene may result in higher rates of horizontal transfer of LTR retrotransposons is investigated, and the env open reading frame is found to be under selective constraint.

Los transposones son elementos genéticos que tienen la capacidad de modificar su posición en el genoma. Como consecuencia, tienen un impacto en la evolución de los genomas inactivando o alterando los genes del huésped y proporcionando nuevas funciones génicas. Los transposones ocupan una fracción importante en todos los genomas resecuenciados. El objetivo del trabajo presentado en esta tesis trata en estudiar los distintos impactos de transposones tanto en genes como en la evolución de los genomas de distintas especies de plantas. En esta tesis, se ha analizado la fracción de transposones en melón y pepino, dos especies muy cercanas. Los resultados sugieren que los transposones han proliferado más en melón, causando un aumento del tamaño del genoma. Los transposones no se encuentran distribuidos habitualmente de forma homogénea y tienden a acumularse en las regiones pericentroméricas heterocromáticas, como es el caso de los genomas de melón y pepino. Curiosamente, los resultados presentados muestran que los transposones han expandido las regiones pericentroméricas en melón, demostrando que los transposones pueden modificar la estructura de los genomas. El número de genomas de referencia de plantas disponibles y el número de variedades resecuenciadas ha crecido exponencialmente permitiendo estudiar la correlación entre las variaciones genéticas y fenotípicas. El propósito del trabajo resumido en la segunda parte de esta tesis consiste en analizar el impacto de los transposones en genomas de especies cultivables, detectando los polimorfismos ocasionados por la presencia o ausencia de transposones en un locus concreto, a través de la comparación de una variedad resecuenciada respecto al genoma de referencia. El análisis de inserciones polimórficas de transposones se ha realizado en tres especies distintas: melón, palmera datilera y Physcomitrella patens. Los resultados obtenidos pueden ayudar a identificar familias de transposones activas recientemente y proporcionar información nueva sobre polimorfismos genéticos que pueden estar ligados a caracteres seleccionados durante la evolución reciente de estas tres especies. Para estudiar el impacto de la transposición en la regulación génica, el trabajo presentado en la tercera parte de esta tesis se centra en la capacidad de los transposones en amplificar y redistribuir sitios de unión a factores de transcripción. Los resultados muestran que algunas familias de MITEs se han amplificado y redistribuido los sitios de unión del factor de transcripción E2F durante la evolución de algunas especies del género Brassica. El objetivo de este trabajo ha sido evaluar el impacto de los sitios de unión a E2F localizados dentro de transposones reprogramando la regulación de genes en la red transcripcional de E2F. Los resultados obtenidos han determinado que los sitios de unión a E2F localizados dentro de transposones tienen capacidad de unirse a los factores de transcripción de E2F in vivo, independientemente de las marcas epigenéticas en la región. Además, los transposones se utilizan como herramienta genética útil para generar colecciones de mutantes en animales y plantas debido a su capacidad de integrar copias en el genoma. En plantas, algunos retrotransposones se integran preferentemente cerca de genes siendo particularmente interesantes para la mutagénesis. De entre todos, el retrotransposón de tabaco Tnt1 se ha utilizado para generar mutantes en distintas especies de plantas. La última parte de esta tesis consiste en analizar la capacidad del retrotransposón de tabaco Tnt1 en transponer en el musgo Physcomitrella patens, ya que se demostró que Tnt1 transpone eficientemente en P. patens y se integra preferentemente cerca de genes. Finalmente, este estudio presenta vectores derivados de Tnt1 diseñados para transponer con alta eficiencia y ser utilizados para generar colecciones de mutantes con inserciones estables en esta especie briofita.
Els transposons són elements genètics que tenen la capacitat de modificar la seva posició dins el genoma. Com a conseqüència, tenen un impacte en l’evolució del genomes inactivant o alterant els gens de l’hoste i proporcionant noves funcions gèniques. Els transposons ocupen una fracció important de tots els genomes seqüenciats. L’objectiu del treball presentat en aquesta tesis consisteix en estudiar els diversos impactes de transposons tant en els gens com en l’evolució dels genomes de diferents espècies de plantes. En aquesta tesis, s’ha analitzat la fracció de transposons en meló i cogombre, dues espècies molt properes. Els resultats suggereixen que els transposons han proliferat més en meló, causant un augment de la mida del genoma. Els transposons no es troben distribuïts habitualment de forma homogènia i tendeixen a acumular-se en les regions pericentromèriques heterocromàtiques, com el cas dels genomes de meló i cogombre. Curiosament, els resultats presentats mostren que els transposons han expandit les regions pericentromèriques en meló, demostrant que els transposons poden modificar l’estructura dels genomes. El número de genomes de referència de plantes disponibles i el número de varietats reseqüenciades ha crescut exponencialment permetent estudiar la correlació entre les variacions genètiques i fenotípiques. El propòsit del treball resumit en la segona part d’aquesta tesis consisteix en analitzar l’impacte dels transposons en genomes d’espècies cultivables detectant els polimorfismes deguts a la presència o absència de transposó en un locus concret, comparant una varietat reseqüenciada respecte al seu genoma de referència. L’anàlisi d’insercions polimòrfiques de transposons s’ha realitzat en tres espècies diferents: meló, palmera datilera i Physcomitrella patens. Els resultats obtinguts poden ajudar a identificar famílies de transposons actives recentment i proporcionar informació nova sobre polimorfismes genètics que poden estar lligats a caràcters seleccionats durant l’evolució recent d’aquestes tres espècies. Per tal d’estudiar l’impacte de la transposició en la regulació gènica, el treball presentat en la tercera part d’aquesta tesis se centra en la capacitat dels transposons en amplificar i redistribuir llocs d’unió a factors de transcripció. Els resultats mostren que algunes famílies de MITEs s’han amplificat i han redistribuït els llocs d’unió del factor de transcripció E2F durant l’evolució d’algunes espècies del gènere Brassica. L’objectiu d’aquest treball és avaluar l’impacte dels llocs d’unió a E2F localitzats dins de transposons reprogramant la regulació de gens de la xarxa transcripcional de E2F. Els resultats obtinguts han determinat que els llocs d’unió a E2F localitzats dins de transposons tenen la capacitat d’unir-se als factors de transcripció de E2F in vivo, independentment de les marques epigenètiques de la regió. A més a més, els transposons s’han convertit en eines genètiques útils per generar col·leccions de mutants en animals i plantes degut a la seva capacitat d’integrar còpies en el genoma. En plantes, alguns retrotransposons s’integren preferentment a prop de gens sent particularment interessants per la mutagènesis. Entre tots ells, el retrotransposó de tabac Tnt1 s’ha utilitzat per generar mutants en diferents espècies de plantes. L’última part d’aquesta tesis consisteix en analitzar la capacitat del retrotransposó de tabac Tnt1 en transposar en la molsa Physcomitrella patens. S’ha demostrat que Tnt1 transposa eficientment en P. patens i s’integra preferentment a prop de gens. Aquest estudi presenta vectors derivats de Tnt1 dissenyats per transposar amb alta eficiència i ser utilitzats per generar col·leccions de mutants amb insercions estables en aquest briòfit.
Transposable elements are genetic elements that have the capacity to modify their position within the genome. As a consequence, they impact the evolution of genomes by inactivating or altering host genes and by providing new gene functions. Transposons account for an important fraction of all sequenced genomes. The goal of the work presented in this dissertation is to investigate the diverse impacts of transposons on gene and genome evolution in different plant species. The transposon content has been analyzed in melon and cucumber, two closely related species. The results suggest that transposons have proliferated to a greater extend in melon, causing an increase of its genome size. Transposable elements are usually not homogenously distributed and tend to accumulate in heterochromatic pericentromeric regions. This is also the case of melon and cucumber genomes. Interestingly, the results presented show that transposons have expanded the pericentromeric regions in melon, showing that transposons can modify the structure of genomes. The number of plant reference genomes made available and the number of varieties resequenced is growing exponentially, and this is allowing to study the correlation between genetic and phenotypic variations. The purpose of the work summarized in the second part of this dissertation is to analyze the impact of transposons in crop genomes by detecting polymorphisms due to the presence or absence of transposon at a given locus, comparing one resequenced variety respect to the reference genome. The analysis of transposon-related polymorphism insertions has been performed in three different species: melon, date palm and Physcomitrella patens. The results obtained can help to identify the transposon families recently active and to provide new information on genetic polymorphisms that can be linked to traits selected during the recent evolution of these three species. In order to study the impact of transposition on gene regulation, the work reported in the third part of this dissertation focuses on the capacity of transposons to amplify and redistribute transcription factor binding sites. The results show that some MITE families have amplified and redistributed the binding sites of E2F transcription factor during Brassica evolution. The goal of this study was to assess the impact of E2F binding sites located within a transposon in reprogramming gene regulation on the E2F transcriptional network. The results obtained have determined that E2F binding sites located within transposons have the capacity to bind E2F transcription factor in vivo, regardless the epigenetic mark context. Moreover, transposons have become a useful genetic tool to generate mutant collections in animals and plants due to the capacity to insert copies into the genome. In plants, some retrotransposons have been shown to integrate preferentially near genes making them particularly interesting for mutagenesis. Among them, the tobacco retrotransposon Tnt1 has been used to generate mutants in different plant species. The last part of this dissertation consists in analyzing the capacity of the tobacco retrotransposon Tnt1 to transpose in the moss Physcomitrella patens. It shows that Tnt1 efficiently transposes in P. patens and inserts preferentially in genic regions. This work presents Tnt1-derived vectors designed for high efficiency transposition that could be used to generate stable insertion mutant collections in this bryophyte species.

Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008.
Committee Chair: Jordan, I. King; Committee Member: Borodovsky, Mark; Committee Member: Bunimovich, Leonid; Committee Member: Choi, Jung; Committee Member: McDonald, John.

BACKGROUND: Comparative evolutionary analysis of whole genomes requires not only accurate annotation of gene space, but also proper annotation of the repetitive fraction which is often the largest component of most if not all genomes larger than 50 kb in size. RESULTS: Here we present the Rice TE database (RiTE-db) - a genus-wide collection of transposable elements and repeated sequences across 11 diploid species of the genus Oryza and the closely-related out-group Leersia perrieri. The database consists of more than 170,000 entries divided into three main types: (i) a classified and curated set of publicly-available repeated sequences, (ii) a set of consensus assemblies of highly-repetitive sequences obtained from genome sequencing surveys of 12 species; and (iii) a set of full-length TEs, identified and extracted from 12 whole genome assemblies. CONCLUSIONS: This is the first report of a repeat dataset that spans the majority of repeat variability within an entire genus, and one that includes complete elements as well as unassembled repeats. The database allows sequence browsing, downloading, and similarity searches. Because of the strategy adopted, the RiTE-db opens a new path to unprecedented direct comparative studies that span the entire nuclear repeat content of 15 million years of Oryza diversity.

Background: Mutator-like transposable elements, a class of DNA transposons, exist pervasively in both prokaryotic and eukaryotic genomes, with more than 10,000 copies identified in the rice genome. These elements can capture ectopic genomic sequences that lead to the formation of new gene structures. Here, based on whole-genome comparative analyses, we comprehensively investigated processes and mechanisms of the evolution of putative genes derived from Mutator-like transposable elements in ten Oryza species and the outgroup Leersia perieri, bridging similar to 20 million years of evolutionary history. Results: Our analysis identified thousands of putative genes in each of the Oryza species, a large proportion of which have evidence of expression and contain chimeric structures. Consistent with previous reports, we observe that the putative Mutator-like transposable element-derived genes are generally GC-rich and mainly derive from GC-rich parental sequences. Furthermore, we determine that Mutator-like transposable elements capture parental sequences preferentially from genomic regions with low methylation levels and high recombination rates. We explicitly show that methylation levels in the internal and terminated inverted repeat regions of these elements, which might be directed by the 24-nucleotide small RNA-mediated pathway, are different and change dynamically over evolutionary time. Lastly, we demonstrate that putative genes derived from Mutator-like transposable elements tend to be expressed in mature pollen, which have undergone de-methylation programming, thereby providing a permissive expression environment for newly formed/transposable element-derived genes. Conclusions: Our results suggest that DNA methylation may be a primary mechanism to facilitate the origination, survival, and regulation of genes derived from Mutator-like transposable elements, thus contributing to the evolution of gene innovation and novelty in plant genomes.

Ma thèse vise à caractériser l’évolution dynamique et l’organisation des génomes des différentes espèces du blé (genres Triticum et Aegilops) en relation avec la prolifération des éléments transposables (TEs) dans leur génome (>80%), les polyploïdisations récurrentes ainsi que la syntenie avec d’autres espèces de la famille des Poaceae. En constituant des sets de séquences génomiques représentatives et en étudiant la variabilité entre des haplotypes des génomes du blé, j’ai caractérisé la dynamique et la prolifération différentielle des TEs qui est la résultante de l’équilibre entre leurs insertions et aussi leurs éliminations actives. Le taux moyen de remplacement de l’espace TEs, mesurant les différences de séquences dues aux insertions et aux délétions entre deux haplotypes, a été ainsi estimé à 86% par million d’années (Ma) et dépasse celles bien documentées du maïs. Les insertions des TEs mais aussi leurs éliminations par recombinaisons illégitimes de l’ADN (pouvant atteindre plusieurs dizaines de kb) ainsi que les recombinaisons génétiques entre haplotypes divergents représentent les principaux mécanismes à la base des changements rapides de l’espace TEs. Sur une échelle d’évolution plus longue (60 Ma), j’ai analysé la conservation des gènes et l’évolution du locus (Ha) entre différentes espèces des Poaceae. J’ai pu ainsi préciser l’émergence du caractère grain tendre et des gènes Ha, comme nouveaux membres de la famille des gènes de Prolamine, dans l’ancêtre commun des Pooideae (blé et Brachypodium, de la tribu des Triticeae et des Brachypodieae) et des Ehrhartoideae (riz), après leur divergence des Panicoideae (maïs, sorgho)
My PhD aims to characterize dynamic evolution and organization of wheat genomes from différent species (Triticum and Aegilops genera) in relation to transposable element (TE) proliferation in their genomes (>80%), polyploidizations and synteny with other Poaceae species. By constituting and comparing representative genomic sequences and analyzing haplotype variability of the wheat genomes, I have characterized dynamics and differential proliferation of TEs, as resulting from the combinations of their insertions and deletions. Mean replacement rate of the TE space, which measures sequence differences due to insertion and removal of TEs between two haplotypes, was estimated to 86% per one million year (My). This is more important than the well-documented haplotype variability found in maize. It was observed that TE insertions and DNA elimination by illegitimate recombination (implicating several ‘tens’ of kb) as well as homologous recombination between divergent haplotypes represent the main molecular basis for rapid change of the TE space. At a longer evolutionary scale (60 My), I have compared gene conservation at the Ha locus region between different Poaceae species. The comparative genome analysis and evolutionary comparison with genes encoding grain reserve proteins of grasses suggest that an ancestral Ha-like gene emerged, as a new member of the Prolamin gene family, in a common ancestor of the Pooideae (wheat and Brachypodium from the Triticeae and Brachypodieae tribes) and Ehrhartoideae (rice), between 60 and 50 My, after their divergence from Panicoideae (Sorghum)

Thesis (Ph. D.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Dept. of Cell Biology and Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

This research focuses on non-Long Terminal Repeat (non-LTR) retrotransposons in the African malaria mosquito, Anopheles gambiae and other mosquito species. An unprecedented diversity of non-LTRs was discovered by genome analysis of the An. gambiae genome assembly. One hundred and four families were found by a reiterative and comprehensive search using the conserved reverse transcriptase domains of known non-LTRs from a number of organisms as the starting queries. These families range in copy number from a few to approximately 2000 and occupy at least 3% of the genome. An. gambiae non-LTRs represent 8 of the 15 previously defined clades, plus two novel clades, Loner and Outcast, raising the total number of known clades to 17. The first invertebrate L1 clade representatives were also found. All clades except one have families with sequence characteristics suggesting recent activity. Juan, a non-LTR of the Jockey clade originally discovered in the mosquito Culex pipiens quinquefasciatus (Mouches et al. 1991), has been implicated in horizontal transfer in three non-sibling species of the Aedes genus (Mouches, Bensaadi, and Salvado 1992). PCR was used to obtain sequences from 18 mosquito species of six genera. Phylogenetic analysis demonstrates predominant vertical inheritance of Juan elements among these species. There is strong evidence from sequence analysis supporting the recent activity of Juan in several divergent species. We hypothesize that the sustained activity (versus quick inactivation) of non-LTRs in mosquitoes may contribute to the diversity we observe in the An. gambiae genome today. Promoter and transcriptional analyses were performed for several families previously identified as potentially active elements based on sequence analysis. RT-PCR results indicate that transcripts are present in An. gambiae cell lines that contain sequences corresponding to 13 of 15 tested non-LTR families. The 5' UTRs of An. gambiae non-LTRs from the I, Jockey, and L1 clades support basal transcription in divergent mosquito cell lines from 3 species. The Jen-1 5'UTR did not support transcription in Ae. aegypti and had low activity in Ae. albopictus. In summary, this research shows that Non-LTRs have been highly successful genomic elements that have flourished in many divergent mosquito species.
Ph. D.

Les éléments transposables (ETs) sont des éléments génétiques mobiles capables de se déplacer et de se multiplier au sein d’un génome. Identifiés dans la plupart des espèces vivantes incluant les bactéries, mais longtemps considérés comme de l’ADN poubelle, aujourd’hui les ETs sont indéniablement des acteurs majeurs impliqués dans l’évolution des gènes, des génomes et des organismes. Si à l’échelle des individus les ETs peuvent avoir des effets délétères pouvant entrainer des maladies, à plus grande échelle ils sont de puissants agents évolutifs impliqués dans la plasticité génomique. Ces « parasites » peuvent également être sources de nouveaux matériels génétiques comme des promoteurs ou même de nouveaux gènes avec de nouvelles fonctions pour l’hôte. Les objectifs majeurs de mon travail de thèse ont été de déterminer les différentes familles d’ETs présentes dans les génomes de poissons, la part que chacune d’entre elles occupe dans ces génomes et enfin de comprendre l’histoire évolutive des familles d’ETs dans les génomes de poissons en comparaison avec les autres génomes de vertébrés. Cette comparaison à grande échelle permettra de comprendre les différentes stratégies évolutives des ETs. D’autre part, j’ai étudié deux gènes de vertébrés, Gin-1 et Gin-2 dérivés d’ETs, dans le but de comprendre leurs origines et évolution au sein des vertébrés ainsi que d’émettre des hypothèses quant à leur fonction moléculaire potentielle encore inconnue. Pour cela, des analyses in silico ont permis de mieux comprendre les origines de ces gènes. Gin-1, présent chez les amniotes, et Gin-2, absent uniquement des mammifères placentaires, dérivent tous deux de transposons GIN
Transposable elements (TEs) are mobile genetic elements - able to move and to multiply within genomes - identified in almost all living organisms including bacteria. Considered as junk DNA for long, nowadays they are undeniably major players of gene, genome and host evolution. TEs can be deleterious causing diseases but these “parasites” can also be source of new genetic materials as promoters or even new genes bringing new functions for hosts. The objectives of my thesis was to determine the presence or not of the different TE families in vertebrate genomes, as well as their respective content to understand their evolutionary history. I performed a large-Scale comparative analysis to highlight the various evolutionary strategies of TEs. I showed that TE content is highly variable in vertebrate genomes, the smallest and the largest being found in fish, and may contribute to their genome sizes especially in fish. These superfamilies underwent differential waves of activity in vertebrate species highlighting TE dynamics. On another hand, I focused on the study of a vertebrate-Specific TE-Derived gene, named Gin-2, to understand its origin, evolution, and its potential function in vertebrates. In silico analyses showed that Gin-2 is a very ancient gene (500 My, only absent from placentals) derived from GIN transposons. Further analyses present a particular expression in brain and gonads during adulthood, while a strong expression during gastrulation suggests a potential role of Gin-2 in zebrafish development. All together, the different analyses contribute to a better view of TE evolution and their evolutionary impacts in vertebrate genomes

Leptosphaeria maculans ‘brassicae’ (Lmb) est un champignon filamenteux de la classe des Dothideomycètes faisant partie du complexe d’espèces Leptosphaeria maculans-Leptosphaeria biglobosa composé d’agents pathogènes des crucifères. Lmb est particulièrement adapté au colza (Brassica napus) et provoque la maladie qui lui est la plus dommageable : la nécrose du collet. Dans le but de mieux comprendre et contrôler cette maladie, l’équipe d’accueil a initié un projet de génomique visant à identifier de façon systématique les gènes impliqués dans le pouvoir pathogène. Les premières données génomiques montraient deux aspects très importants et potentiellement spécifiques de Lmb : (i) tous les gènes d'avirulence caractérisés expérimentalement étaient localisés dans de grandes régions riches en bases AT et composées d'éléments transposables (ET), (ii) ces régions riches en AT préfiguraient une structure génomique particulière, qui, si elle se généralisait à l'ensemble du génome, aurait été totalement inédite chez un micro-organisme eucaryote. La première partie de cette thèse présente la description du génome de Lmb en se focalisant sur sa structure en isochores, résultant d’une invasion du génome par des ET qui ont ensuite été inactivés par un mécanisme de défense spécifique aux champignons ascomycètes, le RIP (Repeat-Induced Point mutation). Puis, l’impact potentiel de cette structure sur la diversification et l’évolution des protéines jouant un rôle clé lors de l’interaction agent pathogène-plante a été évalué, mettant ainsi en avant l’existence d’un génome à « deux vitesses ». Afin de mieux comprendre le rôle potentiel joué par les ET au niveau des capacités d’adaptation de Lmb au colza, une étude de génomique comparative et évolutive de cinq membres du complexe d’espèces a été réalisée. Ce travail montre que Lmb est la seule espèce du complexe dont le génome a été envahi par les ET, et que ces derniers sont impliqués dans (i) des réarrangements intrachromosomiques potentiellement liés à la spéciation entre Lmb et l’espèce la plus proche, (ii) la présence de gènes espèce-spécifiques et (iii) des déplacements dans des régions génomiques très dynamiques de gènes codant des effecteurs. Les travaux constituant cette thèse participent à la généralisation du concept selon lequel un lien fort existe chez les champignons filamenteux phytopathogènes entre ET et gènes impliqués dans la pathogenèse ou l’adaptation à l’hôte
Leptosphaeria maculans ‘brassicae’ (Lmb) is a filamentous ascomycete from class Dothideomycetes. It belongs to the Leptosphaeria maculans-Leptosphaeria biglobosa species complex which comprises pathogens of crucifers. Lmb is specifically adapted to oilseed rape (Brassica napus) and is responsible for the most damaging disease of this crop: “stem canker”. In order to better understand and control the disease, the host team initiated a genomic project aiming at systematically identify genes involved in pathogenicity, analyse genome plasticity and evaluate their incidence on adaptability to host. Preliminary genome data firstly showed that all characterized avirulence genes were localized in large AT-rich regions, mainly composed of Transposable Elements (TEs). In addition, these AT-rich regions were the first hints that the Lmb genome may present a very unusual structure compared to other microorganisms. The first part of this thesis describes the Lmb genome with a special focus on its isochore structure, which is the result of a massive TE invasion of the genome followed by an inactivation of TEs by an ascomycete-specific defense mechanism called RIP (Repeat-Induced Point mutation). The potential impacts of this genome structure on diversification and evolution of proteins involved in the plant-pathogen interaction were assessed and highlighted the existence of a “two speed” genome. To better understand how TEs are involved in adaptation of Lmb towards oilseed rape, a comparative and evolutionary genomic analysis of five members of the species complex was conducted. This study shows that Lmb is the only species of the complex with genome invaded by TEs at such an extent, and that TEs are involved in (i) intrachromosomal rearrangements putatively related to the speciation event between Lmb and its closest relative species, (ii) the presence of species-specific genes, (iii) translocations of effector genes into highly dynamic genomic regions. Our data contribute to the generalization of the “two speed” genome concept in filamentous phytopathogens postulating that highly plastic regions of the genome are enriched in genes involved in niche adaptation and that a strong link exists between TEs and genes involved in pathogenesis or host adaptation

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The mesophragmatica group belongs to the radiation virilis-repleta of the Drosophila subgenus, it was established by Brncic and Koref in 1957. The species of this group present some characteristics of isolated endemic species in some places in Andes. The common ancestral of the species of the group seems to have acquired, for selective pressure, genetics structures better adjusted to each region. Although there is just a few studies involving these species and still have to be done in relation to the presence and evolution of transposable elements in its genomes. In this context, the transposable elements of the families, hobo, Tom/17.6, I, mariner, P, micropia and gypsy had been analyzed in species of Drosophila of the mesophragmatica group using, Dot Blot and PCR. The genomic DNA of species D.viracochi, and one of the D. gasici had presented hybridization for the micropia element when analyzed by Dot Blot. Analysis the presence of the TEs of the families Tom, 17.6, hobo was made by PCR. However, they had not been gotten amplicons for none of these elements. However homologous sequences to element P are found in D. gasici and D. pavani. Using probe of the elements I and mariner they had been carried through Dot Blot not presenting hybridization with genomic DNA of species D. pavani, D. brncici, D. viracochi, D. gasici (three different populations). Genomic DNA of species D. pavani, D. brncici, D. viracochi, D. gasici (three different populations) hybridized with probe of the element gypsy also having amplification for PCR for all the analyzed species. The purify products of PCR had been sequenced. Phylogenetic analysis s confirmed the idea that it has incongruence between the phylogeny of the species and of its TEs due to a standard of complex evolution that involves mechanisms as: random losses, vertical and horizontal transference, ancestral polymorphism, different taxes of evolution. Those mechanisms might be not mutually excludable and probably occur simultaneously.
O grupo mesophragmatica pertence à radiação virilis-repleta do sub-gênero Drosophila, foi estabelecido por Brncic & Koref em 1957. As espécies deste grupo apresentam algumas características de espécies endêmicas isoladas em vários locais nos Andes. O ancestral comum das espécies do grupo parece ter adquirido, por pressão seletiva, estruturas gênicas melhor ajustadas a cada região. Embora existam alguns estudos envolvendo estas espécies pouco se sabe em relação à presença e evolução de elementos transponíveis em seus genomas. Neste contexto, os elementos transponíveis das famílias, hobo, Tom/17.6, I, mariner, P, micropia e gypsy foram analisados em espécies de Drosophila do grupo mesophragmatica usando, Dot Blot e PCR. O DNA genômico das espécies D.viracochi, e uma das linhagens de D. gasici apresentaram hibridização para o elemento micropia quando analisadas por Dot Blot. Análise da presença dos TEs das famílias Tom, 17.6, hobo foi feita por PCR. Porém, não foram obtidos amplicons para nenhum destes elementos. No entanto seqüências homólogas ao elemento P estão presentes em D. gasici e D. pavani. Utilizando sonda dos elementos I e mariner foram realizados Dot Blots não apresentando hibridização com DNA genômico das espécies D. pavani, D. brncici, D. viracochi, D. gasici (três linhagens diferentes). DNA genômico das espécies D. pavani, D. brncici, D. viracochi, D. gasici (três linhagens diferentes) hibridizaram com sonda do elemento gypsy havendo também amplificação por PCR para todos as espécies analisadas. Os produtos de PCR purificados foram seqüenciados. Análise filogenética a partir destas seqüências reforçou a idéia de que haja incongruências entre a filogenia das espécies e de seus TEs devido a um padrão de evolução complexa que envolva mecanismos como: perdas estocásticas, transferência vertical e horizontal, polimorfismo ancestral, diferentes taxas de evolução sendo não mutuamente excludentes e provavelmente ocorram simultaneamente.

Elementos transponíveis (TEs) são sequências de DNA que possuem a capacidade de transposição no genoma hospedeiro. O principal objetivo deste trabalho reside na investigação em torno de possíveis contribuições de TEs nos genomas das algas C. reinhardtii e V. carteri, mais especificamente, na arquitetura dos genes ortólogos nestas espécies. Neste contexto, análises em sílico em larga escala foram realizadas, buscando-se identificar associações entre TEs e os genes ortólogos. Os resultados indicaram que os genes em C. reinhardtii tendem a acumular mais cópias de TEs em relação aos seus ortólogos em V. carteri. C. reinhardtii apresentou maior densidade de cópias de TEs para as regiões flanqueadora 5´ , flanqueadora 3´ e intrônica quando comparada a V. carteri; o inverso foi verificado quando analisada a densidade de TEs nas regiões codificantes. Análises para apurar a distribuição dos elementos em regiões intergênicas e intragênicas foram estabelecidas, nas quais a frequência observada dos elementos foi comparada à frequência esperada segundo a distribuição randômica de TEs no genoma, simulada computacionalmente. Foram constatadas regiões em que a presença dos elementos encontra-se significativamente abaixo do esperado, a exemplo de intervalos adjacentes ao início e ao término dos genes, o que provavelmente reflete a seleção negativa de eventos de integração nestas delimitações, em virtude dos efeitos deletérios associados à disrupção de estruturas de regulação da expressão gênica. De forma geral, nas regiões flanqueadoras 5´ e 3´, foi identificada a tendência de elevação da frequência padronizada de TEs à medida que a classe de distância avaliada se distancia do início e do término do gene, respectivamente. A baixa representatividade dos elementos também foi constatada em regiões intragênicas. O estudo da distribuição de TEs nos íntrons dos genes ortólogos indicou a preservação destas regiões quanto à fixação de TEs, sendo a representatividade abaixo do esperado mais evidente em intervalos adjacentes ao éxon, o que minimiza a chance de ruptura no padrão de splicing dos genes. Em sequências codificantes, a escassez de TEs - esperada devido ao provável efeito deletério destes eventos para a função do gene - foi constatada nos ortólogos das duas espécies. No entanto, inovações decorrentes da integração dos elementos em regiões codificantes podem resultar em efeitos evolutivos positivos, embora estes eventos sejam raros. Nas espécies analisadas foram identificados dois casos, de especial interesse, em que um domínio da sequência peptídica encontra-se localizado em região derivada de TE: o primeiro refere-se ao gene Cre06.g262800, em C. reinhardtii, no qual foi identificado o domínio PHD-finger associadao ao elemento Gypsy-5-LTR_CR; o segundo remete ao gene Vocar20001092m.g, em V. carteri, no qual o domínio zinc knuckle foi reconhecido em região derivada do elemento Gypsy3-LTR_VC. Estes genes constituem exemplos da contribuição de TEs na evolução de sequências codificadoras nas espécies C. reinhardtii e V. carteri, corroborando a hipótese de que os TEs podem contribuir na evolução da arquitetura dos genes, apesar do efeito disruptivo inerente à integração dos mesmos em regiões gênicas.
Transposable elements (TEs) are DNA sequences able to transpose in the host genome. The aim of this study resides in the investigation of TEs contributions in the algae C. reinhardtii and V. carteri genomes, more specifically in the architecture of orthologous genes in these species. In this context, large scale in silico analysis were performed to identify associations between TEs and orthologous genes. The results indicated that genes in the C. reinhardtii specie tend to accumulate more TEs copies than orthologous genes in V. carteri. C. reinhardtii showed higher density of TEs copies in the 5´ flanking, 3´ flanking and intronic regions when compared to V. carteri; the opposite was observed in coding regions. Investigation of the elements distribution in the intergenic and intragenic regions was performed, in which the observed TE frequency was compared to expected TE frequency from the simulated random distribution of the elements in the genome. It was verified regions where TE frequency was significantly lower than expected, as in gene boundaries adjacencies, probably reflecting a negative selection of the TE integration events in these delimitations due to deleterious effects associated with disruption of gene regulatory structures. In general terms, it was observed an increasing standardized frequency in the 5´ and 3´ flanking regions as the distance from gene start and gene end, respectively, increases. TEs underrepresentation was also verified in the intragenic regions. The study of TEs distribution in the introns of orthologous genes revealed the preservation of these structures in relation to TEs fixation, with a stronger underrepresentation near exon, which minimizes the chance of gene splicing pattern disruption. In the coding sequences, the TEs scarcity - expected due the likely deleterious effects to gene function - was verified in the orthologous of both species. However, in rare instances, innovations mediated by TEs integration in the coding regions can lead to positive evolutionary effects. In the species analyzed two instances of particular interest were observed, in which the domain of peptide sequence is located in the region derived from TE. The first one refers to the Cre06.g262800 gene, in the C. reinhardtii specie, which has a PHD-finger domain associated with Gypsy-5-LTR_CR element. The second one refers to the Vocar20001092m.g gene, in V. carteri, in which the zinc knuckle was recognized in region derived from Gypsy3-LTR_VC element. These genes are examples of TEs contributions in the evolution of coding sequences in the C. reinhardtii and V. carteri species, corroborating the hypothesis that TEs can contribute to the evolution of gene architecture, despite the inherent disruptive effect in their integration in the gene regions.

Les éléments transposables (ET) sont des séquences d’ADN capables de catalyser son propre mouvement et d’entrer dans de nouvelles régions du génome. Dans la présente étude, nous avons étudié Helena, un élément LINE qui est à différents stades de son cycle évolutif et donc, il est un bon modèle pour l’étude de la dynamique évolutive des TE. À travers une analyse de bio-informatique dans les douze génomes séquencés de la drosophile nous avons étudié l’évolution de Helena, et nous proposons un scénario possible pour l’évolution de cet élément. Helena est à différents stades de son cycle de vie, allant d’un état complet (D. simulans et D. mojavensis) à très dégradé (D. yakuba, D.erecta, D. ananassae et D. virilis) ou absent (D. pseudoobscura, D. persimilis, D. grimshawi et d. willistoni). L’analyse phylogénétique a montré que Helena était présent chez l’ancêtre commun du genre Drosophila et a été transmis verticalement dans des lignées dérivées. De plus, nous avons détectées des copies intactes uniquement chez D. mojavensis et nous avons étudié plus en détail sa région 5’ (extrémité). Nous avons utilisé un gène rapporteur et confirmé la présence du promoteur interne pour Pol II qui est associé à des modifications épigénétiques de l’histone : hétérochromatine permissive (H3K4me2) et répressive (H3K27me3). Ces « marques bivalents » indiquent que Helena peuvent être exprimés en réponse à un stimulus spécifique. Une étude de l’élément BS, un TE étroitement liée à Helena, a montré que la dynamique évolutive des deux ETs sont très similaires. Les résultats montrent que CET élément, comme Helena, se trouve à différents stades de son cycle évolutif
The transposable elements (TEs) are DNA sequences capable of catalyze its own movement and to enter into new regions of the genome. In the present study we studied Helena, a LINE element that is at different stages of its evolutionary cycle and therefore, it is a good model for studies of TEs evolutionary dynamics. Through bioinformatics analysis of 12 Drosophila species which have their genomes sequenced, we found Helena in different stages of its evolutionary cycle, that varies of at least one full active copy (D. mojavensis) an putatively complete copy, but inactive (D. simulans) to highly degenerate (D. yakuba, D. erecta, D. ananassae and D. virilis) or absent (D. pseudoobscura, D. persimilis, D. willistoni and D. grimshawi) sequences. Phylogenetic analysis showed that Helena was present in the common ancestor of the Drosophila genus and has been vertically transmitted in derived lineages, but lost on some of them. Since a complete highly active copy was observed only in D. mojavensis, we studied in more detail its 5' end region. We used a reporter gene and verified the presence of internal promoter for Pol II that is associated with epigenetic histone modifications for permissive (H3K4me2) and repressive heterochromatin (H3K27me3). These “bivalent marks” indicate that Helena can be expressed in response to specific stimulus. A study of BS element, a TE closely related to Helena, showed that the evolutionary dynamics of both TEs are very similar. Bioinformatics analysis of the 12 Drosophila genomes revealed that BS is also widely variable in the species analyzed regarding to distribution, abundance, degree of degradation and also about their evolutionary cycle

Les éléments transposable (ET) sont des séquences d'ADN qui ont la capacité de se déplacer au sein des génomes. Pour contrebalancer les effets négatifs liés à l'activité des ET, il existe chez leurs hôtes des mécanismes régulant l'activité de transposition. Une fois qu'un ET est régulé, l'accumulation progressive de mutations dans sa séquence conduit fatalement à la perte définitive de son activité de transposition. J'ai cherché au cours de cette thèse à mieux comprendre le succès et le maintien de ces séquences répétées, avec d'une part l'étude des transferts horizontaux (TH) d'ET, un moyen d'échapper aux mécanismes de régulation , et d'autre part l'étude de leur régulation. Dans la première partie de ma thèse, je me suis intéressé à l'étude des TH entre deux espèces proches de drosophiles. Dans cette étude, j'ai développé une nouvelle méthode bioinformatique permettant la détection de séquences transférées horizontalement entre deux génomes eucaryotes qui m'a permis détecter de nombreux TH d'ET. Ce travail m'a aussi conduit à développé une nouvelle méthode de contrôle du taux de faux positifs moyen applicable aux tests multiples unilatéraux. Dans la deuxième partie de ma thèse, j'ai étudié la régulation des ET par la voie des petits ARN, un mécanisme de l'ARN interférence. Dans cette étude, j'ai analysé des données de séquençage de petits ARN, ainsi que d'ARN totaux issues de différentes populations de D. simulans. Ce travail a conduit au développement d'un pipeline d'analyse permettant d'étudier des différences d'expression entre des séquences répétées ainsi que d'une nouvelle procédure de contrôle qualité de ce type de donnée
Transposable elements (TEs) are repeated DNA sequences that are able to move (transpose) within their host genome. To counteract the negative effects of their TEs, regulation mechanisms of the TE transposition are present in the host genome. Once a TE is regulated, the progressive accumulation of mutations in its sequence will inevitably lead to the definitive loss of its transposition capacity. My work during this thesis is was to better understand the succss and the maintaining of these peculiar repeated sequencest, with the study of horizontal transfers (HTs) of TEs enabling them to escape host regulation mechanisms, and the study of this regulation. The first part of my thesis concerns the study of HTs between two closely related drosophila species. I have developed a new bioinformatic method for the detection of HTs between two eukaryotic genomes. The development of this method brought me to work on the unilateral multiple testing problematic for which I have developed a new procedure to control the expected false discovery rate (FDR). The second part of my thesis focuses on the regulation of TEs by the small RNA pathway, an RNA interference mechanism. For this study, I have analyzed sequencing data of small RNAs and total RNAs. For this work, I have developed an analysis pipeline, to study differences of expression between repeated sequences. Some features of the small RNA dataset required the development of a new procedure to parse them. This procedure was extended and implemented in a software to be used for the quality control of next generation sequencing data

Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites. In Chapter 2, I have described the phenotypic effects of the loss of piRNA function in early embryos. The mutations affecting the piRNA pathway are known to cause embryonic lethality. To describe this lethality in detail, I have shown that all the characterized piRNA mutants show compromised zygotic genomic integrity during early embryogenesis. In addition, two piRNA pathway components, Aubergine (Aub) and Armitage (Armi) are also required for telomere resolution during early embryogenesis. Aub and Armi recruit telomeric protection complex proteins, HOAP and HP1, to the telomeric ends and thus avoid activation of the Non-homologous end joining (NHEJ) DNA repair pathway at the telomeres. There are about 120 transposon families in Drosophila melanogaster and piRNA pathway mutations cause activation of many of the resident transposons in the genome. In Chapter 3, I have described the effects of infection by a single transposon, P-element, in naïve strains by introduction through the zygote. Activation of the P-element leads to desilencing of unrelated transposons, causing accumulation of germline DNA damage which is linked to severely reduced fertility in the hybrid females. However, there is partial restoration of fertility as the hybrid progeny age, which correlates with P-element piRNA production and thus P-element silencing. Additionally, a number of transposons mobilize into piRNA generating heterochromatic clusters in the genome, and these insertions are stably inherited in the progeny. Collectively our data shows that piRNA production can be triggered in the adults in an absence of maternal contribution and that piRNAs serve as an adaptive immune system which helps resolve an internal genetic conflict between the host and the parasite. In an effort to understand the phenotypic effects of piRNA dysfunction in Drosophila, we have uncovered new exciting roles for piRNAs in development and presented evidence how transposons can act as architects in restructuring the host genome.

Os elementos de transposição (TEs) se movimentam de um locus para outro no genoma afetando a estrutura e evolução destes. A superfamília de transposases hAT é definida pelos elementos que compartilham os domínios de dimerização e ligação ao DNA com os transposons previamente descritos: hobo, Activator e Tam3. Análises prévias encontraram algumas evidencias da presença genômica e ativação transcricional de TEs relacionados à superfamília hAT em cana-de-açúcar (denominados de família SChAT) e pelo menos três linhagens evolutivas foram postuladas. O objetivo deste trabalho é caracterizar versões genômicas das linhagens de transposons (191 e 257) e linhagem possivelmente domesticada (074). Pretende-se estudar as relações evolutivas, distribuição em gramíneas, identificar os padrões de expressão e propriedades funcionais. Regiões de sintenia foram estabelecidas para estes BACs em Arabidopsis thaliana, Brachypodium distachyon, Sorghum bicolor, Oryza sativa e Zea mays. Elementos relacionados com as três linhagens foram procurados nestes genomas.
Transposable elements (TEs) are able to move from one locus to another within a genome. TE mobilization affects genome structure and evolution. The hAT transposase superfamily is defined as elements that share the dimerization and DNA ligation domains with the previously described hobo, Activator and Tam3 transposon elements. Previous analyses found some genomic and transcriptional evidences of TEs related to hAT superfamily in sugarcane (named SChAT family) and at least three evolutionary lineages were proposed. The aim of this work is to characterize full-length genomic versions of the transposons lineages (191 and 257) and from the domesticated lineage (074). It is proposed to study the evolutionary relationship, distribution along grasses genomes, identify expression patterns and functional capacities of the SChAT elements. Syntenic regions for the BACs containing elements from the three lineages were mapped in Arabidopsis thaliana, Brachypodium distachyon, Sorghum bicolor, Oryza sativa and Zea mays. Related elements were search on the same genomes.

Polymorphisme des cytotypes p, m et intermediaires caracterises par leur capacite a reprimer les anomalies genetiques induites par les elements p de d. M. Etudie en correlation avec des elements p complets et defectifs

Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.

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A ordem Coleoptera é a mais diversificada entre todos os seres vivos, existindo ampla possibilidades de estudos no que diz respeito à diversidade cariotípica e aos mecanismos de diferenciação. As espécies da subtribo Oedionychina (Alticinae; Chrysomelidae) são interessantes para estudos evolutivos, pois possuem cromossomos sexuais gigantes e assinápticos durante a meiose, podendo ser considerados altamente derivados. Assim, o objetivo do presente estudo foi propor os mecanismos moleculares envolvidos no processo de diferenciação e evolução dos cromossomos sexuais em espécies do gênero Omophoita. A análise de mapeamento, utilizando sondas de DNA C0t-1 total (cinética de reassociação de DNA altamente e moderadamente repetitivo) mostrou marcações distribuídas em todos os cromossomos, especialmente nos cromossomos sexuais. A hibridação cruzada entre as espécies produziu um padrão de localização muito semelhante, evidenciando que a maior parte do genoma é compartilhada entre as espécies de Omophoita. Análise em conjunto dos resultados obtidos com bandas C, fluorocromos e C0t-1 mostram que a heterocromatina das espécies em grande parte é composta de DNA repetitivo distribuída ao longo dos cromossomos sexuais e autossomos. O mapeamento cromossômico com sondas de microssatélites (SSRs) mostrou marcações conservadas para os autossomos e diversificadas para os cromossomos sexuais, evidenciando uma diferença de composição de SSRs dos cromossomos sexuais entre as espécies. Os resultados de hibridação com clones de elementos de transposição mostraram alguns padrões semelhantes aos obtidos com SSRs, podendo indicar que ao longo do processo evolutivo das espécies esses elementos estiveram presentes no processo de diferenciação. Considerando todos os resultados, pode se propor uma diferença de constituição nos cromossomos sexuais das espécies e, desta forma, inferir que os DNAs repetitivos tiveram um papel evolutivo na diferenciação desses cromossomos na subtribo.
The order Coleoptera is the most diverse of all living beings, with a wide possibilities of studies with regards to the karyotype diversity and the mechanisms of differentiation. The species of the subtribe Oedionychina (Alticinae; Chrysomelidae) are interesting for evolutionary studies due to the giant sex chromosomes and asynaptic during meiosis, can be considered highly derivate. The objective of this study was to propose the molecular mechanisms involved in the differentiation process and evolution of sex chromosomes in the Omophoita genus. The Mapping analysis using DNA C0t-1 total (reassociation kinetics highly and moderately repetitive DNA) showed marks distributed in all chromosomes, especially in the sex chromosomes. The cross-hybridization among species produced a very similar location pattern, indicating that most of the genome is shared among species Omophoita. Analysis of the results obtained in conjunct with C-bands, fluorochromes and C0t-1 together show that the heterochromatin of the species is largely composed of repetitive DNA distributed throughout the autosomes and sex chromosomes. Chromosome mapping with microsatellite (SSRs) probes showed conserved patterns for autosomes, but diversified to sex chromosomes, showing difference in SSRs composition in the sex chromosomes, of the species. The results of hybridization with transposition element clones showed some similarities patterns to the SSRs markers, which may indicate that throughout the evolutive process of species these elements were present. Considering all results we can propose differences in the constitution of sex chromosomes of the species studied, thus, we can infer an evolutionary role of repetitive DNA in the differentiation of chromosomes in the subtribe.

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The Loricariidae family (Actinopterygii: Siluriformes) is morphologically diverse, has a number close to 900 valid species, distributed in seven subfamilies (Lithogeneinae, Delturinae, Neoplecostominae, Hypoptopomatinae, Loricariinae, Ancistrinae and Hypostominae). However, cytogenetic studies in species of the family show evolutionary trends of karyotype diversification well defined for each of the subfamilies and the diploid number (2n) of 54 chromosomes is considered basal. Among the representatives of the subfamily Loricariinae, the variation of 2n is 36 to 74 chromosomes. Given these data, the Robertsonian rearrangements are the main mechanisms to explain the chromosome number variation in the subfamily. Rineloricaria is the most specious genus of Loricariinae, porting species with 2n = 36 to 2n = 70 chromosomes. However, little is known about what types of repetitive DNAs originate fission and fusion chromosome events. In this study, species of Rineloricaria from different rivers of the Paraná drainage were studied: Rineloricaria latirostris (Laranjinha river, Cinzas basin and Barra Grande river, Ivaí basin); Rineloricaria pentamaculata (Barra Grande and Juruba rivers, Tibagi basin); and, Rineloricaria stellata and Rineloricaria capitonia (Upper Uruguai river). The aim of this study was to characterize the karyotypes of populations/species of Rineloricaria and to check what types of repetitive DNAs may be related to Robertsonian events in the genus. In R. latirostris was detected 2n = 46 chromosomes for both populations, as well as for a triploid specimen from Laranjinha river. Rineloricaria pentamaculata had 2n = 56 chromosomes to populations from Barra Grande and Juruba rivers and a karyomorph in Barra Grande river with 2n = 54 chromosomes. Rineloricaria stellata had 2n = 54 chromosomes, while R. capitonia presented 2n = 64 chromosomes, both from the Uruguai river. The results using the chromosomal markers of 18S rDNA, 5S rDNA and TTAGGGn telomeric probe showed that these repetitive DNAs participated in end to end fusions of the st/a chromosomes in the karyotype diversification of R. latirostris. Vestiges of interstitial telomeric sites (ITS) were also detected in R. pentamaculata, karyomorph of 54 chromosomes from the Barra Grande river, suggesting chromosomal fusion to the diversification of this karyotype. The wide range of 2n between R. stellata and R. capitonia is compatible to the reproductive isolation of syntopic species and the diversification of R. capitonia can be explained by centric fusions. In addition to Robertsonian rearrangements, the pericentric inversions also assisted in the diversification of karyotypic formulas among the species/populations. In situ localization analysis using the transposable element Tc1-Mariner Like probe showed no evidence of the participation of transposon in chromosomal rearrangements and dispersion of multiple sites of 5S rDNA in Rineloricaria. Furthermore, analyzes of the Tc1-Mariner Like sequences showed intense molecular degeneration, especially in transposase domains. These results indicate the absence of activity of these sequences, which must be inert or serve to other genomic functions in the genus. Thus, this study discusses the telomeric instability, repetitive DNAs and the participation of rDNA gene families in karyotype diversification events in Rineloricaria.
A família Loricariidae (Actinopterygii: Siluriformes) é extremamente diversificada morfologicamente, conta com um número próximo a 900 espécies válidas, distribuídas em sete subfamílias (Lithogeneinae, Delturinae, Neoplecostominae, Hypoptopomatinae, Loricariinae, Ancistrinae e Hypostominae). Não obstante, os estudos citogenéticos em representantes da família mostram tendências evolutivas da diversificação cariotípica bem definidas para cada uma das subfamílias, sendo considerado basal o número diploide (2n) de 54 cromossomos. Entre os representantes da subfamília Loricariinae a variação do 2n é de 36 a 74 cromossomos. Diante destes dados, os rearranjos Robertsonianos são os principais mecanismos para explicar a variação cromossômica numérica na subfamília. Rineloricaria é o gênero mais especioso de Loricariinae, com espécies apresentando 2n = 36 até 2n = 70 cromossomos. Contudo, pouco se sabe sobre quais os tipos de DNAs repetitivos originam os eventos de fissão e fusão cromossômica. Neste estudo, foram avaliadas espécies de Rineloricaria de diferentes rios do sistema hidrográfico do Paraná: Rineloricaria latirostris (rio Laranjinha, bacia do rio das Cinzas e rio Barra Grande, bacia do rio Ivaí); Rineloricaria pentamaculata (rio Barra Grande e rio Juruba, bacia do rio Tibagi); e, Rineloricaria stellata e Rineloricaria capitonia (Alto Rio Uruguai). O objetivo foi de caracterizar cariotipicamente as populações/espécies de Rineloricaria estudadas, além de verificar quais os tipos de DNAs repetitivos podem estar relacionados aos eventos Robertsonianos no gênero. Em R. latirostris foi detectado 2n = 46 cromossomos para ambas populações, além de um exemplar triploide para o rio Laranjinha. Rineloricaria pentamaculata apresentou 2n = 56 cromossomos para as populações dos rios Barra Grande e Juruba e um cariomorfo 2n = 54 cromossomos no rio Barra Grande. Rineloricaria stellata apresentou 2n = 54 cromossomos, enquanto R. capitonia detém 2n = 64 cromossomos, ambas do rio Uruguai. Os resultados com marcadores cromossômicos de rDNA 18S, rDNA 5S e sonda TTAGGGn evidenciaram que estes DNAs repetitivos participaram dos eventos de fusão terminal para terminal (end to end fusions) de cromossomos st/a na diversificação cariotípica de R. latirostris. Vestígios de sítios teloméricos intersticiais (ITS) foram evidenciados também em R. pentamaculata, cariomorfo de 54 cromossomos do rio Barra Grande, sugerindo fusão cromossômica para a diversificação deste cariótipo. A ampla variação de 2n entre R. stellata e R. capitonia é compatível para o isolamento reprodutivo das espécies sintópicas e pode ser explicado por fissões cêntricas na diversificação de R. capitonia. Além dos rearranjos Robertsonianos, as inversões pericêntricas também auxiliaram na diversificação de fórmulas cariotípicas entre as espécies/populações. A análise de localização in situ do elemento transponível Tc1-Mariner Like não mostrou evidências da participação deste transposon nos rearranjos cromossômicos e na dispersão dos sítios múltiplos de rDNA 5S em Rineloricaria. Ainda, as análises das sequências Tc1-Mariner Like evidenciaram intensa degeneração molecular, principalmente nos domínios transposase. Estes resultados indicam a ausência de atividade destas sequências, as quais devem ser inertes ou servir para outras funções genômicas no gênero. Desta forma, este estudo discute a instabilidade telomérica, DNAs repetitivos e a participação das famílias gênicas de rDNA nos eventos de diversificação cariotípica em Rineloricaria.

Präeklampsie (PE) ist eine Komplikation, die während der Schwangerschaft auftritt, fast 2-8% aller Schwangerschaften betrifft und human spezifisch ist. PE ist eine der Hauptursachen für den Tod von Mutter und Kind. Eine abnormale Plazentaentwicklung aufgrund einer verminderten Trophoblasteninvasion und einem gestörten Umbau der Spiralarterien trägt zur Pathogenese der PE bei. Klinisch wird die PE durch Bluthochdruck und Proteinurie, auftretendnach der 20. Schwangerschaftswoche, diagnostiziert und kann durch eine Funktionsstörung von Organen begleitet werden. Bei besonders schweren Verläufen ist die frühzeitige Endbindung die letzte Möglichkeit das Überleben der Mutter zu gewährleisten. Das Ziel dieser Studie ist es, weitere Gene zu identifizieren, die durch ERVs in der menschlichen Plazenta spezifisch reguliert werden und in PE dysreguliert sind. Um dieses Ziel zu erreichen, wurde das Transkriptom von primären menschlichen Trophoblastenzellen von 5 gesunden und 5 früh einsetzenden PE-Plazenten mittels RNA-Sequenzierung analysiert. Es wurden 335 Gene identifiziert, welche eine höhere Expression in den Trophoblastenzellen im Vergleich zu anderen Geweben aufwiesen. Zusätzlich zeigten einige der Gene (n=88) eine Co-Regulation der Expression durch retrovirale LTRs (10-kb 5‘ des transcription start side (TSS) des Gens). Hauptinteresse lag hierbei auf den Genen, welche ebenfalls eine Dysregulation in der PE aufwiesen (n = 16). Diese Studie identifizierte EPS8L1, das durch primaten-spezifisches ERV-LTR (MLT1G1) in Trophoblastenzellen reguliert wird, als einen wichtigen Faktor in der Entwicklung der menschlichen Plazenta. EPS8L1 ist in der PE Plazenta dysreguliert und involviert in mehrere Signalwege und die Funktionalität von Trophoblasten wie Invasion, Angiogenese und Redoxhomöostase. Hierdurch führt diese Arbeit zu einem besseren Verständnis der PE und deren human-spezifischer Natur.
Preeclampsia (PE) is a complication that occurs during pregnancy and affects almost 2-8% of all pregnancies and is often regarded as a human-specific disorder.1,2 PE is one of the major causes of maternal and fetal death.1 Failure of the trophoblast cells to invade into the maternal decidua results in the improper remodeling of spiral arteries leading to PE pathogenesis. Clinically, it is diagnosed as a maternal syndrome, diagnosed by the new-onset of hypertension and proteinuria or other end-organ dysfunction after the 20th week of pregnancy. So far, the only effective treatment of the disorder is the removal of the placenta tissue and delivery of the infant. The aim of this study is to identify additional genes that are regulated by the human ERV-LTRs in the human placenta specifically, and are dysregulated in PE. To achieve this aim, the transcriptome of primary human trophoblast cells of 5 healthy and 5 early-onset PE placentas were analyzed by RNA sequencing (RNA-seq). RNA-seq analysis identified genes (n=335) with stronger expression in the trophoblast cells as compared to other human body tissues. Additionally, some of the genes (n=88) showed co-regulation of expression by the human ERV-LTRs in their vicinity (10-kb upstream of transcription start side (TSS) of the gene). Since my interest was to identify the new targets of PE pathogenesis, so I focused on genes (n=16) with dysregulated expression in women presented with PE. This study identified a new gene EPS8L1, regulated by primate-specific ERV-LTR in trophoblast cells that has a predominant role in the human placenta development and demonstrated that its dysregulation affected multiple pathways involved in trophoblast function like invasion, angiogenesis and maintenance of cell redox homeostasis. Furthermore, this study leads to the better understanding of the disease by explaining certain aspects of human-specific nature of PE.

Les conséquences de la spéciation divergente ou réticulée (par hybridation) ont été explorées au cours de la diversification des spartines depuis 6-10MA à travers l’analyse des séquences répétées, de leur expression et régulation. Nous avons montré une corrélation entre éléments transposables, tailles de génome, et relations phylogénétiques, tout en mettant en évidence une dynamique variable des catégories d’éléments transposables ou de séquences satellites selon les lignées, et l’ancienneté des évènements de spéciation. L’abondance des éléments transposables a été mise en relation avec leur niveau d’expression et le rôle des petits ARN dans leur contrôle. Cette régulation se met rapidement en place suite à l’hybridation interspécifique et explique la « quiescence génomique » (absence de prolifération d’éléments transposables) détectée chez l’allododécaploïde récent S. anglica. Les annotations d’éléments transposables et de petits ARNs, les nouveaux transcriptomes de références réalisés chez différentes espèces au cours de ce travail représentent des ressources additionnelles qui permettront d’explorer de manière plus exhaustive la dynamique des génomes de spartines et de comprendre les mécanismes génomiques impliqués dans l’adaptation et l’écologie de ces espèces ingénieurs d’écosystèmes
We explored the consequences of divergent speciation or reticulate evolution (resulting from hybridization) during diversification of the Spartina genus in the last 6-10 MY, based on the analysis of repeated sequences, their expression and regulation. Transposable element amounts, genome size, and phylogenetic relationships were found correlated, although differential dynamics of specific transposable element families or satellite sequences were encountered according to lineages, and to divergence times following the speciation events. The abundance of transposable elements appears related to their level of expression and the role of small RNAs in their control. This regulation is rapidly established following interspecific hybridization and explains the "genomic quiescence" (absence of transposable element “burst”) detected in the recent allododecaploid S. anglica. Annotations of transposable elements and small RNAs, new reference transcriptomes generated for different species during this work represent additional resources that will allow a more comprehensive exploration of the Spartina genome history and dynamics for a better understanding of the genomic mechanisms involved in the adaptation and ecology of these “ecosystem engineers” species

About 40-50% of mammalian genomes are made up of repetitive elements, primarily transposable elements. Transposable elements' activities not only drive genome evolution, they contribute to the creation of novel recombinant repeats. Recombinant repeats have largely remained uncharacterized due to their complexity. Initially, I developed a pipeline for the genome wide identification of recombinant repeats in four different mammals: human, mouse, cow and horse. The pipeline identified 1,336,824 copies, but only 37,830 sequences were able to be clustered into 6,116 families. The majority of the recombinant repeats were simple recombinant repeat families and only a small proportion were complex recombinant repeat families. My analysis showed that recombinant repeat families only covered a small fraction of the genomes examined (0.30% in human, 0.13% in mouse, 0.217% in horse and 0.464% in cow), indicating most of the recombinant repeats were singletons. Further analysis has shown that both classes of RR were created via transposon-into-transposon events, indicating that novel transposable elements are likely to be created via this mechanism. I found that simple recombinant repeats were probably retrotranspositionally active because they contained polyA tails and target site duplications, showing that they integrated into the genome via retrotransposition events. However, complex recombinant repeat families were only replicated via segmental duplications. My analysis showed that complex recombinant repeat families are excellent candidates for the identification of genome segmental duplication regions that cannot be found through standard methods. In addition, I used the RR identification pipeline to annotate possible RR in pig genome. I discovered a novel RR family (LTR2i_SS) that contained > 1,000 copies. Repeat annotation showed that it was a chimeric LTR2_SS that contained ~300bp of un-annotated sequence, only found in the pig genome. Further investigation revealed that some LTR2i_SS flanked β3 proviruses, but these proviruses were unable to replicate autonomously as they did not encode a functional, complete polyprotein. My phylogenetic tree analysis of the LTR2i_SS and LTR2_SS familis suggested that LTR2i_SS was the ancestral form of LTR2_SS. In conclusion, I was able to identify the recombinant repeat distributions in different mammals and determine their most probable origin as TinT events. I have shown that recombinant repeats could serve as an important model to explain the origin of novel transposable elements in genomes, or could be used as markers to identify structural variations, or segmental duplications in different species. However, my data have also shown that we have to be cautious when annotating novel recombinant repeats in genomes, as they could be the ancestral form of other known transposable elements rather than novel forms generated through TinT.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2014

Salmonids are a diverse group of fishes whose common ancestor experienced an evolutionarily important whole genome duplication (WGD) event approximately 90 MYA. This event has shaped the evolutionary trajectory of salmonids, and may have contributed to a proliferation of the repeated DNA sequences known as transposable elements (TEs). In this work I characterized repeated DNA in five salmonid genomes. I found that over half of the DNA within each of these genomes was derived from repeats, a value which is amongst the highest of all vertebrates. I investigated repeats of the most abundant TE superfamily, Tc1-Mariner, and found that large proliferative bursts of this element occurred shortly after the WGD and continued during salmonid speciation, where they have produced dramatic differences in TE content among extant salmonid lineages. This work provides important resources for future studies of salmonids, and advances the understanding of two important evolutionary forces: TEs and WGDs.
Graduate
2019-04-19

碩士
國立交通大學
生物資訊及系統生物研究所
99
LINE-1 and microsatellite are classified into repetitive DNA. They occupy genome in a considerable part. Microsatellite is constituted by simple DNA tract less than ten nucleotides which continuously repeat many times. LINE-1 is the most abundant retrotransposon in the mammalian genome, in the genome. At present, studies suggest that the ORF2 (open reading frame 2) of LINE-1produce microsatellite frequently, but not sure the exact location and many early studies for the microsatellite discussed microsatellite length changes in a short time. We want to explore the location of microsatellite prone position (called length-mutation hotspot) in ORF2 in LINE-1. We also used LINE-1 to find microsatellite and thus observe the orthologous microsatellite in humans, chimpanzees and marmosets. The results show that high-frequency microsatellite region we found was allocated at 5701-5800 bp in LINE-1 ORF2. In this region, the number of interrupted site was larger than other regions. Using BLAST and alignment, the sequences around interrupted site were highly consensus and tended to a restrict region. To find this region, we use 2 steps alignment to get the closest approximately regions to generate microsatellite. We did find a region we called length-mutation hotspot regions. The length of this region was not fixed by the dozens to thousands of nucleotides. Each LINE-1 subfamily of length 16 - 18 bp in length-mutation hotspot was the highest. Analysis of 16 - 18 bp of length-mutation hotspot sequence, we got the sequence of the consensus sequence, called as the standard length-mutation hotspot sequence. Compared standard length-mutation hotspot sequence and length-changed hotspot sequences, we can know which position changed repeat number. After observation of length-changed hotspot region we knew how many in the hotspot region will produce the number of microsatellite, and there were three main microsatellites (TA, TG and CA). In addition, we got expansion time microsatellite in length-mutation hotspot occurred slightly larger than speciation of human and marmoset, about 35 – 40 million years ago. At last, we found a example of orthologs of humans, chimpanzees and marmosets and then we use the dot-plot and alignment to compare.

Transposable elements (TEs) are genetic sequences able to copy or move themselves across their host genome. As TEs move within their host they can act as a source of genetic novelty, and hence are often described as \drivers of evolution". This novelty includes contributing or altering regulatory and coding regions, and promoting non- allelic homologous recombination and, in turn, major structural rearrangements. In some cases, TEs can further contribute to genomic change by jumping between organisms in a process known as horizontal transposon transfer (HTT). HTT is the passing of TEs between organisms by means other than parent to offspring, and has been well described across vertebrates, with multiple events noted in both birds and squamates. Birds are the most diverse class of reptiles, encompassing over 10,000 species, however studies in TE evolution in birds have focused on single lineages. Early findings from the chicken genome led to the assumption that avian TEs are largely stable and inactive. More recent studies have similarly focused on single lineages of birds, revealing some variation in TE activity across birds. In contrast to birds, few studies have explored the evolution of TEs in squamates (lizards and snakes) at a class or family level, instead examining their evolution either across the order or comparing two long diverged species. As such, it is unknown whether patterns seen across all squamates occur at shorter time scales. At lower levels many squamate families are highly diverse, rapidly adapting to new environments and ecological niches. One such family is Hydrophiinae, a family of elapid snakes containing ~100 terrestrial snakes, ~60 marine sea snakes and 6 amphibious sea kraits. In this thesis I investigate the evolution of TEs in two diverse groups of rep- tiles: birds and Australo-Melanesian elapid snakes (Hydrophiinae). I provide the first comprehensive study of TE activity across all orders of birds, focusing on the dominant superfamily, Chicken Repeat 1 (CR1) retrotransposons. By performing comparative genomic analyses I have identified significant variation in the rate of TE expansion both between and within avian orders. Clades including parrots, kiwis and waterfowl show high diversity and large, recent expansions of CR1 retrotransposons, while in various ratites and songbirds CR1s have been near inactive for tens of millions of years. The rest of the chapters focus on the evolution of TEs in hydrophiines, finding repeated HTT events into marine hydrophiines from other marine organisms. TEs in hydrophiines that were acquired via HTT appear to have played a role in their adaptation to the marine environment, with insertions found throughout regulatory regions. In the sea kraits, one horizontally transferred TE has rapidly expanded to make up 8-12% of the sea krait genome in a timespan of just 15-25 million years, the fastest known expansion of TEs in amniotes following a HTT event. Together this thesis presents bioinformatic analyses of two diverse clades of rep- tiles, Aves and Hydrophiine, finding that to truly understand TEs, their evolution and the potential adaptive effects they can cause, we must examine life on both a broad and fine scale.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2021

This thesis studies the long-standing question about the role of transposable elements in host genomes. We put forward and study a hypothesis that transposable elements may act as evolutionary helpers, hastening the adaptation of the host organism to a new environment, while remaining inactive during periods of environmental stability.This hypothesis is analyzed by a modelling approach: we construct and study a computational model of transposable element proliferation, in conditions of environmental stress, then we show that environmental stress causes a burst of transposable element activity, increasing mutability of the organisms, and assisting adaptation.Next, we analyze a mathematical model of transposable element proliferation, deriving closed-form formulas for selection-mutation equilibrium in organisms with certain kinds of transposable elements.The study is augmented by empirical experiments, tying the models to real-world phenomena: by analyzing the role of LINE elements in mediating Nonallelic Homologous Recombination we prove that even inactive transposable elements may exert a significant mutagenic effect on the genome.Finally, we conclude by presenting two tools for mining transposable elements in genomic sequences which we have developed over the course of experiments: TRANScendence, and TIRfinder.
W ramach tej rozprawy zostały przeprowadzone badania nad znanym pytaniem dotyczącym roli elementów transpozonowych w genomach organizmów. Przedkładamy tutaj, i analizujemy hipotezę mówiącą o tym, że elementy transpozonowe mogą działać jako czynnik wspomagający ewolucję organizmu gospodarza, pozwalając mu szybciej dostosować się do nowych warunków środowiskowych, pozostając jednocześnie nieaktywnymi w okresach stabilności środowiska.Hipoteza ta jest analizowana poprzez stworzenie modeli aktywności elementów transpozonowych: w ramach tej pracy zaproponowany został stochastyczny, obliczeniowy model proliferacji elementów transpozonowych w warunkach stresu środowiskowego. Następnie przy jego pomocy pokazane zostało, że stres środowiskowy powoduje wybuch aktywności elementów transpozonowych, zwiększając szybkość mutacji organizmów, i wspomagając je w adaptacji.Dodatkowo, sformułowany i przeanalizowany został matematyczny model proliferacji elementów transpozonowych, oraz wyprowadzone zostały zamknięte formuły opisujące stan równowagi pomiędzy mutacjami i selekcją w organizmach zawierających pewne typy elementów transpozonowych.Badania te zostały poparte wynikami eksperymentalnymi, które wiążą nasze modele z rzeczywistymi zjawiskami: analizujemy rolę elementów LINE w mediowaniu nieallelicznej homologicznej rekombinacji, pokazując że nawet nieaktywne elementy transpozonowe mogą wywierać znaczący mutagenny wpływ na organizm gospodarza. Wreszcie na końcu przedstawione są dwa narzędzia, służące do wykrywania elementów transpozonowych w zsekwencjonowanych genomach: TRANScendence oraz TIRfinder.

Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised.
In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability.
In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations.
This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.