Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Transposable elements (TEs) are DNA regions that can move within and between genomes, causing great impact on the host organisms. The Tc1-Mariner superfamily stands out for being, probably, the DNA transposons superfamily with greater distribution in nature, being ubiquitous in eukaryotes. In part of this work, we characterize elements of the mariner family in Neotropical drosophilids, which were obtained through amplification with degenerated primers. The primers were designed for the catalytic domain region of mariner transposase allowing amplification of a wide range of mariner-like sequences. A sum of twenty-three species have mariner-like sequences belonging to three subfamilies (mellifera, mauritiana and irritans). These elements present a patchy distribution and incongruences with the host phylogeny, suggesting horizontal transmission events between drosophilids and even between drosophilids and species of other families, subfamilies and orders. Moreover, some sequences present open reading frames, conserved catalytic motifs and evidence for the action of a strong purifying selection, which suggest yhat they originated from active elements. In another part of the work, we characterize Paris-like elements (belonging to Tc1 family), through searches in the twelve Drosophila genomes available. These searches, enabled us to find five new Paris-like elements (one in D. ananassae, one in D. pseudoobscura, one in D. persimilis, one in D. mojavensis and one in D. willistoni), with a copy number ranging from two to seven. Three species have putatively active elements. The evolutionary analysis of these elements suggests that they have envolved through vertical transmission associated with some events of stochastic loss in the analysed species. / Elementos transponíveis (TEs) são regiões do DNA que podem se mover dentro e entre genomas, causando grande impacto na evolução dos organismos. A Superfamília Tc1-Mariner se destaca por ser, provavelmente, a superfamília de transposons de DNA com maior distribuição na natureza, sendo ubíqua em eucariotos. Em parte desse trabalho, caracterizamos elementos da família mariner em drosofilideos Neotropicais para os quais obtivemos amplificação com primers degenerados. Os primers foram construídos na região do domínio catalítico da transposase de mariner o que permite amplificar uma ampla gama de sequências relacionadas à mariner. Um total de 23 espécies apresentou sequências relacionadas à mariner pertencentes a três subfamílias (mellifera, mauritiana e irritans). Esses elementos apresentaram uma distribuição descontínua e incongruências com a filogenia das espécies hospedeiras, o que sugere eventos de transmissão horizontal entre drosofilideos e, até mesmo entre drosofilideos e espécies de outra família, superfamília e ordem. Além disso, algumas sequências apresentaram um quadro aberto de leitura, os motivos catalíticos conservados e uma forte seleção purificadora atuando, o que sugere que esses elementos sejam provenientes de elementos ativos. Em outra parte do trabalho, caracterizamos as sequências relacionadas ao elemento Paris (pertencentes à família Tc1), com buscas nos doze genomas de Drosophila disponíveis. Nessas buscas,foram encontrados cinco novos elementos relacionados à Paris (um em D. ananassae, um em D. pseudoobscura, um em D. persimilis, um em D. mojavensis e um em D. willistoni), com um número de cópias variando de dois a sete. Três espécies apresentaram elementos potencialmente ativos. A análise evolutiva desses elementos sugere que estão sendo mantidos por transmissão vertical, com alguns eventos de perda estocástica nas espécies analisadas.

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

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

Transposons sind genetische Elemente, die fähig sind, sich innerhalb des Genoms zu bewegen. Sleeping Beauty (SB) gehört zur Tc1/mariner-Superfamilie von Transposons. SB wurde aus molekularen Fossilien rekonstruiert um u.a. einen sicheren und effizienten Vektor für die Gentherapie zu schaffen. Zu diesem Zweck ist es notwendig, den Mechanismus der SB-Transposition und deren Regulation, die Aktivitäten des Proteins und den Einfluss von Wirtsfaktoren genau zu verstehen. In meiner Arbeit habe ich die einzelnen Schritte des Transpositionsprozesses und die Bildung des sogenannten paired-end complex (PEC) – eine Voraussetzung für die folgenden katalytischen Reaktionen – untersucht. Zusätzlich habe ich versucht, einen in vitro Transpositionstest für SB zu etablieren. SB gehört zur IR/DR-Gruppe der Tc1/mariner-Superfamilie. Im Gegensatz zu mariner-like-Elementen ist die IR/DR-Struktur von SB durch lange IRs mit insgesamt vier Bindestellen für die Transposase gekennzeichnet. Ich habe die Fähigkeit dieser beiden Transposon-Systeme zum Ausschneiden eines Transposonendes ohne die Beteiligung des anderen Endes im PEC getestet. Solche unpräzise Transposition kann zu genomic rearrangements führen. Meine Ergebnisse zeigen, dass SB zwar imstande ist, ein einzelnes Transposonende auszuschneiden, dies geschieht jedoch weit weniger effizient als bei mariner-like-Elementen. Die Unterdrückung unpräziser Transpositionsereignisse ist ein Ergebnis der besseren durch die IR/DR-Struktur bedingten Regulation von SBs Transposition. Die Komplexität der IRs in Kombination mit der zweiteiligen DNA-Bindedomäne von SB kann als Mittel einer raffinierten Regulation des Transpositionsprozesses angesehen werden, welche das Genom vor anormalen Transpositionsereignissen schützt. Die Ergebnisse meiner Arbeit legen ein Modell nahe, in dem die Bildung des PEC während der Transposition von SB ein höchst genau regulierter Prozess ist, der durch die DNA-Protein- und Protein-Protein-Bindeaffinitäten geleitet wird. / Transposons are pieces of DNA able to move within the genomes. Sleeping Beauty is a verterbrate Tc1/mariner transposon reconstructed from molecular fossils to create a safe and efficient vector for gene therapy. For that purpose it is important to deeply understand the mechanism and regulation of the SB transposition, the activities of the transposase and influence of host factors on the process. Therefore, in this project I studied the single steps of the transposition reaction and formation of the paired-end complex (PEC) which is a prerequisite for the subsequent catalytic steps. Additionally, I tried to establish an in vitro transposition assay for Sleeping Beauty that would serve an easy assay for testing the system and probe mechanisms affecting the regulation of transposition activity. Sleeping Beauty belongs to the IR/DR subfamily of the Tc1/mariner-like transposons. In contrast to mariner-like elements the IR/DR structure of SB is characterized by long IRs with four binding sites for the transposase. I compared the ability of the two systems to perform cleavage of the single transposon end without including the second end in the PEC. Such imprecise transposition can lead to genome rearrangements. My results show that SB is capable of single-end cleavage; however, to much lower extent than the mariner-like element. Lower number of imprecise transposition events is a result of better regulation of the SB transposition imposed by the IR/DR stucture. The complexity of the inverted repeats together with the bipartite DNA-binding domain of SB might offer means for more sophisticated regulation of the transposition process, thereby protecting the genome from aberrant transposition events. I propose that complex formation in SB transposition is a strictly regulated ordered assembly process, guided by DNA-protein and protein-protein interaction interfaces of the DNA-binding subdomains. Obtained results allowed me to draw a model how the paired-end complex is formed.

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