Plants as sessile organisms are susceptible to many environmental stresses such as drought, and salinity. They have therefore evolved mechanisms to acclimate and tolerate environmental stresses. Knowledge of the molecular aspects of abiotic stress gleaned from extensive studies in Arabidopsis has provided much information on the complex processes underlying plant response to abiotic stresses. Nevertheless, there is a need for integration of the knowledge gained and a systematic molecular genetic dissection of the complex responses to abiotic stress. In this study in Arabidopsis, comparative expression profiling analysis of progressive (pDr) and moderate (mDr) drought treatments revealed common drought responses, as well as treatment specific signatures responses to drought stress. Under prolonged moderate drought plants develop different mechanisms for acclimation: induction of cell wall loosening at early stage, and a change in hormonal balance (ABA: JA) at late stage of moderate drought. Taking a reverse genetics approach, a MYB transcription factor (MYB109) has been identified as a regulator of growth under drought and salt stress. Global expression profiling showed possible mechanisms of how MYB109 modulates growth under drought conditions: as a regulator of RNA processing and splicing and as a negative regulator of jasmonic acid biosynthesis and signaling. A forward genetics screen for drought and salt tolerance of transposon activation tag (ATag) lines led to the discovery of novel genes, which shed light on unexplored areas of abiotic stress biology. Utilizing this strategy, a potential role for cell wall modification and MATE transporters in response to drought and salt stress has been discovered, which needs further analysis to integrate this information on the role of these biological processes in plant stress biology. / Ph. D.

Transposable elements (TEs) are mobile genetic elements. They are a significant component of many eukaryotic genomes. They are involved in chromosomal rearrangement by serving as substrates for homologous recombination, in creating new genes through a process of TE "domestication", and in modifying and shuffling existing genes by transducing neighboring sequences (Lander et al., 2001). Therefore, both active and inactive TEs are potentially potent agents for genomic change (Kidwell and Lisch, 2001, 2002; Rizzon et al., 2002; Petrov et al., 2003). In the meantime, active TEs are being explored as useful tools for genetic transformation and possible gene drive mechanisms to deliver genes in natural populations (Ashburner et al.,1998; Alphey et al.,2002; Handler and O'Brochta, 2004). My thesis project focuses on AGH1, a novel DNA-mediated TE in Anopheles gambiae and related mosquitoes. I have studied its genomic structure, insertion polymorphism, evolution, and transposition activity. As part of the sequence and structural characterization of AGH1 in the A. gambiae genome, the boundaries of AGH1were determined. The TA target site duplications flanking AGH1 were verified by comparing a genomic sequence that had an AGH1 insertion with the sequence of a corresponding empty site. AGH1 has relatively long, 350bp, TIRs (Terminal inverted repeats). In addition to the transposase ORF (ORF1) that contains a DD34E catalytic motif, it contains an unusual ORF2 with unknown function. Phylogenic analyses clearly suggest that unlike most DD34E transposons that are similar to the Tc1 family, AGH1 belongs to a different clade that is related to the previously characterized fungal TE Ant and protozoan TEC1 and TEC2. Truncated AGH1 and AGH1-related MITE (Miniature inverted-repeat TE) families were also identified. AGH1 insertion polymorphism was studied using 4 natural populations that belong to two molecular forms of A. gambiae, M and S. AGH1 insertions showed considerable differences between M and S forms and the insertions of AGH1 are highly variable in two populations of M. These results are potentially significant in light of the hypothesis that M forms are newly derived incipient species that are only found in West Africa. PCR and sequencing results showed more than 99% sequence identity between AGH1 sequences in A. gambiae, A. arabiensis, and A. melas, which may indicate either purifying selection or recent horizontal transfer. To assess whether AGH1 is currently active, inverse PCR was performed which provided evidence for extrachromosomal circular AGH1 that may be a product of imprecise excision. RT-PCR detected transcripts for both intact and truncated transposase. Preliminary TE display experiments using genomic DNA isolated from different passages of an A. gambiae Sua1B cell line showed possible new insertions and deletions of AGH1 related elements, which may have been mobilized by AGH1. In summary, the structural and genomic characteristics of AGH1 and the phylogenetic relationship between AGH1 and other known transposons in the IS630-Tc1-mariner superfamily have been determined. Significant divergence was shown between M and S forms of A. gambiae according to AGH1 insertion patterns. Observations of high level of insertion polymorphism and low insertion frequency per site in M populations are preliminary indications that AGH1 may be active in some populations. AGH1 has at least been recently transposing and there are also indications for its current activity in A. gambiae cell lines. If AGH1 is indeed active, it has the potential to be used as genetic tools to study mosquito biology and to spread refractory genes into the field populations to help control mosquito-borne diseases. Although a few active DNA transposons have been discovered in different insects and are being used as tools to transform mosquitoes, no DNA active transposons have been reported in mosquitoes. It is our hope that active endogenous DNA transposons may present new features that will help us overcome some of the deficiencies of current transformation tools developed based on exogenous transposons. In addition, the discovery of an active DNA transposon will help us understand how TEs spread in natural populations of mosquitoes, which is critical if we are to use TEs to drive refractory genes into mosquito populations to control vector-borne infectious diseases. The differential insertion patterns of AGH1 in M and S populations are consistent with the hypothesis that the M and S forms of A. gambiae are in the process of incipient speciation. AgH1 showed much higher levels of insertion polymorphisms in two west African populations of the M molecular form compared to two east African S populations. Similarly, the maximum level of chromosomal differentiation is observed in west African dry savannah areas, while a much lower degree of chromosomal polymorphism is observed in east Africa. Therefore our insertion data support the hypothesis that the speciation process is likely to be originated in west Africa, probably as the result of the need of ecological flexibility created by the greater ecological variability of this region. From a biomedical perspective, this type of analysis is critical because the genetic differences between M and S forms may directly impact the effectiveness of mosquito control measure and perhaps disease transmission. / Master of Science

Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus. / The term “transposable elements” (TEs) is based on their ability to change their genomic position. Through insertions, deletions or chromosomal aberrations, their genomic mobility can cause genetic instability. The extent to which they further exert regulatory influence on cellular functions is the subject of current research, as is the resulting question of their overall biological significance. To further pursue these questions the continuation of experimental research is indispensable. In this regard, the Xiphophorus- melanoma-model represents one of the oldest animal models for the study of malignant melanoma. Thanks to its clearly defined genetic background, it is excellently suited for research into melanoma, which continues to be the most lethal of all known forms of skin cancer. The work presented here investigated the role of transposable elements in malignant melanomagenesis of Xiphophorus.

Transposable elements (Transposons; TEs) constitute the majority of DNA in genomes and are a major source of genetic polymorphisms. TEs act as potential regulators of gene expression and lead to phenotypic plasticity in plants and animals. In crops, several TEs were identified to influence alleles associated with important agronomic traits, such as apical dominance in maize and seed number in rice. Crops may harbor more TE-mediated genetic regulations than expected in view of multifunctional TEs in genomes. However, tools that accurately annotate TEs and clarify their associations with agronomic traits are still lacking, which largely limits applications of TEs in crop breeding. Here we 1) evaluate performances of popular tools and strategies to identify TEs in genomes, 2) develop a tool 'DeepTE' to annotate TEs based on deep learning models, and 3) develop a tool 'TE-marker' to identify potential TE-regulated alleles associated with agronomic traits. As a result, we propose a series of recommendations and a guideline to develop a comprehensive library to precisely identify TEs in genomes. Secondly, 'DeepTE' classifies TEs into 15-24 super families according to sequences from plants, metazoans, and fungi. For unknown sequences, this tool can distinguish non-TEs and TEs in plant species. Finally, the 'TE-marker' tool builds a TE-based marker system that is able to cluster rice populations similar to a classical SNP marker approach. This system can also detect association peaks that are equivalent to the ones produced by SNP markers. 'TE-marker' is a novel complementary approach to the classical SNP markers that it assists in revealing population structures and in identifying alleles associated with agronomic traits. / Doctor of Philosophy / Transposable elements (Transposons; TEs) are DNA fragments that can jump and integrate into new positions in the genome. TEs potentially act as regulators of gene expression and alter traits of plants and animals. In crops, several TEs were identified to influence functions of genes that control important agronomic traits, such as branching in maize and seed number in rice. However, tools that identify these associations in the crops are still lacking, which largely limits applications of TEs in crop breeding. Here we evaluated performance of popular tools and strategies that identify TEs, and provide a series of recommendations to efficiently apply these tools to the TE identification. In view of structural and sequence differences, TEs are classified into multiple families. We developed a 'DeepTE' tool to precisely cluster TEs into different families using a deep learning method. Finally, a 'TE-marker' tool was developed to build TE-based genetic markers to identify nearby alleles associated with agronomic traits. Overall, this work could promote the use of TEs as markers in improving quality and yielding crops.

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. While lacking flagella, C. perfringens cells can still migrate across surfaces using a type of gilding motility that involves the formation of filaments of bacteria lined up in an end to end conformation. To discover the gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. Twenty-four mutants with deficiency in gliding motility were identified and one gene, which encodes a homolog of the SagA cell wall-dependent endopeptidase, was further characterized. We also isolated and characterized two hypermotile variants of strain SM101. Compared to wide type cells, the hypermotile cells are longer and video microscopy of their gliding motility suggests they form long, thin filaments that move rapidly away from a colony, analogous to swarmer cells in bacteria with flagella. Whole genome sequencing analysis showed that both mutants have mutations in cell division genes. Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model is presented explaining the principles underlying the hypermotility phenotype. Heat resistant spores are the major route for disease transmission for C. perfringens, which cause food poisoning. To elucidate the molecular mechanisms involved in spore germination as well as to identify attractive targets for development of germination inhibitors to kill spores, we combined 1D-SDS-PAGE and MALDI-TOF-MS/MS to map the whole spore inner membrane proteome, both from dormant and germinated spores. As the first comprehensive spore inner membrane proteome study, we identified 494 proteins in total and 119 are predicted to be membrane-associated proteins. Among those membrane-associated proteins, 71 changed at least two-fold in abundance after germination. This study provides the first comprehensive list of the spore inner membrane proteins that may be involved in germination of the C. perfringens spore and their relative levels during germination. / Ph. D.

Os elementos de transposicão (TEs) são sequências genéticas móveis. Sua capacidade mutagênica faz deles uma importante fonte de variabilidade nos genomas. Outro importante papel dos TEs na evolução dos genomas é o de doadores de domínios protéicos na formação de novos genes. 276 clones de cDNA homólogos a TEs foram previamente identificados no banco de dados do SUCEST (projeto de sequenciamento de etiquetas expressas de cana-de-acúcar). Neste trabalho nós realizamos o sequenciamento completo de 156 destes clones e a classificação e caracterização de suas sequências comparando-as com bancos de dados. Foram identificadas 9 diferentes famílias de transposons e 11 diferentes famílias de retrotransposons. As famílias mais representadas entre os transposons foram MuDr e hAT (que engloba os elementos do tipo Ac e Tam3), para os quais foram identificados 43 e 32 clones de cDNA, respectivamente. Entre os retrotransposons, a família mais representada foi Hopscotch, apresentando 25 clones de cDNA. Após esta análise global, o foco das investigações voltou-se para os cDNAs do tipo hAT. Uma análise comparativa destes cDNAs revelou que as sequências homólogas a hAT estão distribuídas em dois grupos. O grupo I, é composto por sequências com alta conservação no nível de nucleotídeos, está presente no genoma de todas as gramíneas analisadas (híbridos e parentais da cana-de-acúcar, milho e arroz) com um baixo número de cópias, teve a sua expressão detectada em folhas, raízes e mais intensamente em calos cana. Além disso, apresenta alta similaridade de sequências com transposases domesticadas descritas na literatura. O grupo II, por sua vez, é composto por sequências mais heterogêneas, que apresentam similaridade com os transposons originais que constituem a superfamília hAT: hobo (de Drosophila melanogaster), Ac (de Zea mays) e Tam3 (de Antirrhinum majus). Sua distribuição é restrita ao genoma de Saccharum, com um número de cópias maior que o grupo I. Um ensaio de PCR-Inversa identificou terminações inversas repetidas (TIRs) para o cDNA TE221 do grupo II. A partir de iniciadores desenhados sobre estas TIRs foi possível recuperar dois elementos, de 3,5kb e 4,2kb, respectivamente, e um MITE de 250 pb, todos homólogos a hAT. Este resultado demonstrou que a estratégia utilizada para recuperar elementos do genoma da cana-de-açúcar a partir do cDNA TE221 mostrou-se eficiente. Homólogos aos grupos I e II de cana-de-acúcar foram identificados em bancos de dados de milho, arroz e arabidopsis. Estes dados sugerem que a separação dos dois grupos ocorreu antes da divergência entre as classes Monocotiledonea e Eudicotiledonea. Com base nos resultados aqui apresentados sugerimos que um transposon ancestral do tipo hAT, presente nas angiospermas anteriormente à separação de Monocotiledonea e Eudicotiledonea, teve sua transposase capturada na formação de um gene com função celular. A partir do evento da domesticação, estas transposases seguiram dois caminhos evolutivos distintos, um como gene funcional e outro como um transposon tradicional. Estas duas formas de transposase do tipo hAT podem ser encontradas no genoma da cana-de-acúcar, representadas pelos elementos dos grupos I e II, respectivamente. / Transposable elements (TEs) are mobile genetic sequences. Their mutagenic capacity makes them important sources of variation in the genomes. These elements have another important evolutionary role as donors of functional protein domains in the formation of new genes. 276 cDNA clones homologous to TEs were previously identified in the Brazilian Sugarcane Expressed Sequence Tag Project (SUCEST) databases. In this work, we have obtained the full sequences of 156 for these clones. These sequences were compared with Genbank database. We have identified 9 families of transposons and 11 families of retrotransposons. The most representative families found amongst the transposons were MuDr and hAT (wich encompass Ac and Tam3), with 43 and 32 cDNAs, respectively. Amongst the retrotransposons, the most representative family was Hopscotch, with 25 cDNAs. After this global analysis, we have focused our investigation in the hAT-like cDNAs. A comparative analysis of these cDNAs has revealed a profile of two distinct groups. Group I is composed of sequences with high conservation at nucleotide level, it is present in the genome of all grasses analysed (hybrids and parentals of sugarcane, maize and rice) with low copy number, it is expressed in leaves and roots of sugarcane, and more intensely in callus. In addition, group I sequences have clustered with domesticated transposases. The group II is composed of more heterogeneous sequences similar with the original elements that constitute the hAT superfamily: hobo (from Drosophilla melanogaster), Ac (from Zea mays) and Tam3 (from Antirrhinum majus). This group was shown to be restricted to the genome of Saccharum, with higher copy number than group one. Inverse-PCR assays has identified terminal inverted repeats (TIRs) to the cDNA TE221 from group II. Primers based on the sequences of the TIRs allowed us to recover three elements hAT-like from sugarcane’s genomic DNA: one of 3,5kb and another of 4,2kb, and a MITE of 250 bp. These results corroborate the strategy applied in order to recover elements from the sugarcane´s genome. Sequences homologous to both sugarcane group I and group II were found also in maize and rice, as well as in arabidopsis databases. These data suggest that the divergence of the two groups occured before the separation between the classes Monocotiledonea and Eudicotiledonea. Based on our results, we suggest the existence of an ancestral transposon hAT-like, present in angiosperms before the separation between Monocotiledonea and Eudicotiledonea, of which the transposase was captured to compose a new gene with some cellular function. Since the domestication event, these transposases followed distinct evolutive pathways, one as a regular gene and another as a bona fide transposon. These two forms of hAT-like transposases could be found in the sugarcane’s genome, represented by the elements from groups I and II, respectively.

Os elementos de transposicão (TEs) são sequências genéticas móveis. Sua capacidade mutagênica faz deles uma importante fonte de variabilidade nos genomas. Outro importante papel dos TEs na evolução dos genomas é o de doadores de domínios protéicos na formação de novos genes. 276 clones de cDNA homólogos a TEs foram previamente identificados no banco de dados do SUCEST (projeto de sequenciamento de etiquetas expressas de cana-de-acúcar). Neste trabalho nós realizamos o sequenciamento completo de 156 destes clones e a classificação e caracterização de suas sequências comparando-as com bancos de dados. Foram identificadas 9 diferentes famílias de transposons e 11 diferentes famílias de retrotransposons. As famílias mais representadas entre os transposons foram MuDr e hAT (que engloba os elementos do tipo Ac e Tam3), para os quais foram identificados 43 e 32 clones de cDNA, respectivamente. Entre os retrotransposons, a família mais representada foi Hopscotch, apresentando 25 clones de cDNA. Após esta análise global, o foco das investigações voltou-se para os cDNAs do tipo hAT. Uma análise comparativa destes cDNAs revelou que as sequências homólogas a hAT estão distribuídas em dois grupos. O grupo I, é composto por sequências com alta conservação no nível de nucleotídeos, está presente no genoma de todas as gramíneas analisadas (híbridos e parentais da cana-de-acúcar, milho e arroz) com um baixo número de cópias, teve a sua expressão detectada em folhas, raízes e mais intensamente em calos cana. Além disso, apresenta alta similaridade de sequências com transposases domesticadas descritas na literatura. O grupo II, por sua vez, é composto por sequências mais heterogêneas, que apresentam similaridade com os transposons originais que constituem a superfamília hAT: hobo (de Drosophila melanogaster), Ac (de Zea mays) e Tam3 (de Antirrhinum majus). Sua distribuição é restrita ao genoma de Saccharum, com um número de cópias maior que o grupo I. Um ensaio de PCR-Inversa identificou terminações inversas repetidas (TIRs) para o cDNA TE221 do grupo II. A partir de iniciadores desenhados sobre estas TIRs foi possível recuperar dois elementos, de 3,5kb e 4,2kb, respectivamente, e um MITE de 250 pb, todos homólogos a hAT. Este resultado demonstrou que a estratégia utilizada para recuperar elementos do genoma da cana-de-açúcar a partir do cDNA TE221 mostrou-se eficiente. Homólogos aos grupos I e II de cana-de-acúcar foram identificados em bancos de dados de milho, arroz e arabidopsis. Estes dados sugerem que a separação dos dois grupos ocorreu antes da divergência entre as classes Monocotiledonea e Eudicotiledonea. Com base nos resultados aqui apresentados sugerimos que um transposon ancestral do tipo hAT, presente nas angiospermas anteriormente à separação de Monocotiledonea e Eudicotiledonea, teve sua transposase capturada na formação de um gene com função celular. A partir do evento da domesticação, estas transposases seguiram dois caminhos evolutivos distintos, um como gene funcional e outro como um transposon tradicional. Estas duas formas de transposase do tipo hAT podem ser encontradas no genoma da cana-de-acúcar, representadas pelos elementos dos grupos I e II, respectivamente. / Transposable elements (TEs) are mobile genetic sequences. Their mutagenic capacity makes them important sources of variation in the genomes. These elements have another important evolutionary role as donors of functional protein domains in the formation of new genes. 276 cDNA clones homologous to TEs were previously identified in the Brazilian Sugarcane Expressed Sequence Tag Project (SUCEST) databases. In this work, we have obtained the full sequences of 156 for these clones. These sequences were compared with Genbank database. We have identified 9 families of transposons and 11 families of retrotransposons. The most representative families found amongst the transposons were MuDr and hAT (wich encompass Ac and Tam3), with 43 and 32 cDNAs, respectively. Amongst the retrotransposons, the most representative family was Hopscotch, with 25 cDNAs. After this global analysis, we have focused our investigation in the hAT-like cDNAs. A comparative analysis of these cDNAs has revealed a profile of two distinct groups. Group I is composed of sequences with high conservation at nucleotide level, it is present in the genome of all grasses analysed (hybrids and parentals of sugarcane, maize and rice) with low copy number, it is expressed in leaves and roots of sugarcane, and more intensely in callus. In addition, group I sequences have clustered with domesticated transposases. The group II is composed of more heterogeneous sequences similar with the original elements that constitute the hAT superfamily: hobo (from Drosophilla melanogaster), Ac (from Zea mays) and Tam3 (from Antirrhinum majus). This group was shown to be restricted to the genome of Saccharum, with higher copy number than group one. Inverse-PCR assays has identified terminal inverted repeats (TIRs) to the cDNA TE221 from group II. Primers based on the sequences of the TIRs allowed us to recover three elements hAT-like from sugarcane’s genomic DNA: one of 3,5kb and another of 4,2kb, and a MITE of 250 bp. These results corroborate the strategy applied in order to recover elements from the sugarcane´s genome. Sequences homologous to both sugarcane group I and group II were found also in maize and rice, as well as in arabidopsis databases. These data suggest that the divergence of the two groups occured before the separation between the classes Monocotiledonea and Eudicotiledonea. Based on our results, we suggest the existence of an ancestral transposon hAT-like, present in angiosperms before the separation between Monocotiledonea and Eudicotiledonea, of which the transposase was captured to compose a new gene with some cellular function. Since the domestication event, these transposases followed distinct evolutive pathways, one as a regular gene and another as a bona fide transposon. These two forms of hAT-like transposases could be found in the sugarcane’s genome, represented by the elements from groups I and II, respectively.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Transposable elements (TEs) are DNA fragments that can move within and between genomes causing great impact on the evolution of organisms. The hAT superfamily belongs to class II, subclass I of TEs and despite having originally been described in insects and plants, it is now known that these elements have a wide distribution and are diverse in many groups of higher organisms. Phylogenetic studies show that this superfamily is very ancient and its occurrence in these groups may be related to events of horizontal transfer (HT). This work was performed in order to estimate possible cases of hATs elements' HT. The elements homo1 and howilli2 described by Ortiz and Loreto in 2009 in D. willistoni and D. mojavensis species were searched in Neotropical Drosophila genomes using PCR with degenerate primers and because they are very similar elements and described in distantly related species, it was suggested that these elements might be being transmitted horizontally. Amplification was detected in 18 of the 34 species analyzed and these elements showed a patch distribution and incongruities with the TEs and host species' phylogeny, suggesting possible cases of HT. In addition, the estimated divergence of sequences found showed that these elements are or recently were active in the genomes that were investigated. Thus, our results demonstrate that these elements could be in an expansion process in Neotropical drosophilid genomes due to the large amount of HT events observed. / Elementos transponíveis (TEs) são fragmentos de DNA que podem se mover dentro e entre genomas, causando grande impacto na evolução dos organismos. A superfamília hAT pertence a classe II, subclasse I dos TEs e apesar de originalmente terem sido descritos em insetos e plantas, hoje se sabe que esses elementos possuem uma ampla distribuição e são diversos em muitos grupos de organismos superiores. Estudos filogenéticos demonstram que esta superfamília é muito antiga e que sua ocorrência nesses grupos pode estar relacionada a eventos de transferência horizontal (TH). Este trabalho foi realizado com a finalidade de estimar possíveis casos de TH entre elementos hATs. Os elementos homo1 e howilli2, descritos por Ortiz e Loreto em 2009, nas espécies D. willistoni e D. mojavensis, foram buscados nos genomas de Drosophila Neotropicais por meio de PCR com primers degenerados, e por se tratarem de elementos muito similares e descritos em espécies distantemente relacionadas, foi sugerido que esses elementos poderiam estar sendo transmitidos horizontalmente. Das 34 espécies analisadas, houve amplificação em 18, e esses elementos apresentaram uma distribuição descontínua e incongruências com a filogenia das espécies hospedeiras, o que sugere eventos de TH. Além disso, a estimativa de divergência das sequências encontradas revelou que esses elementos são ou foram ativos muito recentemente nos genomas em que foram investigados. Sendo assim, nossos resultados demonstram que esses elementos podem estar em processo de expansão nos genomas de drosofilídeos Neotropicais devido a grande quantidade de eventos de TH que foram observados.

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.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy Ganta / Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment. This method aided in identifying the genomic locations of four E. chaffeensis mutants and also was valuable in confirming four other sites mapped previously by the rescue cloning method. This is the first mutational analysis study in the genome of an Ehrlichia species. Mapping the genomic transposon insertion sites is the first critical step needed for the continued research to define the importance of the mutations in understanding the pathogenesis caused by the organism.