Dermatófitos são um grupo de fungos intimamente relacionados, que tem a capacidade de invadir tecidos queratinizados como pele, cabelos e unhas de homens e outros animais causando dermatofitoses. Os agentes envolvidos nessas infecções pertencem aos gêneros Trichophyton, Microsporum ou Epidermophyton e, de acordo com seu habitat natural, são classificados em espécies geofílicas, zoofílicas ou antropofílicas. A maior incidência de dermatofitoses é causada pelo gênero Trichophyton, sendo T. rubrum a espécie mais prevalente em infecções de pele e unhas em humanos. Devido à severidade e longevidade destas infecções, e à resistência ao tratamento, o estudo de fatores envolvidos na interação patógeno-hospedeiro, na resistência dos dermatófitos a agentes antifúngicos e na manutenção do processo infeccioso são de grande relevância. Por análises morfológicas, fisiológicas e de expressão gênica, comparamos cinco dermatófitos cujos genomas foram sequenciados por iniciativa do Broad Institute, Microsporum canis, Trichophyton equinum, Trichophyton interdigitale, Trichophyton rubrum e Trichophyton tonsurans. Cultivos em queratina, mimetizando o processo infeccioso, foram utilizados para analisar o envolvimento dos dermatófitos na interação patógeno-hospedeiro e manutenção do processo infeccioso. Também expusemos as espécies a concentrações subinibitórias de agentes terapêuticos, de modo a verificar a resposta destes fungos a diferentes drogas. Observamos que o acúmulo de transcritos dos genes relacionados à virulência em dermatófitos avaliados durante o crescimento em queratina sugere que a maquinaria metabólica com atividade de formação da parede celular do fungo, metabolização do substrato e adesão ao hospedeiro ativa nos períodos iniciais de infecção. Contudo, um padrão de expressão correlacionado à similaridade das sequências genômicas não foi observado nas condições testadas. Também não se observa correlação direta entre o nicho preferencial dos dermatófitos e os níveis transcricionais em resposta à queratina de origem animal. Analisamos três genes envolvidos na resistência a múltiplas drogas (MDR) durante crescimento na presença de drogas com atividade antifúngica. Nossos dados sugerem que os genes MDR atuam sinergicamente em dermatófitos, e podem atuar de forma compensatória quando em presença de drogas antifúngicas, o que pode ser uma importante causa de falhas no tratamento. Nossos resultados fornecem evidências de que a expressão dos genes analisados não se correlaciona com as relações filogenéticas entre estes dermatófitos, visto que apesar da íntima relação entre o conteúdo genético e organização do genoma, os níveis transcricionais destes genes são diferentes entre as espécies. Assim, diferenças na adaptação a nichos específicos e a progressão da doença entre os dermatófitos podem ser explicadas por diferentes perfis de transcrição do gene. / Dermatophytes are a group of closely related fungi, which have the ability to invade keratinized tissues, such as skin, hair, and nails of both human and animal hosts causing dermatophytosis. The agents involved in these infections belong to the genera Trichophyton, Microsporum or Epidermophyton and, according to their natural habitat, are classified as geophilic, zoophilic or anthropophilic species. The higher incidence of dermatophytosis is caused by the genera Trichophyton, being the specie T. rubrum the most prevalent causative of human skin and nail infections. Because of the severity and longevity of these infections and their resistance to treatment, the study of the factors involved in host-pathogen interaction, in resistance of dermatophytes to antifungal agents, and in maintenance of the infection is relevant. Through morphological, physiological and gene expression analysis we compared five dermatophytes, whose genomes were sequenced by initiative of the Broad Institute: Microsporum canis, Trichophyton equinum, Trichophyton interdigitale, T. rubrum and Trichophyton tonsurans. Growth in keratin, which mimetize the infectious process, was used to analyze the involvement of dermatophytes in host-pathogen interaction and maintenance of the infectious process. We also exposed the species to subinibitory concentrations of therapeutic agents to verify the response of these fungi to different drugs. We observed that the accumulation of transcripts of genes related to virulence in dermatophytes evaluated during growth in keratin, suggest that the metabolic machinery with activity on fungal cell wall formation, substrate metabolization, and host adhesion is activated in early stages of infection. However, an expression pattern correlating to genomic sequence similarity was not observed in the conditions tested. We also did not observe a direct correlation between the preferential niche of these dermatophytes and the transcriptional levels in response to the keratin from animal origin. We analyze three genes involved in multidrug resistance (MDR) during growth in the presence of drugs with antifungal activity. Our data suggest that MDR genes act synergistically in dermatophytes, and they may compensate for one another when challenged with antifungal drugs, which can be an important cause of therapeutic failure. We provide evidence that the expression of the analyzed genes does not correlate with the phylogeny of these dermatophytes since, in spite of the different species being highly related in gene content and genome organization, the transcription level of these genes is different among these species. Thus, differences in adaptation to a specific niche and disease progression among dermatophytes would be explained by different gene transcription profiles.

L’Identification rapide et la classification microbienne précise sont cruciales en microbiologie médicale pour la surveillance de la santé humaine et animale, établir un diagnostic clinique approprié et choisir des mesures thérapeutiques et de contrôle optimales. Cependant, les seuils universels utilisés pour la définition des espèces ne sont pas applicables à de nombreux genres bactériens. C'est notamment le cas des espèces du genre Rickettsia, qui expriment peu de caractéristiques phénotypiques distinctives. Compte tenu de la disponibilité des séquences de près de 100 génomes de Rickettsia, nous avons voulu évaluer une gamme de paramètres taxonomiques basés sur l’analyse des séquences génomiques afin de mettre au point des recommandations pour la classification des isolats au niveau de l’espèce et du genre. En comparant le degré de similarité des séquences de 78 génomes de Rickettsia et 61 génomes de 3 genres étroitement apparentés en utilisant 4 paramètres génomiques, nous avons montré que les outils taxonomiques basés sur les séquences génomiques sont simples à utiliser et rapides, et permettent une classification taxonomique fiable et reproductible des isolats de rickettsies avec des seuils spécifiques. Les résultats obtenus nous ont permis d'élaborer des recommandations pour la classification des isolats de rickettsies au niveau du genre et de l'espèce. À l'aide de la taxono-génomique, nous avons également pu décrire 17 nouvelles espèces bactériennes associées à l'homme. L'utilisation des outils génomiques est donc parfaitement adaptée à la classification taxonomique et peut changer radicalement notre vision de la taxonomie et de l'évolution bactérienne à l'avenir. / Rapid identification and precise microbial classification are crucial in medical microbiology for human and animal health monitoring, appropriate clinical diagnosis and selection of optimal therapeutic and control measures. Indeed, the universal used for the definition of species are not applicable to many bacterial genera. This is particularly true of species of the genus Rickettsia which are strictly intracellular alpha-proteobacteria that express few phenotypic characteristics. Given the availability of genomic sequences of nearly 100 rickettsial genomes, we wanted to evaluate a range of taxonomic parameters based on genomic sequence analysis, to develop guidelines for the classification of Rickettsia isolates at the genus and species levels. By comparing the degree of similarity of the sequences of 78 genomes from Rickettsia species and 61 genomes from 3 closely related genera using several genomic parameters, we have shown that genome-based taxonomic tools are simple to use and fast, and allow for a reliable and reproducible taxonomic classification of isolates within species of the genus Rickettsia, with specific thresholds. The obtained results enabled us to develop guidelines for classifying rickettsial isolates at the genus and species levels. Using taxono-genomics, we have also been able to describe 17 new human-associated bacterial species on the basis of a combination of genomic analysis and phenotypic properties. The use of genomic tools is therefore perfectly adapted to taxonomic classification and can dramatically change our vision of taxonomy and bacterial evolution in the future.

Bacillus thuringiensis subsp. israelensis é uma bactéria gram positiva, amplamente utilizada no controle de insetos Diptera, família Culicidae, tais como Aedes aegypti e Culex quinquefasciatus. O sequenciamento de genomas completos de estirpes de B. thuringiensis tem permitido a identificação e caracterização de proteínas inseticidas, assim como a realização de estudos de genômica comparativa, permitindo evidenciar diferenças funcionais e filogenéticas. Neste contexto, o presente estudo teve como objetivo realizar análises genômicas de quatro isolados de B. thuringiensis subsp. israelensis que apresentam diferentes toxicidades para larvas de A. aegypti e C. quinquefasciatus. O sequenciamento dos genomas dos isolados T0124, T0131, T0137 e T0139 foi realizado a partir da plataforma MiSeq-Ilumina, e a montagem e anotação foram feitas utilizando ferramentas do programa de bioinformática Geneious. As análises resultaram em cromossomos com tamanhos aproximados de 5.414 Kb, conteúdo G+C de 35,2% e 5.358 regiões codificantes. Com relação ao conteúdo extracromossomal, foram montados três plasmídeos menores de 5,4; 6,8 e 7,6 Kb, e três plasmídeos maiores de 127, 235 e 359 Kb para todos os isolados, e estes foram enumerados de 1 a 6. A anotação revelou que apenas os plasmídeos de número 4, que correspondem ao pBtoxis, possuem proteínas inseticidas, sendo elas Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa, Cyt2Ba e Cyt1Ca. A análise comparativa entre os genomas demonstra que os cromossomos T0124, T0131, T0137 e T0139 apresentam sequências colineares e com 99% de identidade, e seus respectivos plasmídeos 1, 2, 3, 4, 5 e 6 compartilham todas as suas regiões codificantes, incluindo aquelas relacionadas a δ-endotoxinas. A comparação de sequências de enterotoxinas, metaloproteases e fosfolipases demonstra que estes também são conservados entre os genomas. Na análise filogenética, os cromossomos T0124, T0131, T0137 eT0139 agruparam-se juntamente com os genomas dos isolados AM65-52 e HD-789 disponíveis no GenBank, sugerindo uma estreita relação genética entre estirpes de B. thuringiensis subsp. israelensis. Os isolados T0124, T0131, T0137 e T0139 apresentam diferentes concentrações letais para larvas de A. aegypti e C. quinquefasciatus, e a adição de dados proteômicos aos dados genômicos, aqui obtidos, poderão auxiliar na compreensão destas diferenças de toxicidade. Contudo as análises comparativas entre os genomas dos isolados T0124, T0131, T0137 e T0139 mostram alto grau de conservação genética a nível de nucleotídeos de B. thuringiensis subsp. israelensis. / Bacillus thuringiensis subsp. israelensis is a gram-positive bacterium widely used to control insects of the order Diptera, such as Aedes aegypti (Diptera: Culicidae) and Culex quinquefasciatus (Diptera: Culicidae). The sequencing of complete genomes of B. thuringiensis strains has allowed the identification and characterization of insecticidal proteins, as well as the comparative genomic studies, allowing the identification of functional and phylogenetic differences. In this context, the present study aimed to perform genomic analysis of four isolates of B. thuringiensis subsp. israelensis having different toxicities for larvae of A. aegypti and C. quinquefasciatus larvae. Sequencing of genomes of the isolates T0124, T0131, T0137 and T0139 was performed from the MiSeq-Ilumina platform, and assembly and annotation were done using tools from the Geneious bioinformatics program. The analyzes resulted in chromosomes with approximate sizes of 5,414 Kb, G + C content of 35.2% and 5,358 coding regions. Regarding extrachromosomal content, three smaller plasmids of 5.4, 6.8 and 7.6 Kb, and three larger plasmids of 127, 235 and 359 Kb were assembled for all the isolates, and these were enumerated from 1 to 6. The annotation revealed that only the plasmids of number 4, corresponding to pBtoxis, possess insecticidal proteins, being Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa, Cyt2Ba and Cyt1Ca. Comparative analysis between the genomes demonstrates that chromosomes T0124, T0131, T0137 and T0139 have collinear sequences and 99% identity, and their respective plasmids 1, 2, 3, 4, 5 and 6 share all of their coding regions, including those related to δ-endotoxins. Comparison of enterotoxin, metalloprotease and phospholipase sequences shows that these are conserved among the genomes. In the phylogenetic analysis, chromosomes T0124, T0131, T0137 and T0139 were grouped together with the genomes of the isolates AM65-52 and HD-789 available in GenBank, suggesting a close genetic relation between strains of B. thuringiensis subsp. israelensis. The isolates T0124, T0131, T0137 and T0139 present different lethal concentrations to larvae of A. aegypti and C. quinquefasciatus, and the addition of proteomic data to the genomic data obtained here may help to understand these differences in toxicity. However, the comparative analyzes between the genomes of isolates T0124, T0131, T0137 and T0139 show a high degree of genetic conservation at the nucleotide level of B. thuringiensis subsp. israelensis.

Cela fait plus de quarante ans que la plupart des modifications des ARNt ont été découvertes mais ce n’est que récemment que les gènes correspondants ont pu être identifié. La modification N6-threonylcarbamoyl adénosine (t6A) est universelle et se trouve à la position 37, adjacente de l’anticodon, dans de nombreux ARNt. Les quatre gènes responsables de la synthèse de cette modification chez les bactéries furent découverts par des approches de génomique comparative mais uniquement deux de ces gènes sont universels, TsaC/Sua5 et TsaD/Kae1/Qri7. Des travaux récents ont révélé qu'il existait différentes voies enzymatiques pour la synthèse de cette modification selon domaine de la vie, les organelles et les espèces considérés. L'étude de ces variations est toujours en cours de caractérisation.Ce travail a identifié quatre autres protéines requises pour la synthèse de t6A dans les ARNt cytoplasmiques de levure (Bud32, Pcc1, Cgi121 et Gon7) et établi que seuls Sua5 et Qri7 sont requis pour modifier les ARNt mitochondriaux. La même enzyme, Sua5, effectue la première étape de la synthèse de t6A à la fois dans le cytoplasme et les mitochondries. Cette protéine peut être localisée dans les deux compartiments grâce à l’utilisation de sites d’initiation de la traduction différents. Cette étude a montré qu’une machinerie de synthèse minimale est requise pour la synthèse de t6A dans les mitochondries, potentiellement similaire à la machinerie présente dans le dernier ancêtre commun. Les rôles de cette modification complexe in vivo semblent également varier. Par exemple, t6A est indispensable chez les procaryotes, mais pas dans la levure. Les causes des phénotypes pléïotropes observés lors de la diminution ou l'absence de t6A ne sont pas encore entièrement comprises. Nous avons pu élucider certains des rôles joués par la modification t6A, en effectuant une analyse globale des erreurs de traduction observées en absence de cette modification par analyse des profils ribosomaux. Par exemple, il semble que la présence de t6A permet aux ARNt rares de concurrencer plus efficacement les ARNt abondants. La complexité et la diversité des voies de synthèse combiné à l’importance fonctionnelle et évolutive de cette modification ont fait de t6A une “décoration” des ARNt particulièrement fascinante à étudier. / The modification of tRNA has a rich literature of biochemical analysis going back more than 40 years; however, the genes responsible for the modifications have only been recently identified. Comparative genomic analysis has allowed for the identification of the genes in bacteria, and subsequent characterization of the enzymes, responsible for the modification N6-threonylcarbamoyladenosine (t6A) located at position 37, adjacent to the anticodon of tRNAs. While the modification is present in all domains of life, only two of the four enzymes responsible for biosynthesis machinery are conserved. In Eukaryotes, both cytoplasmic and mitochondrial tRNAs are modified with t6A, and previously only the two universally conserved members of the cytoplasmic t6A synthesis pathway, TsaC/Sua5 and TsaD/KaeI/Qri7 were known. Recent progress on deciphering the t6A synthesis pathways has revealed that different solutions have been adopted in different kingdoms, species, and organelles, and these variant pathways are still being characterized.This investigation identified the other four proteins required for cytoplasmic synthesis (Bud32, Pcc1, Cgi121, Gon7), and determined that only Sua5 and Qri7 are required for mitochondrial synthesis of t6A in yeast. The same enzyme, Sua5, performs the first step of t6A synthesis in both the cytoplasm and the mitochondria. It is targeted to both the cytoplasm and the mitochondria through the use of alternative, in-frame AUG translational start sites. This study showed that a minimum synthesis machinery is responsible for mitochondrial t6A, implicating a core set of enzymes from the LUCA.The roles of this complex modification in vivo also seem to vary. For example, t6A is essential in prokaryotes, but not in yeast. The causes of the observed pleiotropic phenotypes triggered by the reduction or absence of t6A synthesis enzymes are not yet fully understood. This work used ribosome profiling to map all translation errors occurring when t6A was absent. By examining ribosomal occupancy of every codon, this work indicates that t6A is helping rare tRNAs compete with high copy tRNAs. The complexity and diversity of the t6A pathway combined with the functional and evolutionary importance of this modification have made t6A a particularly fascinating “decoration” of tRNA to study.

Cana-de-açúcar é uma planta agrícola com grande importância econômica para o Brasil. Os cultivares modernos de cana-de-açúcar apresentam uma alta complexidade genômica, devido ao seu genoma ser poliploide e com número cromossômico variando entre 100-120. As plantas de cana-de-açúcar estão sujeitas ao ataque de diversos tipos de patógenos, e sua tolerância depende da ação de genes de resistência. Estes podem desencadear uma cascata de reações ou interagir diretamente com uma molécula efetora, resultando em tolerância por parte da planta à presença do patógeno. O genoma dos cultivares moderno apresentam inúmeros elementos de transposição posicionados em diferentes regiões do genoma. Devido a essa abundância, os elementos de transposição estão sendo usados como marcadores moleculares, por exemplo, RBIP (Retrotransposon-Based Insertion Polymorphism), que identifica polimorfismos baseado na inserção de elementos do tipo retrotransposons. Esta técnica é capaz de diferenciar a presença e a ausência de um elemento de transposição em uma determinada região do genoma. Para a aplicação em organismos poliploides foi desenvolvida a técnica RBIP-qPCR, permitindo dosar a presença e ausência de um elemento de transposição em um organismo poliploide. Sendo assim este trabalho tem como objetivo aplicar a técnica de RBIP-qPCR em elementos de transposição alocados adjacentes a genes de resistência a fim de, posteriormente, verificar a existência de associação entre genes de resistência e elementos de transposição. Para isso foram analisadas três famílias de genes de resistência, I2C-2, Xa21 e Pti1. Foram realizadas análises de composição genômica de cada uma das regiões, genômica comparativa entre as variedades de R570 e SP80-3280 para a região genômica que contém o gene I2C-2, além de uma análise de expressão de genes e elementos de transposição vizinhos neste loco. A partir destes estudos foi possível selecionar um elemento de transposição, scDEL 5.1, para aplicar a técnica de RBIP-qPCR. Os resultados das análises de reconhecimento do ambiente genômico determinou a escolha de um elemento para aplicação da técnica. Verificamos que na ausência de scDEL 5.1 a metodologia RBIPqPCR foi eficaz em todos os genomas testados. As análises de presença de scDEL 5.1 no loco revelaram que elementos com elevado número de cópias no genoma não são adequados para a aplicação da metodologia. / Sugarcane is an agricultural plant with great economic importance for Brazil. The modern cultivars of sugarcane present a high genomic complexity, due to their genome being polyploid and with chromosome number varying between 100-120. Sugarcane plants are targets of several types of pathogens, and their tolerance depends on the action of resistance genes. These can trigger a cascade of reactions or interact directly with an effector molecule, resulting in tolerance by the plant to the presence of the pathogen. The genome of modern cultivars presents several transposable elements allocated in different regions of the genome. Due to this abundance, transposable elements are being used as molecular markers, such as RBIP (Retrotransposon-Based Insertion Polymorphism), which identifies polymorphisms based on the insertion of retrotransposon. This technique is able to differentiate the presence and absence of a transposable element in a particular region of the genome. For the application in polyploid organisms the RBIP-qPCR technique was developed, allowing measuring the presence and absence of a transposable element in a polyploid organism. Therefore, the objective of this work is to apply the RBIP-qPCR technique to on a transposable element localized adjacent to resistance genes in order to, latterly, verify the existence of association between resistance genes and transposable elements. For this, three families of resistance genes, I2C-2, Xa21 and Pti1 were analyzed. Genomic composition analyzes of each region, comparative genomics between the R570 and SP80-3280 varieties for the genomic region containing the I2C-2 gene, as well as an analysis of genes and elements expression neighbors at this locus were performed. From these studies it was possible to select a transposition element, scDEL 5.1, to apply the RBIP-qPCR technique. The results of the genomic environment recognition analysis determined the choice the transposable element for the application of the technique. We verified that in the absence of scDEL 5.1 the RBIP-qPCR methodology was effective in all genomes tested. Analysis of the presence of scDEL 5.1 in the locus revealed that elements with high copy number in the genome are not suitable for the application of the methodology.

Les Poaceae aussi appelées Graminées forment une importante famille botanique regroupant près de 12 000 espèces en plus de 700 genres dont les céréales. Cette famille présente un intérêt économique majeur car elle est importante dans la nutrition humaine et animale. De ce fait, cette famille a été très étudiée en génomique comparée depuis les années 1990 révélant une grande conservation de la structure de leur génome depuis leur divergence d’un ancêtre commun. Avec le séquençage de Brachypodium distachyon en 2009, nous avons réalisé l’analyse de son génome par l’identification de douze blocs de synténie avec les génomes séquencés du riz, du sorgho et du maïs ainsi que sept blocs de duplications partagées entre ces graminées. Ces données nous ont permis de suggérer que les cinq chromosomes modernes de Brachypodium sont issus de l’ancêtre commun des graminées constitué de douze chromosomes et ayant subi sept fusions au cours de l’évolution. Ces travaux nous ont permis de confirmer un possible génome ancêtre des graminées constitué de cinq chromosomes porteurs de près de 10 000 gènes et d’une taille minimale de près de 35Mb. Ensuite, sur la base des résultats de génomique comparée, nous nous sommes intéressés à l’évolution des différentes familles de micro-ARN (miARN). La comparaison de ces ARN non-codants réalisée pour le riz, le sorgho, le maïs et Brachypodium montre une conservation de cette famille chez les graminées avec 50% d’orthologues et 20% de paralogues. Sur la base des résultats de paléogénomique, nous avons proposé une modélisation de l’évolution des miARN qui corrobore l’hypothèse d’une origine très ancienne de ce mécanisme de « gene silencing ». Au-delà des nouvelles connaissances fondamentales générées au cours de ce travail de thèse sur l’évolution des génomes de graminées, les résultats que nous avons obtenus ont des applications potentielles dans le domaine de l’amélioration variétale, comme avec par exemple la possibilité de définir des marqueurs moléculaires de type COS (Conserved Orthologous Set). Ces marqueurs COS ont été mis en oeuvre pour l’étude de caractères agronomiques d’intérêt dans des espèces dont le génome n’est pas encore complètement séquencé comme le blé. / Poaceae also called Grasses are an important botanical family consisting in nearly 12,000 species in over 700 genres including cereals. This family is of major economic interest because it comprises cereals that are among the most important crops for human and animal nutrition. This family has been extensively studied in comparative genomics since the 1990s and showed a high degree of gene conservation among species since they diverged from a common ancestor. With the sequencing of Brachypodium distachyon in 2009, we performed an analysis of its genome by the identification of twelve synteny blocks with the sequenced genomes of rice, sorghum and maize and seven duplications blocks shared with these last grass species. These data allowed us to suggest the five chromosomes of Brachypodium are from the common ancestor composed of twelve chromosomes and having undergone seven fusions during the evolution. This work allowed us to confirm a possible grass ancestor with five chromosomes carrying almost 10,000 genes with a size of 35Mb. Then, based on these comparative genomics results, we studied more particularly the evolution of different families of microRNAs (miRNAs). The comparison of non-coding RNA from rice, sorghum, maize and Brachypodium showed conservation into this family for the grass species with 50% of orthologs and 20% of paralogs. Based on the paleogenomics results, we proposed an evolutionary scenario of miRNA genes, which supports the hypothesis of an ancient origin of this gene silencing mechanism in plants. Beyond the fundamental knowledge generated on the evolution of grass genomes during this PhD, these results have potential applications in breeding, for example with the possibility to identify COS (Conserved Orthologous Set) molecular markers. Such COS markers have been used for the study of agronomic traits in species not completely sequenced as wheat.

.

The process of evolution is of both scientific and medical interest. This thesis presents several studies using complete genomic reference sequences, comparative genomic data, and intraspecific diversity data to study the two key processes of evolution: mutation and selection. Large duplications, deletions, inversions, and translocations of DNA contribute to genomic variation both between and within species. Human chromosomes 15 and 17 contain a high percentage of dispersed, recently duplicated sequences. Examination of the relationships between these sequences showed that the majority of all duplications within each chromosome could be linked through core sequences that are prone to duplication. Comparison to orthologous sequences in other mammals allowed a reconstruction of the ancestral state of the human chromosomes, revealing that regions of rearrangement specific to the human lineage are highly enriched in chromosome-specific duplications. Comparison to copy number variation data from other studies also shows that these regions are enriched in current human structural variation. One specific region, the MAPT locus at 17q21.31, known to contain an inversion polymorphism in Europeans, was resequenced completely across both human orientation haplotypes and in chimpanzee and orangutan, revealing complex duplication structures at the inversion breakpoints, with the human region being more complex than chimpanzee or orangutan. Fluorescent in-situ hybridization analysis of human, chimpanzee, and orangutan chromosomes showed inversion polymorphisms of independent origin in all three species, demonstrating that this region has been a hotspot of genomic rearrangement for at least twelve million years. These results reveal a mechanistic relationship between sequence duplication and rearrangement in the great apes. We also generated a draft sequence of the chimpanzee genome and compared it to that of the human. Among other findings, this showed that CpG dinucleotides contribute 25% of all single base mutations, with a rate of mutation ~10-fold that of other bases, and that the male mutation rate in great apes is ~5-6 times the female rate, a higher ratio than had been observed in comparisons of primates and rodents. We detected six regions of probable recent positive selection in humans with a statistical method relying on chimpanzee sequence to control for regional variation in mutation rates. Finally, resequencing of several lines of domestic chicken and comparison to the reference chicken genome identified a number of gene deletions fixed in domestic lines and also several potential selective sweeps. Of particular interest are a missense mutation in TSHR nearly fixed in all domestic chickens and a partial deletion of SH3RF2 fixed in a high growth line. The TSHR mutation may play a role in relaxation of seasonal reproduction. A high-resolution QTL mapping experiment showed that the SH3RF2 deletion is significantly associated with increased growth. This work provides important new insights into the mechanics of evolutionary change at both the single nucleotide and structural level and identifies potential targets of natural and artificial selection in humans and chickens.

This research project presents a comprehensive functional analysis of a complex prokaryotic signal transduction system and the mechanisms underlying its evolution. The chemotaxis system regulates motility in prokaryotes and is their most complex signal transduction system. The system has been extensively characterized experimentally, but recent studies have created new questions about the function and origin of this system. Comparative genomics analyses are well-suited for studying the chemotaxis system since it is present in taxonomically diverse organisms. The first aim of this project is to understand the evolutionary history of the chemotaxis system that has resulted in the diversity of chemotaxis systems that have been experimentally. The results reveal three functional families of chemotaxis systems that regulate flagellar motility, type IV pili motility, and non-motility outputs. The flagellar family shows extensive diversity with 10 conserved classes that have variable accessory proteins, and these classes show a co-evolutionary relationship with flagella. The second aim of this project is to analyze the molecular evolution of chemotaxis system components and utilize that information to predict the contact sites involved in protein-protein interactions. The analysis supports that there is evolutionary pressure at the amino acid sequence level to maintain protein-protein interactions. From this observation, a method to predict the contact sites of protein-protein interactions from sequence information alone was developed and validated by experimental and structural information.

Non-coding RNAs (ncRNAs) are transcripts that function directly as RNA molecule without ever being translated to protein. The transcriptional output of eukaryotic cells is diverse, pervasive, and multi-layered. It consists of spliced as well as unspliced transcripts of both protein-coding messenger RNAs and functional ncRNAs. However, it also contains degradable non-functional by-products and artefacts - certainly a reason why ncRNAs have long been wrongly disposed as transcriptional noise. Today, RNA-controlled regulatory processes are broadly recognized for a variety of ncRNA classes. The thermoresponsive ROSE ncRNA (repression of heat shock gene expression) is only one example of a regulatory ncRNA acting at the post-transcriptional level via conformational changes of its secondary structure. Bioinformatics helps to identify novel ncRNAs in the bulk of genomic and transcriptomic sequence data which are produced at ever increasing rates. However, ncRNA annotation is unfortunately not part of generic genome annotation pipelines. Dedicated computational searches for particular ncRNAs are veritable research projects in their own right. Despite best efforts, ncRNAs across the animal phylogeny remain to a large extent uncharted territory. This thesis describes a comprehensive collection of exploratory bioinformatic field studies designed to de novo predict ncRNA genes in a series of computational screens and in a multitude of newly sequenced genomes. Non-coding RNAs can be divided into subclasses (families) according to peculiar functional, structural, or compositional similarities. A simple but eligible and frequently applied criterion to classify RNA species is length. In line, the thesis is structured into two parts: We present a series of pilot-studies investigating (1) the short and (2) the long ncRNA repertoire of several model species by means of state-of-the-art bioinformatic techniques. In the first part of the thesis, we focus on the detection of short ncRNAs exhibiting thermodynamically stable and evolutionary conserved secondary structures. We provide evidence for the presence of short structured ncRNAs in a variety of different species, ranging from bacteria to insects and higher eukaryotes. In particular, we highlight drawbacks and opportunities of RNAz-based ncRNA prediction at several hitherto scarcely investigated scenarios, as for example ncRNA prediction in the light of whole genome duplications. A recent microarray study provides experimental evidence for our approach. Differential expression of at least one-sixth of our drosophilid RNAz predictions has been reported. Beyond the means of RNAz, we moreover manually compile sophisticated annotation of short ncRNAs in schistosomes. Obviously, accumulating knowledge about the genetic material of malaria causing parasites which infect millions of humans world-wide is of utmost scientific interest. Since the performance of any comparative genomics approach is limited by the quality of its input alignments, we introduce a novel light-weight and performant genome-wide alignment approach: NcDNAlign. Although the tool is optimized for speed rather than sensitivity and requires only a minor fraction of CPU time compared to existing programs, we demonstrate that it is basically as sensitive and specific as competing approaches when applied to genome-wide ncRNA gene finding and analysis of ultra-conserved regions. By design, however, prediction approaches that search for regions with an excess of mutations that maintain secondary structure motifs will miss ncRNAs that are unstructured or whose structure is not well conserved in evolution. In the second part of the thesis, we therefore overcome secondary structure prediction and, based on splice site detection, develop novel strategies specifically designed to identify long ncRNAs in genomic sequences - probably the open problem in current RNA research. We perform splice site anchored gene-finding in drosophilids, nematodes, and vertebrate genomes and, at least for a subset of obtained candidate genes, provide experimental evidence for expression and the existence of novel spliced transcripts undoubtedly confirming our approach. In summary, we found evidence for a large number of previously undescribed RNAs which consolidates the idea of non-coding RNAs as an abundant class of regulatory active transcripts. Certainly, ncRNA prediction is a complex task. This thesis, however, rationally advises how to unveil the RNA complement of newly sequenced genomes. Since our results have already established both subsequent computational as well as experimental studies, we believe to have enduringly stimulated the field of RNA research and to have contributed to an enriched view on the subject.