L’inférence de génomes ancestraux est une étape essentielle pour l’étude de l’évolution des génomes. Connaissant les génomes d’espèces éteintes, on peut proposer des mécanismes biologiques expliquant les divergences entre les génomes des espèces modernes. Diverses méthodes visant à résoudre ce problème existent, se classant parmis deux grandes catégories : les méthodes de distance et les méthodes de synténie. L’état de l’art des distances génomiques ne permettant qu’un certain répertoire de réarrangements pour le moment, les méthodes de synténie sont donc plus appropriées en pratique. Nous proposons une méthode de synténie pour la reconstruction de génomes ancestraux basée sur une définition relaxée d’adjacences de gènes, permettant un contenu en gène inégal dans les génomes modernes causé par des pertes de gènes de même que des duplications de génomes entiers (DGE). Des simulations sont effectuées, démontrant une capacité de former une solution assemblée en un nombre réduit de régions ancestrales contigües par rapport à d’autres méthodes tout en gardant une bonne fiabilité. Des applications sur des données de levures et de plantes céréalières montrent des résultats en accord avec d’autres publications, notamment la présence de fusion imbriquée de chromosomes pendant l’évolution des céréales. / Ancestral genome inference is a decisive step for studying genome evolution. Knowing genomes from extinct species, one can propose biological mecanisms explaining divergences between extant species genomes. Various methods classified in two categories have been developped : distance based methods and synteny based methods. The state of the art of distance based methods only permit a certain repertoire of genomic rearrangements, thus synteny based methods are more appropriate in practice for the time being. We propose a synteny method for ancestral genome reconstruction based on a relaxed defenition of gene adjacencies, permitting unequal gene content in extant genomes caused by gene losses and whole genome duplications (WGD). Simulations results demonstrate our method’s ability to form a more assembled solution rather than a collection of contiguous ancestral regions (CAR) with respect to other methods, while maintaining a good reliability. Applications on data sets from yeasts and cereal species show results agreeing with other publications, notably the existence of nested chromosome fusion during the evolution of cereals.

Vias redox são importantes reguladores da homeostase e sinalização celular, mas o entendimento dos mecanismos desses processos é incompleto. Tiol-proteínas como a dissulfeto isomerase proteica (PDI) podem ser moduladores dessas vias. A PDI(PDIA1) é o protótipo da família das PDIs, cuja função canônica é o enovelamento redox de proteínas no retículo endoplasmático. Além disso, PDI exerce regulação de NADPH oxidases, as principais fontes de oxidantes celulares, e é necessária para ativação de RhoGTPases, organização do citoesqueleto e migração de células vasculares. No estudo de mecanismos pelos quais a PDI regula RhoGTPases, mostramos, em redes computacionais e em experimentos de co-imunoprecitação, associação entre PDIA1 e o regulador de RhoGTPases RhoGDIalfa. Além disso, identificamos forte proximidade entre os genes codificando estas proteínas. Neste estudo, caracterizamos o perfil e implicações desta sintenia gênica.A análise bioinformática pelos programs Ensembl, NCBI e UCSC evidencia um padrão de sintenia entre diferentes isoformas destas duas famílias: PDIA1 (P4HB), PDIA2 (PDIP) e PDIA8 (Erp27) são vizinhos, respectivamente, a RhoGDIbeta, RhoGDIy e RHOGDIalfa, com correspondentes regiões intergênicas de 7.1, 2.9 e 0.14 kb em distintos cromossomos em H. sapiens. O padrão dessa sintenia foi fortemente conservado emC. elegans, alguns peixes e uniformemente em anfíbios, répteis, aves e mamíferos. Leveduras expressam no mesmo cromossomo , porém em locais distantes (i.emacrossintenia) ortólogos da PDIA1 e RhoGDI?, mas não expressam outras PDIs e RhoGDIssintênicasnos eucariotos complexos. No entanto, sintenia entre PDI e RhoGDI foi também observada na planta A. thaliana, sem evidência de um ancestral comum. Os pares sintênicos associam-se a blocos vizinhos conservados, porém diversos para cada par, enquanto cada bloco contem um gene codificando um distinto regulador da PP1 (fosfatase proteica-1). Análise filogenética mostrou topologia semelhante entre as duas famílias.Análise dos dados do estudo ENCODE e predição pelo Softberry identificou sítios de ligação a fatores de transcrição comuns entre os distintos pares, cuja ontologia indicou principalmente desenvolvimento, processos metabólicos e resposta imune. O estudo de possíveis implicações funcionais dessa sintenia mostrou que manipulações da expressão proteica de PDIA1 não promovem mudança consistente na expressão proteica de RhoGDIalfa, tanto in vitro (silenciamento da PDI por siRNA e superexpressão por vetor lentiviral induzível) como in vivo (camundongo transgênico com superexpressão constitutiva da PDIA1). No entanto, as mudanças da expressãogênica de ambos os genes na camada íntima de artérias carótidas de camundongo durante remodelamento induzido por fluxo foram fortemente correlacionadas. Experimentos de coimunoprecipitação e co-localização à microscopia confocal sugeriram interação física entre PDIA1 e RhoGDIAalfa. Deste modo, estes dados mostram um intrigante padrão de conservação evolutiva da proximidade gênica entre PDIs e RhoGDIs, não usual em eucariotos. Genes sintênicos frequentemente codificam proteínas que tendem a interagir física e/ou funcionalmente. Com efeito, nosso dados sugerem co-regulação e interação física entre PDIA1 e RhoGDIAalfa, corroborando a convergência entre essas proteínas como possível mecanismo envolvido na regulação redox do citoesqueleto pela PDIA1 / Redox pathways are important regulators of homeostasis and cell signaling, but the understanding of the mechanisms of these processes is incomplete. Thiol proteins such as protein disulfide isomerase (PDI) can be modulators of these pathways. PDI (PDIA1) is the prototype of the family of PDIs whose canonical function is a redox protein folding in the endoplasmic reticulum. In addition, PDI exerts regulatory NADPH oxidase, the main sources of cellular oxidant, and is required for activation RhoGTPases, cytoskeletal organization and migration of vascular cells. In the study of mechanisms by which regulates PDI RhoGTPases, we showed in computer networks and co-imunoprecitation experiments association between PDIA1 and the regulator of RhoGTPases, RhoGDI?. In addition, we identified strong proximity of the genes encoding these proteins. In this study, we characterize the profile and implications of this synteny. .A bioinformatic analysis by programs Ensembl, NCBI and UCSC shows a pattern of synteny between different isoforms of these two families: PDIA1 (P4HB), PDIA2 (PDIP) and PDIA8 (Erp27) are neighbors , respectively RhoGDIalfa, and RhoGDIy RHOGDIbeta with corresponding intergenic regions 7.1, 2.9 and 0:14 kb in different chromosomes of H. sapiens. The pattern of this synteny was strongly maintained in C. elegans, some fish and evenly amphibians, reptiles, birds and mammals. Yeasts express on the same chromosome, but in distant places (i.e macrosintenia) orthologs of PDIA1 and RhoGDI?, but do not express other syntenics PDIs and RhoGDIs in complex eukaryotes. However, synteny between PDI and RhoGDI was also observed in the plant A. thaliana, no evidence of a common ancestor. The syntenic pairs are associated with the stored neighboring blocks, but different for each pair, while each block contains a gene encoding a regulator of distinct PP1 (protein phosphatase-1). Phylogenetic analysis showed similar topology between the two famílias. The identified binding sites common transcription factors between different pairs, which mainly indicated ontology development, metabolic and immune response. The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDI, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDIalfa, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). However, changes of gene expression of both genes in the intima of mouse carotid arteries during remodeling induced by flow were strongly correlated. Immunoprecipitation experiments and co-location to confocal microscopy suggested physical interaction between PDIA1 and RhoGDIAalfa. Thus, these data show an intriguing pattern of evolutionary conservation of gene proximity between POIs and RhoGDIs not common in eukaryotes. sintênicos genes often encode proteins that tend to interact physically and / or functionally. Indeed, our data suggest co-regulation and physical interaction between PDIA1 and RhoGDIAalfa, supporting the convergence of these proteins as a possible mechanism involved in redox regulation of cytoskeleton by PDIA1

HMMs de perfil são um método poderoso para modelar a diversidade de sequências biológicas e constituem uma abordagem muito sensível para a detecção de ortólogos remotos. Uma potencial aplicação de tais modelos é a detecção de vírus emergentes e novos elementos genéticos móveis. Nosso grupo desenvolveu recentemente o GenSeed-HMM, um programa que emprega HMMs de perfil como sementes para montagem progressiva de genes-alvo, utilizando tanto dados genômicos como metagenômicos. No presente trabalho foi desenvolvido o TABAJARA, um programa para o desenho racional de HMMs de perfil. Partindo de um alinhamento de múltiplas sequências, o TABAJARA é capaz de encontrar blocos que são (1) conservados ou (2) discriminativos para dois ou mais grupos de sequências. O programa utiliza diferentes métricas para atribuir pontuações posição-específicas ao longo de todo o alinhamento e utiliza então uma janela deslizante para encontrar as regiões com maiores pontuações. Blocos de alinhamento selecionados são então extraídos e utilizados para construir HMMs de perfil. Para validar o método, o programa TABAJARA foi empregado para a construção de modelos para vírus do gênero Flavivirus e para fagos da família Microviridae. Em ambos os grupos virais foi possível se obter modelos de ampla abrangência, capazes de detectar todos os membros de um respectivo grupo taxonômico, e modelos de abrangência mais restrita, específicos para espécies distintas de Flavivirus (ex. DENV, ZIKV ou YFV) ou subfamílias de Microviridae (ex. Alpavirinae, Gokushovirinae e Pichovirinae). Em outra validação, foram utilizadas sequências da endonuclease Cas1 para se obter modelos capazes de diferenciar CRISPRs de casposons, esses últimos representando uma superfamília de transposons de DNA autossintetizantes, os quais originaram o sistema de imunidade CRISPR-Cas de procariotos. O TABAJARA conseguiu gerar modelos específicos de Cas1 derivada de casposons, permitindo sua diferenciação em relação aos seus ortólogos de CRISPRs. No presente trabalho foi desenvolvido ainda o HMM-Prospector, uma ferramenta que utiliza um conjunto de HMMs de perfil para a triagem de dados de sequenciamento genômico ou metagenômico. O programa informa quais são os modelos mais reconhecidos pelas leituras, sob valores de corte de pontuação definidos pelo usuário, assim como quantas leituras são detectadas por cada modelo. Com esta informação, os modelos mais relevantes podem ser utilizados como sementes em montagens progressivas com o programa GenSeed-HMM, dentro de uma abordagem integrada para a construção de modelos e sua aplicação. Finamente, foi desenvolvido o e-Finder, um aplicativo genérico para a detecção e extração de elementos multigênicos a partir de genomas ou metagenomas montados utilizando HMMs de perfil. O e-Finder executa buscas de similaridade entre os HMMs de perfil e as sequências traduzidas dos dados montados e checa, em seguida, se os critérios de sintenia pré-definidos foram atendidos, incluindo o número mínimo de genes, a ordem dos genes e as distâncias intergênicas. As sequências dos elementos são então extraídas, as regiões codificantes (ORFs) identificadas e traduzidas conceitualmente em sequências completas de proteínas. Para validar esta ferramenta, foram empegados dois estudos de caso, profagos da família Microviridae e casposons, utilizando-se HMMs de perfil específicos, construídos com o programa TABAJARA. Em ambos os casos, o e-Finder foi executado usando-se a base de dados PATRIC, um repositório com mais de 135.000 genomas de bactérias e arqueias. Foram identificados um total de 91 contigs positivos para casposons a partir de 79 genomas distintos. No caso dos Microviridae, foram encontrados 104 profagos candidatos, estendendo o conhecimento da gama de hospedeiros bacterianos. Em ambos os casos, análises filogenéticas confirmaram a correta atribuição taxonômica das sequências positivas. Os programas desenvolvidos neste trabalho podem ser utilizados isoladamente ou em combinação para detectar e discriminar sequências conhecidas ou remotamente relacionadas. Juntamente com o GenSeed-HMM, estes programas constituem um conjunto integrado de ferramentas com potencial aplicação na busca de novos vírus e elementos genéticos móveis, bem como em qualquer outra tarefa relacionada à detecção e/ou discriminação de subgrupos de famílias de sequências nucleotídicas ou proteicas / Profile HMMs are a powerful way of modeling sequence diversity and constitute a very sensitive approach to detect remote orthologs. A potential application of such models is the detection of emerging viruses and novel mobile genetic elements. Our group has recently developed GenSeed-HMM, a tool that employs profile HMMs as seeds for gene-targeted progressive assembly using either genomic or metagenomic data. In this work we developed TABAJARA, a program for the rational design of profile HMMs. Starting from a multiple sequence alignment, TABAJARA is able to find blocks that are either (1) conserved across all sequences or (2) discriminative for two or more specific groups of sequences. The program uses different metrics to ascribe position-specific scores along the whole alignment and then uses a sliding-window to find top-scoring regions. Selected alignment blocks are then extracted and used to build profile HMMs. To validate the method, we employed TABAJARA to construct models for viruses of the Flavivirus genus and phages of the Microviridae family. In both viral groups we were able to obtain wide-range models, able to detect all members of the respective taxonomic group, and models that are specific to particular Flavivirus species (e.g. DENV, ZIKV or YFV) or Microviridae subfamilies (e.g. Alpavirinae, Gokushovirinae and Pichovirinae). In another validation, we used sequences of the endonuclease Cas1 to obtain models capable of differentiating CRISPRs from casposons, the latter elements representing a superfamily of self-synthesizing DNA transposons that originated the prokaryotic CRISPR-Cas immunity. TABAJARA succeeded to generate models specific to casposon-derived Cas1, enabling their differentiation from CRISPR orthologs. We also developed HMM-Prospector, a tool that can use a batch of profile HMMs to screen genomic or metagenomic sequencing data, reporting which profile HMMs are mostly recognized under user-defined score cutoff values, and how many reads are detected by each model. With this information, the most relevant models can be used as seeds in progressive assemblies with GenSeed-HMM program, providing an integrated approach for model construction and application. Finally, we developed e-Finder, a generic application for detecting and extracting multigene elements from assembled genomes or metagenomes using profile HMMs. e-Finder runs similarity searches of profile HMMs against translated sequences of the assembled data and then checks if pre-defined syntenic criteria have been fulfilled, including minimum number of genes, gene order and intergenic distances. Element sequences are then extracted, their ORFs identified and conceptually translated into full-length protein sequences. To validate the tool, we employed two distinct case studies, prophages of the Microviridae family and casposons, using specific profile HMMs constructed by TABAJARA. In both cases, we executed e-Finder using the PATRIC database, a repository with over 135,000 bacterial and archaeal genomes. We identified in total 91 casposon-positive contigs from 79 distinct genomes. In the case of Microviridae, we found a total of 104 provirus candidates, extending the known range of bacterial hosts. In both cases, phylogenetic analyses confirmed the correct taxonomic assignment of the positive sequences. The programs developed in this work can be used alone or in combination to detect and discriminate known or distantly related sequences. Together with GenSeed-HMM, these programs provide an integrated toolbox with potential application in the search of novel viruses and mobile genetic elements, as well as in any other task related to the detection and/or discrimination of subgroups of DNA or protein sequences.

L’inférence de génomes ancestraux est une étape essentielle pour l’étude de l’évolution des génomes. Connaissant les génomes d’espèces éteintes, on peut proposer des mécanismes biologiques expliquant les divergences entre les génomes des espèces modernes. Diverses méthodes visant à résoudre ce problème existent, se classant parmis deux grandes catégories : les méthodes de distance et les méthodes de synténie. L’état de l’art des distances génomiques ne permettant qu’un certain répertoire de réarrangements pour le moment, les méthodes de synténie sont donc plus appropriées en pratique. Nous proposons une méthode de synténie pour la reconstruction de génomes ancestraux basée sur une définition relaxée d’adjacences de gènes, permettant un contenu en gène inégal dans les génomes modernes causé par des pertes de gènes de même que des duplications de génomes entiers (DGE). Des simulations sont effectuées, démontrant une capacité de former une solution assemblée en un nombre réduit de régions ancestrales contigües par rapport à d’autres méthodes tout en gardant une bonne fiabilité. Des applications sur des données de levures et de plantes céréalières montrent des résultats en accord avec d’autres publications, notamment la présence de fusion imbriquée de chromosomes pendant l’évolution des céréales. / Ancestral genome inference is a decisive step for studying genome evolution. Knowing genomes from extinct species, one can propose biological mecanisms explaining divergences between extant species genomes. Various methods classified in two categories have been developped : distance based methods and synteny based methods. The state of the art of distance based methods only permit a certain repertoire of genomic rearrangements, thus synteny based methods are more appropriate in practice for the time being. We propose a synteny method for ancestral genome reconstruction based on a relaxed defenition of gene adjacencies, permitting unequal gene content in extant genomes caused by gene losses and whole genome duplications (WGD). Simulations results demonstrate our method’s ability to form a more assembled solution rather than a collection of contiguous ancestral regions (CAR) with respect to other methods, while maintaining a good reliability. Applications on data sets from yeasts and cereal species show results agreeing with other publications, notably the existence of nested chromosome fusion during the evolution of cereals.

Vias redox são importantes reguladores da homeostase e sinalização celular, mas o entendimento dos mecanismos desses processos é incompleto. Tiol-proteínas como a dissulfeto isomerase proteica (PDI) podem ser moduladores dessas vias. A PDI(PDIA1) é o protótipo da família das PDIs, cuja função canônica é o enovelamento redox de proteínas no retículo endoplasmático. Além disso, PDI exerce regulação de NADPH oxidases, as principais fontes de oxidantes celulares, e é necessária para ativação de RhoGTPases, organização do citoesqueleto e migração de células vasculares. No estudo de mecanismos pelos quais a PDI regula RhoGTPases, mostramos, em redes computacionais e em experimentos de co-imunoprecitação, associação entre PDIA1 e o regulador de RhoGTPases RhoGDIalfa. Além disso, identificamos forte proximidade entre os genes codificando estas proteínas. Neste estudo, caracterizamos o perfil e implicações desta sintenia gênica.A análise bioinformática pelos programs Ensembl, NCBI e UCSC evidencia um padrão de sintenia entre diferentes isoformas destas duas famílias: PDIA1 (P4HB), PDIA2 (PDIP) e PDIA8 (Erp27) são vizinhos, respectivamente, a RhoGDIbeta, RhoGDIy e RHOGDIalfa, com correspondentes regiões intergênicas de 7.1, 2.9 e 0.14 kb em distintos cromossomos em H. sapiens. O padrão dessa sintenia foi fortemente conservado emC. elegans, alguns peixes e uniformemente em anfíbios, répteis, aves e mamíferos. Leveduras expressam no mesmo cromossomo , porém em locais distantes (i.emacrossintenia) ortólogos da PDIA1 e RhoGDI?, mas não expressam outras PDIs e RhoGDIssintênicasnos eucariotos complexos. No entanto, sintenia entre PDI e RhoGDI foi também observada na planta A. thaliana, sem evidência de um ancestral comum. Os pares sintênicos associam-se a blocos vizinhos conservados, porém diversos para cada par, enquanto cada bloco contem um gene codificando um distinto regulador da PP1 (fosfatase proteica-1). Análise filogenética mostrou topologia semelhante entre as duas famílias.Análise dos dados do estudo ENCODE e predição pelo Softberry identificou sítios de ligação a fatores de transcrição comuns entre os distintos pares, cuja ontologia indicou principalmente desenvolvimento, processos metabólicos e resposta imune. O estudo de possíveis implicações funcionais dessa sintenia mostrou que manipulações da expressão proteica de PDIA1 não promovem mudança consistente na expressão proteica de RhoGDIalfa, tanto in vitro (silenciamento da PDI por siRNA e superexpressão por vetor lentiviral induzível) como in vivo (camundongo transgênico com superexpressão constitutiva da PDIA1). No entanto, as mudanças da expressãogênica de ambos os genes na camada íntima de artérias carótidas de camundongo durante remodelamento induzido por fluxo foram fortemente correlacionadas. Experimentos de coimunoprecipitação e co-localização à microscopia confocal sugeriram interação física entre PDIA1 e RhoGDIAalfa. Deste modo, estes dados mostram um intrigante padrão de conservação evolutiva da proximidade gênica entre PDIs e RhoGDIs, não usual em eucariotos. Genes sintênicos frequentemente codificam proteínas que tendem a interagir física e/ou funcionalmente. Com efeito, nosso dados sugerem co-regulação e interação física entre PDIA1 e RhoGDIAalfa, corroborando a convergência entre essas proteínas como possível mecanismo envolvido na regulação redox do citoesqueleto pela PDIA1 / Redox pathways are important regulators of homeostasis and cell signaling, but the understanding of the mechanisms of these processes is incomplete. Thiol proteins such as protein disulfide isomerase (PDI) can be modulators of these pathways. PDI (PDIA1) is the prototype of the family of PDIs whose canonical function is a redox protein folding in the endoplasmic reticulum. In addition, PDI exerts regulatory NADPH oxidase, the main sources of cellular oxidant, and is required for activation RhoGTPases, cytoskeletal organization and migration of vascular cells. In the study of mechanisms by which regulates PDI RhoGTPases, we showed in computer networks and co-imunoprecitation experiments association between PDIA1 and the regulator of RhoGTPases, RhoGDI?. In addition, we identified strong proximity of the genes encoding these proteins. In this study, we characterize the profile and implications of this synteny. .A bioinformatic analysis by programs Ensembl, NCBI and UCSC shows a pattern of synteny between different isoforms of these two families: PDIA1 (P4HB), PDIA2 (PDIP) and PDIA8 (Erp27) are neighbors , respectively RhoGDIalfa, and RhoGDIy RHOGDIbeta with corresponding intergenic regions 7.1, 2.9 and 0:14 kb in different chromosomes of H. sapiens. The pattern of this synteny was strongly maintained in C. elegans, some fish and evenly amphibians, reptiles, birds and mammals. Yeasts express on the same chromosome, but in distant places (i.e macrosintenia) orthologs of PDIA1 and RhoGDI?, but do not express other syntenics PDIs and RhoGDIs in complex eukaryotes. However, synteny between PDI and RhoGDI was also observed in the plant A. thaliana, no evidence of a common ancestor. The syntenic pairs are associated with the stored neighboring blocks, but different for each pair, while each block contains a gene encoding a regulator of distinct PP1 (protein phosphatase-1). Phylogenetic analysis showed similar topology between the two famílias. The identified binding sites common transcription factors between different pairs, which mainly indicated ontology development, metabolic and immune response. The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDI, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDIalfa, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). However, changes of gene expression of both genes in the intima of mouse carotid arteries during remodeling induced by flow were strongly correlated. Immunoprecipitation experiments and co-location to confocal microscopy suggested physical interaction between PDIA1 and RhoGDIAalfa. Thus, these data show an intriguing pattern of evolutionary conservation of gene proximity between POIs and RhoGDIs not common in eukaryotes. sintênicos genes often encode proteins that tend to interact physically and / or functionally. Indeed, our data suggest co-regulation and physical interaction between PDIA1 and RhoGDIAalfa, supporting the convergence of these proteins as a possible mechanism involved in redox regulation of cytoskeleton by PDIA1

Background: Transfer RNAs (tRNAs) are ubiquitous in all living organism. They implement the genetic code so that most genomes contain distinct tRNAs for almost all 61 codons. They behave similar to mobile elements and proliferate in genomes spawning both local and non-local copies. Most tRNA families are therefore typically present as multicopy genes. The members of the individual tRNA families evolve under concerted or rapid birth-death evolution, so that paralogous copies maintain almost identical sequences over long evolutionary time-scales. To a good approximation these are functionally equivalent. Individual tRNA copies thus are evolutionary unstable and easily turn into pseudogenes and disappear. This leads to a rapid turnover of tRNAs and often large differences in the tRNA complements of closely related species. Since tRNA paralogs are not distinguished by sequence, common methods cannot not be used to establish orthology between tRNA genes. Results: In this contribution we introduce a general framework to distinguish orthologs and paralogs in gene families that are subject to concerted evolution. It is based on the use of uniquely aligned adjacent sequence elements as anchors to establish syntenic conservation of sequence intervals. In practice, anchors and intervals can be extracted from genome-wide multiple sequence alignments. Syntenic clusters of concertedly evolving genes of different families can then be subdivided by list alignments, leading to usually small clusters of candidate co-orthologs. On the basis of recent advances in phylogenetic combinatorics, these candidate clusters can be further processed by cograph editing to recover their duplication histories. We developed a workflow that can be conceptualized as stepwise refinement of a graph of homologous genes. We apply this analysis strategy with different types of synteny anchors to investigate the evolution of tRNAs in primates and fruit flies. We identified a large number of tRNA remolding events concentrated at the tips of the phylogeny. With one notable exception all phylogenetically old tRNA remoldings do not change the isoacceptor class. Conclusions: Gene families evolving under concerted evolution are not amenable to classical phylogenetic analyses since paralogs maintain identical, species-specific sequences, precluding the estimation of correct gene trees from sequence differences. This leaves conservation of syntenic arrangements with respect to "anchor elements" that are not subject to concerted evolution as the only viable source of phylogenetic information. We have demonstrated here that a purely synteny-based analysis of tRNA gene histories is indeed feasible. Although the choice of synteny anchors influences the resolution in particular when tight gene clusters are present, and the quality of sequence alignments, genome assemblies, and genome rearrangements limits the scope of the analysis, largely coherent results can be obtained for tRNAs. In particular, we conclude that a large fraction of the tRNAs are recent copies. This proliferation is compensated by rapid pseudogenization as exemplified by many very recent alloacceptor remoldings.

Leaf rust caused by Puccinia hordei is an important disease of barley (Hordeum vulgare) in many regions of the world. Yield losses up to 62% have been reported in susceptible cultivars. The Rph5 gene confers resistance to the most prevalent races (8 and 30) of barley leaf rust in the United States. Therefore, the molecular mapping of Rph5 is of great interest. Genetic studies were performed by analysis of 93 and 91 F2 plants derived from the crosses 'Bowman' (rph5) x 'Magnif 102' (Rph5) and 'Moore' (rph5) x Virginia 92-42-46 (Rph5), respectively. Linkage analysis positioned the Rph5 locus to the extreme telomeric region of the short arm of barley chromosome 3H at 0.2 cM proximal to RFLP marker VT1 and 0.5 cM distal from RFLP marker C970 in the Bowman x Magnif 102 population. Synteny between rice chromosome 1 and barley chromosome 3 was employed to saturate the region within the sub-centimorgan region around Rph5 using sequence-tagged site (STS) markers that were developed based on barley expressed sequence tags (ESTs) syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising distal region of the rice chromosome 1S. Five rice PAC clones were used as queries to blastn 370,258 barley ESTs. Ninety four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 10 EST-based STS markers were incorporated into the 'Bowman' x 'Magnif 102' high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, co-segregate with Rph5. Genes, represented by these markers, are putative candidates for Rph5. Results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley EST resources for marker saturation of targeted barley genomic region. / Ph. D.

Le génome bactérien est classiquement pensé comme constitué de “chromosomes”, éléments génomiques essentiels pour l’organisme, stables et à évolution lente, et de “plasmides”, éléments génomiques accessoires, mobiles et à évolution rapide. La distinction entre plasmides et chromosomes a récemment été mise en défaut avec la découverte dans certaines lignées bactériennes d’éléments génomiques intermédiaires, possédant à la fois des caractéristiques de chromosomes et de plasmides. Désignés par le terme de “chromosomes secondaires”, “mégaplasmides” ou “chromid”, ces éléments sont dispersés parmi les lignées bactériennes et sont couramment décrits comme des plasmides adaptés et modifiés. Cependant, leur véritable nature et les mécanismes permettant leur intégration dans le génome stable reste à caractériser. En utilisant les protéines liées aux Systèmes de Transmission de l’Information Génétique (STIG) comme variables descriptives des éléments génomiques bactériens (ou réplicons), une étude globale de génomique comparative a été conduite sur l’ensemble des génomes bactériens disponibles. A travers l’analyse de l’information contenue dans ce jeu de données par différentes approches analytiques, il apparait que les STIG constituent des marqueurs pertinents de l’état d’intégration des réplicons dans le génome stable, ainsi que de leur origine évolutive, et que les Réplicons Extra-Chromosomiques Essentiels (RECE) témoignent de la diversité des mécanismes génétiques et des processus évolutifs permettant l’intégration de réplicons dans le génome stable, attestant ainsi de la continuité du matériel génomique. / The genome of bacteria is classically separated into essential, stable and slow evolving replicons (chromosomes) and accessory, mobile and rapidly evolving replicons (plasmids). This paradigm is being questioned since the discovery of extra-chromosomal essential replicons (ECERs), be they called ”megaplasmids”, ”secondary chromosomes” or ”chromids”, which possess both chromosomal and plasmidic features. These ECERs are found in diverse lineages across the bacterial phylogeny and are generally believed to be modified plasmids. However, their true nature and the mechanisms permitting their integration within the sable genome are yet to be formally determined. The relationships between replicons, with reference to their genetic information inheritance systems (GIIS), were explored under the assumption that the inheritance of ECERs is integrated to the cell cycle and highly constrained in contrast to that of standard plasmids. A global comparative genomics analysis including all available of complete bacterial genome sequences, was performed using GIIS functional homologues as parameters and applying several analytical procedures. GIIS proved appropriate in characterizing the level of integration within the stable genome, as well as the origins, of the replicons. The study of ECERs thus provides clues to the genetic mechanisms and evolutionary processes involved in the replicon stabilization into the essential genome and the continuity of the genomic material.

No description available.