Genome Biology and Evolution of the Animal Kingdom

Estudi realitzat a partir d’una estada a la Institut J.W. Jenkinson Laboratory for Evolution and Development of the University of Oxford, Regne Unit, entre 2010 i 2012. He estat membre del laboratori del Professor Peter W.H. Holland com a becari post-doctoral Beatriu de Pinós des de setembre de 2010 al setembre de 2012. El nostre projecte de recerca se centra en l'anàlisi genòmic comparatiu del Regne Animal, tot explorant el contingut dels genomes a través de totes les branques de l'arbre dels animals. Totes les referències a les meves publicacions durant aquest post-doc es poden trobar a http://about.me/jordi_paps. Crec que el nombre i la qualitat dels resultats del meu post-doc, un total de 8 publicacions incloent dos articles a la prestigiosa revista Nature, són prova de l'èxit d'aquest post-doc. Prof Peter W. H. Holland (Departament de Zoologia de la Universitat d'Oxford) i jo som coautors de tres articles de genòmica comparativa, resultats directes d'aquest projecte: 1) comparació de families gèniques entre vertebrats invertebrats (Briefings in Functional Genomics), 2) el genoma de l'ostra (publicat a la revista Nature), i 3) els genomes de 6 platihelmints paràsits (acceptat també a Nature). A més, tenim altres 2 treballs en preparació. Un d'ells analitza l'evolució, expressió i funció dels gens Hox al a la tènia Hymenolepis. El perfil fi d'aquests gens clau del desenvolupament esclareix els canvis d'estil de vida dels organismes. A més, durant aquest últim post-doc he participat en diverses col•laboracions, incloent anàlisi de gens d'envelliment a cucs plans, un estudi sobre la filogènia del grup Gastrotricha, una revisió de l'evolució phylum Platyhelminthes, així com un capítol d'un llibre sobre l'evolució dels animals bilaterals. Finalment, gràcies a la beca Beatriu de Pinós, el Prof. Peter W.H. Holland m'ha convidat a formar part del seu equip com un investigador post-doctoral en el seu projecte ERC Advance actual sobre duplicacions genòmiques.

SGP-1: prediction and validation of homologous genes based on sequence alignments

Conventional methods of gene prediction rely on the recognition of DNA-sequence signals, the coding potential or the comparison of a genomic sequence with a cDNA, EST, or protein database. Reasons for limited accuracy in many circumstances are species-specific training and the incompleteness of reference databases. Lately, comparative genome analysis has attracted increasing attention. Several analysis tools that are based on human/mouse comparisons are already available. Here, we present a program for the prediction of protein-coding genes, termed SGP-1 (Syntenic Gene Prediction), which is based on the similarity of homologous genomic sequences. In contrast to most existing tools, the accuracy of SGP-1 depends little on species-specific properties such as codon usage or the nucleotide distribution. SGP-1 may therefore be applied to nonstandard model organisms in vertebrates as well as in plants, without the need for extensive parameter training. In addition to predicting genes in large-scale genomic sequences, the program may be useful to validate gene structure annotations from databases. To this end, SGP-1 output also contains comparisons between predicted and annotated gene structures in HTML format. The program can be accessed via a Web server at http://soft.ice.mpg.de/sgp-1. The source code, written in ANSI C, is available on request from the authors.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Kabuverdianu or Cape Verdean and Kryol, Or Guinea-Bissao (Creole Portugues)

This chpter is dedicated the comparative Syntatic analysis of two portuguese-based creoles.

Death and resurrection of the human IRGM gene

Immunity-related GTPases (IRG) play an important role in defense against intracellular pathogens. One member of this gene family in humans, IRGM, has been recently implicated as a risk factor for Crohn's disease. We analyzed the detailed structure of this gene family among primates and showed that most of the IRG gene cluster was deleted early in primate evolution, after the divergence of the anthropoids from prosimians ( about 50 million years ago). Comparative sequence analysis of New World and Old World monkey species shows that the single-copy IRGM gene became pseudogenized as a result of an Alu retrotransposition event in the anthropoid common ancestor that disrupted the open reading frame (ORF). We find that the ORF was reestablished as a part of a polymorphic stop codon in the common ancestor of humans and great apes. Expression analysis suggests that this change occurred in conjunction with the insertion of an endogenous retrovirus, which altered the transcription initiation, splicing, and expression profile of IRGM. These data argue that the gene became pseudogenized and was then resurrected through a series of complex structural events and suggest remarkable functional plasticity where alleles experience diverse evolutionary pressures over time. Such dynamism in structure and evolution may be critical for a gene family locked in an arms race with an ever-changing repertoire of intracellular parasites.

Molecular evolution of the Drosophila olfactory system

The olfactory system is an attractive model to study the genetic mechanisms underlying evolution of the nervous system. This sensory system mediates the detection and behavioural responses to an enormous diversity of volatile chemicals in the environment and displays rapid evolution, as species acquire, modify and discard olfactory receptors and circuits to adapt to new olfactory stimuli. Drosophilids provide an attractive model to study these processes. The availability of 12 sequenced genomes of Drosophila species occupying diverse ecological niches provides a rich resource for genomic analyses. Moreover, one of these species, Drosophila melanogaster, is amenable to a powerful combination of genetic and electrophysiological analyses. D. melanogaster has two distinct families of olfactory receptors to detect odours, the well-characterised Odorant Receptors (ORs) and the recently identified lonotropic Receptors (IRs). In my thesis, I have provided new insights into the genetic mechanisms underlying olfactory system evolution through three distinct, but interrelated projects. First, I performed a comparative genomic analysis of the IR repertoire in 12 sequenced Drosophila species, which has revealed that the olfactory IRs are highly conserved across species. By contrast, a large fraction of IRs that are not expressed in the olfactory system - and which may be gustatory receptors - are much more variable in sequence and gene copy number. Second, to identify ligands for IR expressing olfactory sensory neurons, I have performed an electrophysiological screen in D. melanogaster using a panel of over 160 odours. I found that the IRs respond to a number of amines, aldehydes and acids, contrasting with the chemical specificity of the OR repertoire, which is mainly tuned to esters, alcohols and ketones. Finally, the identification of ligands for IRs in this species allowed me to investigate in detail the molecular and functional evolution of a tandem array of IRs, IR75a/IR75b/IR75c, in D. sechellia. This species is endemic to the Seychelles archipelago and highly specialised to breed on the fruits of Morinda citrifolia, which is repulsive and toxic for other Drosophila species. These studies led me to discover that receptor loss, changes in receptor specificity and changes in receptor expression have likely played an important role during the evolution of these IRs in D. sechellia. These changes may explain, in part, the unique chemical ecology of this species. - Le système olfactif est un excellent modèle pour étudier les mécanismes génétiques impliqués dans l'étude de l'évolution du système nerveux. Ce système sensoriel permet la détection de nombreux composés volatils présents dans l'environnement et est à la base des réponses comportementales. Il est propre à chaque espèce et évolue rapidement en modifiant ou en éliminant des récepteurs et leurs circuits olfactifs correspondants pour s'adapter à de nouvelles odeurs. Pour étudier le système olfactif et son évolution, nous avons décidé d'utiliser la drosophile comme modèle. Le séquençage complet de 12 souches de drosophiles habitant différentes niches écologiques permet une analyse génomique conséquente. De plus, l'une de ces espèces Drosophila melanogaster permet la combinaison d'analyses génétiques et électrophysiologiques. En effet, D. melanogaster possède 2 familles distinctes de récepteurs olfactifs qui permettent la détection d'odeurs: les récepteurs olfactifs (ORs) étant les mieux caractérisés et les récepteurs ionotropiques (IRs), plus récemment identifiés. Au cours de ma thèse, j'ai apporté des nouvelles connaissances qui m'ont permis de mieux comprendre les mécanismes génétiques à la base de l'évolution du système olfactif au travers de trois projets différents, mais interdépendants. Premièrement, j'ai réalisé une analyse génomique comparative de l'ensemble des IRs dans les 12 souches de drosophiles séquencées jusqu'à présent. Ceci a montré que les récepteurs olfactifs IRs sont hautement conservés parmi l'ensemble de ces espèces. Au contraire, une grande partie des IRs qui ne sont pas exprimés dans le système olfactif, et qui semblent être des récepteurs gustatifs, sont beaucoup plus variables dans leur séquence et dans le nombre de copie de gènes. Deuxièmement, pour identifier les ligands des récepteurs IRs exprimés par les neurones sensoriels olfactifs, j'ai réalisé une étude électrophysiologique chez D. melanogaster e η testant l'effet de plus de 160 composés chimiques sur les IRs. J'ai trouvé que les IRs répondent à un nombre d'amines, d'aldéhydes et d'acides, contrairement aux récepteurs olfactifs ORs qui eux répondent principalement aux esthers, alcools et cétones. Finalement, l'identification de ligands pour les IRs dans ces espèces m'a permis d'étudier en détail l'évolution fonctionnelle et moléculaire des IR75a/IR75b/IR75c dans D. sechellia. Cette espèce est endémique de l'archipel des Seychelles et se nourrit spécifiquement du fruit Morinda citrifolia qui est répulsif et toxique pour d'autres souches de drosophiles. Ces études m'ont poussé à découvrir que, la perte de IR75a, le changement dans la spécificité de IR75b ainsi que le changement dans l'expression de IR75c ont probablement joué un rôle important dans l'évolution des IRs chez D. sechellia. Ces changements peuvent expliquer, en partie, l'écologie chimique propre à cette espèce. Résumé français large public Le système olfactif permet aux animaux de détecter des milliers de molécules odorantes, les aidant ainsi à trouver de la nourriture, à distinguer si elle est fraîche ou avariée, à trouver des partenaires sexuels, ainsi qu'à éviter les prédateurs. Selon l'environnement et le mode de vie des espèces, le système olfactif doit détecter des odeurs très diverses ; en effet, un moustique qui recherche du sang humain pour se nourrir doit détecter des odeurs bien différentes d'une abeille qui recherche des fleurs. Dans ma thèse, j'ai essayé de comprendre comment les systèmes olfactifs d'une espèce évoluent pour s'adapter aux exigences induites par son environnement. Un très bon modèle pour étudier cela est la drosophile dont les différentes espèces se nichent dans des habitats très divers. Pour ce faire, j'ai étudié les récepteurs olfactifs de différentes espèces de la drosophile. Ces récepteurs sont des protéines qui se lient à des odeurs spécifiques. Lorsqu'ils se lient, ils activent un neurone qui envoie un signal électrique au cerveau. Ce signal est ensuite traité par ce dernier qui indique à la mouche si l'odeur est attractive ou répulsive. J'ai identifié les récepteurs olfactifs de plusieurs espèces de drosophile et étudié s'il y avait des différences entre elles. La plupart des récepteurs sont similaires entre les espèces, cependant dans l'une d'entre elles, certains récepteurs sont différents. Ce fait est particulièrement intéressant car cette espèce de drosophile se nourrit de fruits que les autres espèces n'apprécient pas. Comme nous ne savons pas quels récepteurs se lient à quelles odeurs, j'ai testé un grand nombre de composants odorants. Ceci m'a permis de constater que, effectivement, certains changements produits dans ces récepteurs expliquent pourquoi cette espèce aime particulièrement ces fruits. En outre, mes résultats contribuent à mieux comprendre les changements génétiques qui sont impliqués dans l'évolution du système olfactif.

Oxidative phosphorylation flexibility in the liver of mice resistant to high-fat diet-induced hepatic steatosis.

OBJECTIVE To identify metabolic pathways that may underlie susceptibility or resistance to high-fat diet-induced hepatic steatosis. RESEARCH DESIGN AND METHODS We performed comparative transcriptomic analysis of the livers of A/J and C57Bl/6 mice, which are, respectively, resistant and susceptible to high-fat diet-induced hepatosteatosis and obesity. Mice from both strains were fed a normal chow or a high-fat diet for 2, 10, and 30 days, and transcriptomic data were analyzed by time-dependent gene set enrichment analysis. Biochemical analysis of mitochondrial respiration was performed to confirm the transcriptomic analysis. RESULTS Time-dependent gene set enrichment analysis revealed a rapid, transient, and coordinate upregulation of 13 oxidative phosphorylation genes after initiation of high-fat diet feeding in the A/J, but not in the C57Bl/6, mouse livers. Biochemical analysis using liver mitochondria from both strains of mice confirmed a rapid increase by high-fat diet feeding of the respiration rate in A/J but not C57Bl/6 mice. Importantly, ATP production was the same in both types of mitochondria, indicating increased uncoupling of the A/J mitochondria. CONCLUSIONS Together with previous data showing increased expression of mitochondrial β-oxidation genes in C57Bl/6 but not A/J mouse livers, our present study suggests that an important aspect of the adaptation of livers to high-fat diet feeding is to increase the activity of the oxidative phosphorylation chain and its uncoupling to dissipate the excess of incoming metabolic energy and to reduce the production of reactive oxygen species. The flexibility in oxidative phosphorylation activity may thus participate in the protection of A/J mouse livers against the initial damages induced by high-fat diet feeding that may lead to hepatosteatosis.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea.

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-kappaB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-kappaB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.

Le dialogisme intertextuel des contes des Grimm

Ute Heidmann Le dialogisme intertextuel des contes des Grimm Préalables pour une enquête à mener « Le caractère le plus important de l'énoncé, ou en tous les cas le plus ignoré, est son dialogisme, c'est-à-dire sa dimension intertextuelle », constate Todorov en référence à la conception dialogique du langage proposée par Bakthine. Cet article introductif postule que ce constat s'applique aussi aux contes des Grimm. En partant des recherches déjà menées sur Apulée, Straporola, Basile, Perrault, La Fontaine et Lhéritier*, il présente des concepts (réponse intertextuelle, reconfiguration générique et scénographie en trompe-l'oeil) dont il illustre l'efficacité pour l'analyse des Kinder- und Hausmärchen. L'analyse de la préface de 1812 montre que les Grimm créent une scénographie pour légitimer le genre des Kinder- und Hausmärchen en les présentant comme des contes "d'origine" qui auraient "poussé" comme des plantes dans leur région et qu'ils n'auraient fait que "collecter". Cette scénographie en trompe-l'oeil permet de dissimuler le fort impact des contes européens et notamment français sur les Kinder- und Hausmärchen. Leurs commentaires paratextuels permettent en revanche de retracer ces dialogues intertextuels qui ne se limitent pas à imiter les "voix déjà présentes dans le choeur complexe" des narrateurs des contes déjà racontés, mais qui créent des effets de sens nouveaux et significativement différents en guise de réponse aux "histoires ou contes du passé", comme l'avaient déjà fait Charles Perrault avant eux. *(dans Féeries 8 et Textualité et intertextualité des contes, Editions Classiques Garnier 2010) "The most important feature of the utterance, or at least the most neglected, is its dialogism, that is, its intertextual dimension" states Todorov in reference to Bakthin's dialogical conception of human speech. Ute Heidmann's introductory essay argues that this applies also to the Grimm's tales. Extending her former theoretical and intertextual investigation on Apuleius, Straporala, Basile, Perrault, La Fontaine and Lhéritier*, she proposes a series of conceptual options (as intertextual response, scenography, trompe l'oeil, generic reconfiguration, discursive strategy) that can efficiently be used for the work on the Kinder- und Hausmärchen, gesammelt durch die Brüder Grimm. The article shows how the Grimms skilfully construct a highly suggestive scenography and topography for the new generic form thus creating the idea of a genuine tale, having grown naturally in the earth of their own region and how it is efficiently used to dissimulate the strong impact of the European and namely the French fairy tales on the Grimm's tales. The extensive paratextual commentaries are shown to serve the same purpose. Once these strategies are "deconstructed" as such, the way is free to trace the very complex intertextual dialogues with already existing Italian, French, German tales, that underlie the Kinder- und Hausmärchen. Comparative textual analysis can then make us discover, that these dialogues are from just "imitating" "the many other voices already present in the complex chorus" of fairy tale writers and narrators: they actually create new and different meaning by responding to them. * (in Féeries 8, Textualité et intertextualité des contes, Classiques Garnier 2010)

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Differences based on fine motor behaviour in Parkinson's patients compared to an age matched control group in proprioceptive and visuo-proprioceptive test conditions

Personality differences based on fine motor precision performance were studied in early stage Parkinson's patients and an age-matched control group under two different test conditions: proprioceptive + visual information and proprioceptive information alone. A comparative data analysis for deviations of three measured movement types (transversal, frontal and sagittal) was done for both hands (dominant and non-dominant) with relation to personality dimensions. There were found significant differences between the two groups in decision making dimension and emotionality. After splitting the data for gender subgroups, some significant differences were found for men but not for women. The differences in fine motor task performance varied, being better in some directions for the Parkinson"s patients and worse in others. The findings may suggest that medication has both positive and negative effects on motor performance and provoke personality changes, being more pronounced in men.

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.