Hidden diversity in honey bee gut symbionts detected by single-cell genomics.

Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification.

Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

Molecular evidence for ancient asexuality in timema stick insects.

Asexuality is rare in animals in spite of its apparent advantage relative to sexual reproduction, indicating that it must be associated with profound costs [1-9]. One expectation is that reproductive advantages gained by new asexual lineages will be quickly eroded over time [3, 5-7]. Ancient asexual taxa that have evolved and adapted without sex would be "scandalous" exceptions to this rule, but it is often difficult to exclude the possibility that putative asexuals deploy some form of "cryptic" sex, or have abandoned sex more recently than estimated from divergence times to sexual relatives [10]. Here we provide evidence, from high intraspecific divergence of mitochondrial sequence and nuclear allele divergence patterns, that several independently derived Timema stick-insect lineages have persisted without recombination for more than a million generations. Nuclear alleles in the asexual lineages displayed significantly higher intraindividual divergences than in related sexual species. In addition, within two asexuals, nuclear allele phylogenies suggested the presence of two clades, with sequences from the same individual appearing in both clades. These data strongly support ancient asexuality in Timema and validate the genus as an exceptional opportunity to attack the question of how asexual reproduction can be maintained over long periods of evolutionary time.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

PhylomeDB v4: zooming into the plurality of evolutionary histories of a genome

Phylogenetic trees representing the evolutionary relationships of homologous genes are the entry point for many evolutionary analyses. For instance, the use of a phylogenetic tree can aid in the inference of orthology and paralogy relationships, and in the detection of relevant evolutionary events such as gene family expansions and contractions, horizontal gene transfer, recombination or incomplete lineage sorting. Similarly, given the plurality of evolutionary histories among genes encoded in a given genome, there is a need for the combined analysis of genome-wide collections of phylogenetic trees (phylomes). Here, we introduce a new release of PhylomeDB (http://phylomedb.org), a public repository of phylomes. Currently, PhylomeDB hosts 120 public phylomes, comprising >1.5 million maximum likelihood trees and multiple sequence alignments. In the current release, phylogenetic trees are annotated with taxonomic, protein-domain arrangement, functional and evolutionary information. PhylomeDB is also a major source for phylogeny-based predictions of orthology and paralogy, covering >10 million proteins across 1059 sequenced species. Here we describe newly implemented PhylomeDB features, and discuss a benchmark of the orthology predictions provided by the database, the impact of proteome updates and the use of the phylome approach in the analysis of newly sequenced genomes and transcriptomes.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

Control of gene expression in melanocytes and RPE by conserved distal regulatory elements

Résumé Durant le développement embryonnaire, les cellules pigmentaires des mammifères se développent à partir de deux origines différentes : les melanocytes se développent à partir de la crête neurale alors que les cellules de la rétine pigmentaire (RP) ont une origine neuronale. Un grand nombre de gènes sont impliqués dans la pigmentation dont les gènes de la famille tyrosinase à savoir Tyr, Tyrp1 et Dct. Certaines études ont suggéré que les gènes de la pigmentation sont régulés de manière différentielle dans les mélanocytes et dans la RP. Dans ce travail, les gènes de la famille tyrosinase ont été étudiés comme modèle de la régulation des gènes de la pigmentation par des éléments régulateurs agissant à distance. II a été montré que le promoteur du gène Tyrp1pouvait induire l'expression d'un transgène uniquement dans la RP alors que ce gène est aussi exprimé dans les mélanocytes comme le montre le phénotype des souris mutantes pour Tyrp1. Ce résultat suggère que les éléments régulateurs du promoteur sont suffisants pour l'expression dans la RP mais pas pour l'expression dans les mélanocytes. J'ai donc cherché à identifier la séquence qui régule l'expression dans les mélanocytes. Un chromosome artificiel bactérien (CAB) contenant le gène Tyrp1 s'est avéré suffisant pour induire l'expression dans les mélanocytes, comme démontré par la correction du phénotype mutant. La séquence de ce CAB contient plusieurs régions très conservées qui pourraient représenter de nouveaux éléments régulateurs. Par la suite, j'ai focalisé mon analyse sur une séquence située à -I5 kb qui s'est révélée être un amplificateur spécifique aux mélanocytes comme démontré par des expériences de cultures cellulaire et de transgenèse. De plus, une analyse poussée de cet élément a révélé que le facteur de transcription Sox 10 représentait un transactivateur de cet amplificateur. Comme pour Tyrp1, la régulation du gène tyrosinase est contrôlée par différents éléments régulateurs dans les mélanocytes et la RP. Il a été montré que le promoteur de tyrosinase n'était pas suffisant pour une forte expression dans les mélanocytes et la RP. De plus, l'analyse de la région située en amont a révélé la présence d'un amplificateur nécessaire à l'expression dans les mélanocytes à la position -15 kb. Cet amplificateur n'est toutefois pas actif dans la RP mais agit comme un répresseur dans ces cellules. Ces résultats indiquent que certains éléments nécessaires à l'expression dans les deux types de cellules pigmentaires sont absents de ces constructions. Comme pour Tyrp1, j'ai en premier lieu démontré qu'un CAB était capable de corriger le phénotype albinique, puis ai inséré un gène reporter (lacZ) dans le CAB par recombinaison homologue et ai finalement analysé l'expression du reporter en transgenèse. Ces souris ont montré une expression forte du lacZ dans les mélanocytes et la RP, ce qui indique que le CAB contient les séquences régulatrices nécessaires à l'expression correcte de tyrosinase. Afin de localiser plus précisément les éléments régulateurs, j'ai ensuite généré des délétions dans le CAB et analysé l'expression du lacZ en transgenèse. La comparaison de séquences génomiques provenant de différentes espèces a permis par la suite d'identifier des régions représentant de nouveaux éléments régulateurs potentiels. En utilisant cette approche, j'ai identifié une région qui se comporte comme un amplificateur dans la RP et qui est nécessaire à l'expression de tyrosinase dans ce tissu. De plus, j'ai identifié les facteurs de transcription Mitf et Sox10 comme transactivateurs de l'amplificateur spécifique aux mélanocytes situé à -15 kb. L'identification et la caractérisation des ces éléments régulateurs des gènes tyrosinase et Tyrp1confirme donc que la régulation différentielle des gènes dans les mélanocytes et la RP est liée à des éléments régulateurs séparés. Summary Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. A large set of genes are involved in pigmentation, including the members of the tyrosinase gene family, namely tyrosinase, Tyrp1 and Dct. Previous studies have suggested that pigmentation genes are differentially regulated in melanocytes and RPE. In this work, the tyrosinase gene family was used as a model for studying the involvement of distal regulatory elements in pigment cell-specific gene expression. The promoter of the Tyrp1 gene has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. I thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1 b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. I further focused on a sequence located at -15 kb which I identified as amelanocyte-specific enhancer as shown by cell culture and transgenic mice. In addition, further functional analysis identified the transcription factor Sox10 as being able to bind and transactivate this enhancer. As for Tyrp1, tyrosinase gene regulation is mediated by different cis-regulatory elements in melanocytes and RPE. It was shown that the tyrosinase promoter was not sufficient to confer strong and specific expression in melanocytes and RPE. Moreover, analysis of tyrosinase upstream sequence, revealed the presence of a specific enhancer at position -15 kb which was necessary to confer strong expression in melanocytes. This enhancer element however failed to act as an enhancer in the RPE, but rather repressed expression. This indicates that some regulatory elements required for tyrosinase expression in both RPE and melanocytes are still missing from these constructs. As for Tyrp1, I first demonstrated that a BAC containing the Tyr gene is able to rescue the Tyr c (albino) phenotype in mice, then I inserted a lacZ reporter gene in the BAC by homologous recombination, and finally analysed the pattern of lacZ expression in transgenic mice. These mice showed strong lacZ expression in both RPE and melanocytes, indicating that the BAC contains the regulatory sequences required for proper tyrosinase expression. In order to localize more precisely these regulatory elements, I have then generated several deletions in the BAC and analysed lacZ expression in transgenic mice. Multi-species comparative genomic analysis then allowed identifying conserved sequences that potentially represent novel regulatory elements. Using this experimental approach, I identified a region that behaves as a RPE-specific enhancer and that is required for tyrosinase expression in the retina] pigment epithelium. In addition, I identified the transcription factors Mitf and Sox l0 as being transactivators of the melanocyte-specific enhancer located at -l5 kb. The identification and characterization of these tyrosinase and Tyrp1 distal regulatory element supports the idea that separate regulatory sequences mediate differential gene expression in melanocytes and RPE.

Origin and genome evolution of polyploid green toads in Central Asia: evidence from microsatellite markers.

Polyploidization, which is expected to trigger major genomic reorganizations, occurs much less commonly in animals than in plants, possibly because of constraints imposed by sex-determination systems. We investigated the origins and consequences of allopolyploidization in Palearctic green toads (Bufo viridis subgroup) from Central Asia, with three ploidy levels and different modes of genome transmission (sexual versus clonal), to (i) establish a topology for the reticulate phylogeny in a species-rich radiation involving several closely related lineages and (ii) explore processes of genomic reorganization that may follow polyploidization. Sibship analyses based on 30 cross-amplifying microsatellite markers substantiated the maternal origins and revealed the paternal origins and relationships of subgenomes in allopolyploids. Analyses of the synteny of linkage groups identified three markers affected by translocation events, which occurred only within the paternally inherited subgenomes of allopolyploid toads and exclusively affected the linkage group that determines sex in several diploid species of the green toad radiation. Recombination rates did not differ between diploid and polyploid toad species, and were overall much reduced in males, independent of linkage group and ploidy levels. Clonally transmitted subgenomes in allotriploid toads provided support for strong genetic drift, presumably resulting from recombination arrest. The Palearctic green toad radiation seems to offer unique opportunities to investigate the consequences of polyploidization and clonal transmission on the dynamics of genomes in vertebrates.

Thymus-independent generation of NK1+ T cells in vitro from fetal liver precursors.

NK1.1+TCR alpha beta+ (NK1+) T cells are an unusual subset of mouse TCR alpha beta+ cells found primarily in adult thymus and liver. In contrast to conventional TCR alpha beta+ cells, NK1+ T cells have a TCR repertoire that is highly skewed to V alpha14 and to Vbeta8, -7, and -2. The developmental origin and ligand specificity of NK1+ T cells are controversial. We show here that NK1+ T cells with a typically biased V alpha and V beta repertoire develop in cytokine-supplemented suspension cultures of fetal liver established from either normal or athymic mice. Furthermore, NK1+ T cell development in fetal liver cultures is abrogated in beta2m-deficient mice (which lack MHC class I and other related molecules) and can be partially inhibited by the presence of anti-CD1 mAbs. Moreover, mixing experiments indicate that recombination-deficient SCID fetal liver cells can reconstitute NK1+ T cell development in beta2m-deficient fetal liver cultures. Collectively, our data demonstrate that NK1+ T cells can develop extrathymically from fetal liver precursors and that a beta2m-associated ligand (putatively CD1) present on nonlymphoid cells is essential for their positive selection and/or expansion.

Organisation of genetic variation in multinucleate arbuscular mycorrhizal fungi

Les Champignons Endomycorhiziens Arbusculaires (CEA) forment une symbiose racinaire avec environ 80% des espèces connues de plantes vasculaires. Ils occupent une position écologique très importante liée aux bénéfices qu'ils confèrent aux plantes. Des études moléculaires effectuées sur des gènes ribosomaux ont révélé un très grand polymorphisme, tant à l'intérieur des espèces qu'entre celles-ci. Ces champignons étant coenocytiques et multinucléés, l'organisation de cette variabilité génétique intraspécifique pourrait avoir différentes origines. Ce travail se propose d'examiner l'organisation et l'évolution de cette variabilité. Sur la base de fossiles, l'existence des CEA remonte à au moins 450 millions d'années. Cette symbiose peut donc être considérée comme ancienne. Les premières données moléculaires n'indiquant pas de reproduction sexuée, une hypothèse fut élaborée stipulant que les CEA seraient des asexués ancestraux. La première partie de cette thèse (chapitre 2) met en évidence l'existence de recombinaison dans différents CEA mais montre également que celle-ci est insuffisante pour purger les mutations accumulées. La reproduction étant essentiellement asexuée, on peut prédire que les nombreux noyaux ont probablement divergé génétiquement. En collaboration avec M. Hijri nous avons pu vérifier cette hypothèse (chapitre 2). Dans le chapitre 3 j'ai cherché à comprendre si le polymorphisme était également présent dans une population naturelle du CEA Glomus intraradices au niveau intraspécifique, ce qui n'avait encore jamais été examiné. En comparant les empreintes génétiques d'individus obtenus chacun à partir d'une spore mise en culture, j'ai clairement démontré que d'importantes différences génétiques existent entre ceux-ci. Un résultat similaire, portant sur des traits quantitatifs d'individus de la même population, a été trouvé par A. Koch. Les deux études en ensemble montre que le polymorphisme génétique dans cette population est suffisamment grand pour être important au niveau écologique. Dans le chapitre 4, j'ai cherché a examiner le polymorphisme des séquences du gène BiP au sein d'un individu. C'est la première étude qui examine la diversité génétique du génome de CEA avec un autre marqueur que l'ADN ribosomique. J'ai trouvé 31 types de séquences différentes du gène BiP issu d'un isolat de G. intraradices mis en culture à partir d'une seule spore. Cette variation n'était pas restreinte à des zones sélectivement neutres du BiP. Mes résultats montrent qu'il y a un grand nombre de variants non-fonctionnels, proportionnellement au faible nombre de copies attendues par noyau. Ceci va dans le sens d'une partition de l'information génétique entre les noyaux.<br/><br/>Arbuscular mycorrhizal fungi (AMF) are root symbionts with about 80% of all known species of vascular land plants. AMF are ecologically important because of the benefits that they confer to plants. Molecular studies on AMF showed that rDNA sequences were highly variable between species and within species. Because AMF are coenocytic and multinucleate there are several possibilities how this intraspecific genetic variation could be organized. Therefore, the organization and evolution of this variation in AMF were investigated in the present work. Based on fossil records the AMF symbiosis has existed for 450 Million years and is therefore considered ancient. First molecular data indicated no evident sexual reproduction and gave rise to the hypothesis that AMF might be ancient asexuals. The first part of this thesis (Chapter 2) shows evidence for recombination in different AMF but also indicates that it has not been frequent enough to purge accumulated mutations. Given asexual reproduction, it has been predicted that the many nuclei in AMF should diverge leading to genetically different nuclei. This hypothesis has been confirmed by an experiment of M. Hijri and is also included in chapter 2 as the results were published together. In chapter 3 I then investigated whether intraspecific genetic variation also exists in a field population of the AMF Glomus intraradices. Comparing genetic fingerprints of individuals derived from single spores I could clearly show that large genetic differences exist. A similar result, based on quantitative genetic traits, was found for the same population by A. Koch. The two studies taken together show that the genetic variation observed in the population is high enough to be of ecological relevance. Lastly, in chapter 4, I investigated within individual genetic variation among BiP gene sequences. It is the first study that has analyzed genetic diversity in the AMF genome in a region of DNA other than rDNA. I found 31 sequence variants of the BiP gene in one G. intraradices isolate that originated from one spore. Genetic variation was not only restricted to selectively neutral parts of BiP. A high number of predicted non-functional variants compared to a likely low number of copies per nucleus indicated that functional genetic information might even be partitioned among nuclei. The results of this work contribute to our understanding of potential evolutionary strategies of ancient asexuals, they also suggest that genetic differences in a population might be ecologically relevant and they show that this variation even occurs in functional regions of the AMF genome.

Fabrication and characterization of silicon position sensitive particle detectors

Position sensitive particle detectors are needed in high energy physics research. This thesis describes the development of fabrication processes and characterization techniques of silicon microstrip detectors used in the work for searching elementary particles in the European center for nuclear research, CERN. The detectors give an electrical signal along the particles trajectory after a collision in the particle accelerator. The trajectories give information about the nature of the particle in the struggle to reveal the structure of the matter and the universe. Detectors made of semiconductors have a better position resolution than conventional wire chamber detectors. Silicon semiconductor is overwhelmingly used as a detector material because of its cheapness and standard usage in integrated circuit industry. After a short spread sheet analysis of the basic building block of radiation detectors, the pn junction, the operation of a silicon radiation detector is discussed in general. The microstrip detector is then introduced and the detailed structure of a double-sided ac-coupled strip detector revealed. The fabrication aspects of strip detectors are discussedstarting from the process development and general principles ending up to the description of the double-sided ac-coupled strip detector process. Recombination and generation lifetime measurements in radiation detectors are discussed shortly. The results of electrical tests, ie. measuring the leakage currents and bias resistors, are displayed. The beam test setups and the results, the signal to noise ratio and the position accuracy, are then described. It was found out in earlier research that a heavy irradiation changes the properties of radiation detectors dramatically. A scanning electron microscope method was developed to measure the electric potential and field inside irradiated detectorsto see how a high radiation fluence changes them. The method and the most important results are discussed shortly.

Evolution based on domain combinations: the case of glutaredoxins

Background: Protein domains represent the basic units in the evolution of proteins. Domain duplication and shuffling by recombination and fusion, followed by divergence are the most common mechanisms in this process. Such domain fusion and recombination events are predicted to occur only once for a given multidomain architecture. However, other scenarios may be relevant in the evolution of specific proteins, such as convergent evolution of multidomain architectures. With this in mind, we study glutaredoxin (GRX) domains, because these domains of approximately one hundred amino acids are widespread in archaea, bacteria and eukaryotes and participate in fusion proteins. GRXs are responsible for the reduction of protein disulfides or glutathione-protein mixed disulfides and are involved in cellular redox regulation, although their specific roles and targets are often unclear. Results: In this work we analyze the distribution and evolution of GRX proteins in archaea,bacteria and eukaryotes. We study over one thousand GRX proteins, each containing at least one GRX domain, from hundreds of different organisms and trace the origin and evolution of the GRX domain within the tree of life. Conclusion: Our results suggest that single domain GRX proteins of the CGFS and CPYC classes have, each, evolved through duplication and divergence from one initial gene that was present in the last common ancestor of all organisms. Remarkably, we identify a case of convergent evolution in domain architecture that involves the GRX domain. Two independent recombination events of a TRX domain to a GRX domain are likely to have occurred, which is an exception to the dominant mechanism of domain architecture evolution.

Review. Characterization and selection of hexaploid wheats containing resistance to Heterodera avenae or Mayetiola destructor introgressed from Aegilops

Cereal cyst nematode (CCN, Heterodera avenae) and Hessian fly (HF, Mayetiola destructor) are two major pests affecting wheat crops worldwide including important cereal areas of Spain. Aegilops ventricosa and Ae. triuncialis were used as donors in a strategy to introduce resistance genes (RG) for these pests in hexaploid wheat (Triticum aestivum L.). Two 42 chromosomes introgression lines have been derived from Ae. ventricosa: H-93-8 and H-93-33 carrying genes Cre2 and H27 conferring resistance to CCN and HF, respectively. Line TR-3531 with 42 chromosomes has been derived from Ae. triuncialis and carries RGs conferring resistance for CCN (Cre7) and for HF (H30). Alien material has been incorporated in lines H-93 by chromosomal substitution and recombination, while in line TR-3531 homoeologous recombination affecting small DNA fragments has played a major role. It has been demonstrated that Cre2, Cre7, H27 and H30 are major single dominant genes and not allelic of other previously described RGs. Biochemical and molecular-biology studies of the defense mechanism triggered by Cre2 and Cre7 have revealed specific induction of peroxidase and other antioxidant enzymes. In parallel to these basic studies advanced lines carrying resistance genes for CNN and/or HF have been developed. Selection was done using molecular markers for eventually «pyramiding» resistance genes. Several isozyme and RAPD markers have been described and, currently, new markers based on transposable elements and NBS-LRR sequences are being developed. At present, two advanced lines have already been included at the Spanish Catalogue of Commercial Plant Varieties.

Sex-chromosome evolution of Palearctic tree frogs in space and time

Sexual reproduction is nearly universal in eukaryotes and genetic determination of sex prevails among animals. The astonishing diversity of sex-determining systems and sex chromosomes is yet bewildering. Some taxonomic groups possess conserved and dimorphic sex chromosomes, involving a functional copy (e.g. mammals' X, birds' Z) and a degenerated copy (mammals' Y, birds' W), implying that sex- chromosomes are expected to decay. In contrast, others like amphibians, reptiles and fishes yet maintained undifferentiated sex chromosomes. Why such different evolutionary trajectories? In this thesis, we empirically test and characterize the main hypotheses proposed to prevent the genetic decay of sex chromosomes, namely occasional X-Y recombination and frequent sex-chromosome transitions, using the Palearctic radiation of Hyla tree frogs as a model system. We take a phylogeographic and phylogenetic approach to relate sex-chromosome recombination, differentiation, and transitions in a spatial and temporal framework. By reconstructing the recent evolutionary history of the widespread European tree frog H. arborea, we showed that sex chromosomes can recombine in males, preventing their differentiation, a situation that potentially evolves rapidly. At the scale of the entire radiation, X-Y recombination combines with frequent transitions to prevent sex-chromosome degeneration in Hyla: we traced several turnovers of sex-determining system within the last 10My. These rapid changes seem less random than usually assumed: we gathered evidences that one chromosome pair is a sex expert, carrying genes with key role in animal sex determination, and which probably specialized through frequent reuse as a sex chromosome in Hyla and other amphibians. Finally, we took advantage of secondary contact zones between closely-related Hyla lineages to evaluate the consequences of sex chromosome homomorphy on the genetics of speciation. In comparison with other systems, the evolution of sex chromosomes in Hyla emphasized the existence of consistent evolutionary patterns within the chaotic diversity of flexibility of cold-blooded vertebrates' sex-determining systems, and provides insights into the evolution of recombination. Beyond sex-chromosome evolution, this work also significantly contributed to speciation, phylogeography and applied conservation research. -- La reproduction sexuée est quasi-universelle chez les eucaryotes et le sexe est le plus souvent déterminé génétiquement au sein du règne animal. L'incroyable diversité des systèmes de reproduction et des chromosomes sexuels est particulièrement étonnante. Certains groupes taxonomiques possèdent des chromosomes sexuels dimorphiques et très conservés, avec une copie entièrement fonctionnelle (ex : le X des mammifères, le Z des oiseaux) et une copie dégénérée (ex : le Y des mammifères, le W des oiseaux), suggérant que les chromosomes sexuels sont voués à se détériorer. Cependant les chromosomes sexuels d'autres groupes tels que les amphibiens, les reptiles et les poissons sont pour la plupart indifférenciés. Comment expliquer des trajectoires évolutives si différentes? Au cours de cette thèse, nous avons étudié empiriquement les processus évolutifs pouvant maintenir les chromosomes sexuels intacts, à savoir la recombinaison X-Y occasionnel ainsi que les substitutions fréquentes de chromosomes sexuels, en utilisant les rainettes Paléarctiques du genre Hyla comme modèle d'étude. Nous avons adopté une approche phylogéographique et phylogénétique pour appréhender les événements de recombinaison, de différenciation et de transitions de chromosomes sexuels dans un contexte spatio-temporel. En retraçant l'histoire évolutive récente de la rainette verte H. arborea, nous avons mis en évidence que les chromosomes sexuels pouvaient recombiner chez les mâles, empêchant ainsi leur différenciation, et que ce processus avait le potentiel d'évoluer très rapidement. A l'échelle plus globale de la radiation, il apparait que les phénomènes de recombinaison X-Y soient également accompagnés de substitutions de chromosomes sexuels, et participent de concert au maintien de chromosomes sexuels intacts dans les populations: le système de détermination du sexe des rainettes a changé plusieurs fois au cours des 10 derniers millions d'années. Ces transitions fréquentes ne semblent pas aléatoires: nous avons identifié une paire de chromosomes qui présente des caractéristiques présageant d'une spécialisation dans le déterminisme du sexe (notamment car elle possède des gènes importants pour cette fonction), et qui a été réutilisée plusieurs fois comme tel chez les rainettes ainsi que d'autres amphibiens. Enfin, nous avons étudié l'hybridation entre différentes espèces dans leurs zones de contact, afin d'évaluer si l'absence de différenciation entre X et Y jouaient un rôle dans les processus génétiques de spéciation. Outre son intérêt pour la compréhension de l'évolution des chromosomes sexuels, ce travail contribue de manière significative à d'autres domaines de recherche tels que la spéciation, la phylogéographie, ainsi que la biologie de la conservation.

A single epidermal stem cell strategy for safe ex vivo gene therapy.

There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.