Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.

The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming.

We present a high-quality (>100× depth) Illumina genome sequence of the leaf-cutting ant Acromyrmex echinatior, a model species for symbiosis and reproductive conflict studies. We compare this genome with three previously sequenced genomes of ants from different subfamilies and focus our analyses on aspects of the genome likely to be associated with known evolutionary changes. The first is the specialized fungal diet of A. echinatior, where we find gene loss in the ant's arginine synthesis pathway, loss of detoxification genes, and expansion of a group of peptidase proteins. One of these is a unique ant-derived contribution to the fecal fluid, which otherwise consists of "garden manuring" fungal enzymes that are unaffected by ant digestion. The second is multiple mating of queens and ejaculate competition, which may be associated with a greatly expanded nardilysin-like peptidase gene family. The third is sex determination, where we could identify only a single homolog of the feminizer gene. As other ants and the honeybee have duplications of this gene, we hypothesize that this may partly explain the frequent production of diploid male larvae in A. echinatior. The fourth is the evolution of eusociality, where we find a highly conserved ant-specific profile of neuropeptide genes that may be related to caste determination. These first analyses of the A. echinatior genome indicate that considerable genetic changes are likely to have accompanied the transition from hunter-gathering to agricultural food production 50 million years ago, and the transition from single to multiple queen mating 10 million years ago.

Extensive gene traffic on the mammalian X chromosome.

Mammalian sex chromosomes have undergone profound changes since evolving from ancestral autosomes. By examining retroposed genes in the human and mouse genomes, we demonstrate that, during evolution, the mammalian X chromosome has generated and recruited a disproportionately high number of functional retroposed genes, whereas the autosomes experienced lower gene turnover. Most autosomal copies originating from X-linked genes exhibited testis-biased expression. Such export is incompatible with mutational bias and is likely driven by natural selection to attain male germline function. However, the excess recruitment is consistent with a combination of both natural selection and mutational bias.

Global Genomic Diversity of Human Papillomavirus 6 Based on 724 Isolates and 190 Complete Genome Sequences.

Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Role of retinoic acid signaling pathway in endoderm antero-posterior patterning

Summary : During vertebrate embryonic development, the endoderm gives rise to the digestive tract and associated organs such as thyroid, lung, liver and pancreas. Earlier studies have shown that extracellular signals coming from the lateral plate mesoderm pattern the endoderm along the antero-posterior axis specifying different organ primordia. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. In this study, we focused on the role of the retinoic acid (RA) signaling pathway in the regionalization of the future gut tube along the main body axis. We show that the RA-synthesizing enzyme Raldh2 is expressed in mesoderm close to the endoderm during gastrulation and during somitogenesis. During the same period, all retinoic acid receptors (RARs), which directly activate gene transcription, are expressed in endoderm suggesting that endoderm can be responsive to RA. Activation or inhibition of RA signaling was achieved by adding RA or RAR inhibitors tither on beads or in the medium to cultured chick embryos. Branchial arch (BA) endoderm markers were shifted posteriorly upon depletion of RA at gastrulation, but were not shifted after this stage. Conversely, exposure to exogenous RA repressed the most-anterior BA markers and shifted more posterior BA markers anteriorly. This suggests that graded levels of RA activity in the foregut define gene boundaries and expression levels. The posterior foregut and midget markers Pdxl and CdxA require RA for their expression, but elevated RA does not shift their expression domain along the antero-posterior axis. In addition, we investigated if RA signaling pathway interacts with other signaling pathways to pattern the endoderm. Although both RA and FGFs block anterior foregut marker expression, our experiments suggest that FGF signaling does not depend on RA in anterior endoderm. To validate our chick data in mammalians and evaluate whether RA acts directly on endoderm, we have further generated a conditional loss-of-function system in the mouse, which is still under examination.

Differential transgene expression profiles in rat brain, using rAAV2/1 vectors with tetracycline-inducible and cytomegalovirus promoters.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for the safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors (recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1) using the tetracycline-inducible (TetON) expression cassette in comparison with the cytomegalovirus (CMV) promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although green fluorescent protein (GFP) was expressed mainly in neurons with both vectors, the relative proportions of DARPP-32-positive projection neurons and parvalbumin-positive interneurons were, respectively, 13:1 and 2:1 for the CMV and TetON vectors. DARP32-positive neurons projecting to the globus pallidus were strongly GFP positive with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV vector but poorly by the TetON vector. Numerous GFP-positive cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP-positive neurons were observed with the CMV vector but not the TetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-TetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase-positive neurons by the TetON vector whereas with the CMV vector, GFP-positive cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-TetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

Solving ambiguities in contig assembly of Idiomarina loihiensis L2TR chromosome by in silico analyses.

Nucleotide composition analyses of bacterial genomes such as cumulative GC skew highlight the atypical, strongly asymmetric architecture of the recently published chromosome of Idiomarina loihiensis L2TR, suggesting that an inversion of a 600-kb chromosomal segment occurred. The presence of 3.4-kb inverted repeated sequences at the borders of the putative rearrangement supports this hypothesis. Reverting in silico this segment restores (1) a symmetric chromosome architecture; (2) the co-orientation of transcription of all rRNA operons with DNA replication; and (3) a better conservation of gene order between this chromosome and other gamma-proteobacterial ones. Finally, long-range PCRs encompassing the ends of the 600-kb segment reveal the existence of the reverted configuration but not of the published one. This demonstrates how cumulative nucleotide-skew analyses can validate genome assemblies.

Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Evolutionary dynamics of coding and non-coding transcriptomes.

Gene expression changes may underlie much of phenotypic evolution. The development of high-throughput RNA sequencing protocols has opened the door to unprecedented large-scale and cross-species transcriptome comparisons by allowing accurate and sensitive assessments of transcript sequences and expression levels. Here, we review the initial wave of the new generation of comparative transcriptomic studies in mammals and vertebrate outgroup species in the context of earlier work. Together with various large-scale genomic and epigenomic data, these studies have unveiled commonalities and differences in the dynamics of gene expression evolution for various types of coding and non-coding genes across mammalian lineages, organs, developmental stages, chromosomes and sexes. They have also provided intriguing new clues to the regulatory basis and phenotypic implications of evolutionary gene expression changes.

Lausannevirus, a giant amoebal virus encoding histone doublets.

Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co-culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae-infecting viruses.

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.

Resolving the ortholog conjecture: orthologs tend to be weakly, but significantly, more similar in function than paralogs.

The function of most proteins is not determined experimentally, but is extrapolated from homologs. According to the "ortholog conjecture", or standard model of phylogenomics, protein function changes rapidly after duplication, leading to paralogs with different functions, while orthologs retain the ancestral function. We report here that a comparison of experimentally supported functional annotations among homologs from 13 genomes mostly supports this model. We show that to analyze GO annotation effectively, several confounding factors need to be controlled: authorship bias, variation of GO term frequency among species, variation of background similarity among species pairs, and propagated annotation bias. After controlling for these biases, we observe that orthologs have generally more similar functional annotations than paralogs. This is especially strong for sub-cellular localization. We observe only a weak decrease in functional similarity with increasing sequence divergence. These findings hold over a large diversity of species; notably orthologs from model organisms such as E. coli, yeast or mouse have conserved function with human proteins.

Comparative genomics of epidemic versus sporadic Staphylococcus aureus strains does not reveal molecular markers for epidemicity.

Staphylococcus aureus, especially when it is methicillin resistant, has been recognised as a major cause of nosocomial and community-acquired infections. It has also been shown that certain strains were able to cause clonal epidemics whereas others showed a more incidental occurrence. On the basis of this behavioural distinction, a genetic feature underlying this difference in epidemicity can be assumed. Understanding the difference will not only contribute to the development of markers for the identification of epidemic strains but will also shed light on the evolution of clones. Genomes of strains from two independent collections (n=18 and n=10 strains) were analysed. Both collections were composed of carefully selected, genetically diverse strains with clinically well-defined epidemic and sporadic behaviour. Comparative genome hybridisation (CGH) was performed using an Agilent array for one collection (up to 11 probes per open reading frame - ORF), and an Affymetrix array for the other (up to 30 probes per ORF). Presence and absence information of probe homologues and ORFs was taken for analysis of molecular variance (AMOVA) at the strain and behaviour levels. Not a single probe showed 100% concordant differences between epidemic and sporadic strains. Moreover, probe differences between groups were always smaller than those within groups. This was also true, when the analysis was focussed on presence versus absence of ORF's or when probe information was transformed into allelic profiles. These findings present strong evidence against the presence or absence of a single common specific genetic factor differentiating epidemic from sporadic S. aureus clones.

Are invasive marsh frogs (Pelophylax ridibundus) replacing the native P. lessonae /P. esculentus hybridogenetic complex in Western Europe? Genetic evidence from a field study.

The water-frog L-E system, widespread in Western Europe, comprises the pool frog Pelophylax lessonae and the hybridogenetic edible frog P. esculentus, which originated from hybridization between pool frogs and marsh frogs (P. ridibundus). In P. esculentus, the lessonae (L) genome is eliminated during meiosis and has to be gained anew each generation from a P. lessonae partner, while the ridibundus (R') genome is transmitted clonally. It therefore accumulates deleterious mutations, so that R'R' offspring from P. esculentus x P. esculentus crosses are normally unviable. This system is now threatened by invasive P. ridibundus (RR) imported from Eastern Europe and the Balkans. We investigated the genetic interactions between invasive marsh frogs and native water frogs in a Swiss wetland area, and used genetic data collected in the field to validate several components of a recently postulated mechanism of species replacement. We identified neo-ridibundus individuals derived from crosses between invasive ridibundus and native esculentus, as well as newly arisen hybridogenetic esculentus lineages stemming from crosses between invasive ridibundus (RR) and native lessonae (LL). As their ridibundus genomes are likely to carry less deleterious mutations, such lineages are expected to produce viable ridibundus offspring, contributing to species replacement. However, such crosses with invasive ridibundus only occurred at a limited scale; moreover, RR x LL crosses did not induce any introgression from the ridibundus to the lessonae genome. We did not find any ridibundus stemming from crosses between ancient esculentus lineages. Despite several decades of presence on the site, introduced ridibundus individuals only represent 15% of sampled frogs, and their spatial distribution seems shaped by specific ecological requirements rather than history of colonization. We therefore expect the three taxa to coexist stably in this area.

Native homing endonucleases can target conserved genes in humans and in animal models.

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.