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1.  Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the evolutionary dynamics of bracoviruses 
Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.
PMCID: PMC3758192  PMID: 23938757
polydnavirus; bracovirus; parasitoid wasp; obligatory mutualism; comparative genomics
2.  Extensive Transcription Analysis of the Hyposoter didymator Ichnovirus Genome in Permissive and Non-Permissive Lepidopteran Host Species 
PLoS ONE  2014;9(8):e104072.
Ichnoviruses are large dsDNA viruses that belong to the Polydnaviridae family. They are specifically associated with endoparasitic wasps of the family Ichneumonidae and essential for host parasitization by these wasps. We sequenced the Hyposoter didymator Ichnovirus (HdIV) encapsidated genome for further analysis of the transcription pattern of the entire set of HdIV genes following the parasitization of four different lepidopteran host species. The HdIV genome was found to consist of at least 50 circular dsDNA molecules, carrying 135 genes, 98 of which formed 18 gene families. The HdIV genome had general features typical of Ichnovirus (IV) genomes and closely resembled that of the IV carried by Hyposoter fugitivus. Subsequent transcriptomic analysis with Illumina technology during the course of Spodoptera frugiperda parasitization led to the identification of a small subset of less than 30 genes with high RPKM values in permissive hosts, consisting with these genes encoding crucial virulence proteins. Comparisons of HdIV expression profiles between host species revealed differences in transcript levels for given HdIV genes between two permissive hosts, S. frugiperda and Pseudoplusia includens. However, we found no evident intrafamily gene-specific transcription pattern consistent with the presence of multigenic families within IV genomes reflecting an ability of the wasps concerned to exploit different host species. Interestingly, in two non-permissive hosts, Mamestra brassiccae and Anticarsia gemmatalis (most of the parasitoid eggs were eliminated by the host cellular immune response), HdIV genes were generally less strongly transcribed than in permissive hosts. This suggests that successful parasitism is dependent on the expression of given HdIV genes exceeding a particular threshold value. These results raise questions about the mecanisms involved in regulating IV gene expression according to the nature of the lepidopteran host species encountered.
PMCID: PMC4130501  PMID: 25117496
3.  KIF1A missense mutations in SPG30, an autosomal recessive spastic paraplegia: distinct phenotypes according to the nature of the mutations 
The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases characterised by progressive spasticity in the lower limbs. The nosology of autosomal recessive forms is complex as most mapped loci have been identified in only one or a few families and account for only a small percentage of patients. We used next-generation sequencing focused on the SPG30 chromosomal region on chromosome 2q37.3 in two patients from the original linked family. In addition, wide genome scan and candidate gene analysis were performed in a second family of Palestinian origin. We identified a single homozygous mutation, p.R350G, that was found to cosegregate with the disease in the SPG30 kindred and was absent in 970 control chromosomes while affecting a strongly conserved amino acid at the end of the motor domain of KIF1A. Homozygosity and linkage mapping followed by mutation screening of KIF1A allowed us to identify a second mutation, p.A255V, in the second family. Comparison of the clinical features with the nature of the mutations of all reported KIF1A families, including those reported recently with hereditary sensory and autonomic neuropathy, suggests phenotype–genotype correlations that may help to understand the mechanisms involved in motor neuron degeneration. We have shown that mutations in the KIF1A gene are responsible for SPG30 in two autosomal recessive HSP families. In published families, the nature of the KIF1A mutations seems to be of good predictor of the underlying phenotype and vice versa.
PMCID: PMC3355258  PMID: 22258533
hereditary spastic paraplegia; motor neuron disease; hereditary sensory and autonomic neuropathy; next-generation sequencing; SPG30; KIF1A
4.  TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum 
Nature genetics  2011;43(3):189-196.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as interact in trans with other disease-causing genes, and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of disorders.
PMCID: PMC3071301  PMID: 21258341
5.  High Level of Structural Polymorphism Driven by Mobile Elements in the Hox Genomic Region of the Chaetognath Spadella cephaloptera 
Little is known about the relationships between genome polymorphism, mobile element dynamics, and population size among animal populations. The chaetognath species Spadella cephaloptera offers a unique perspective to examine this issue because they display a high level of genetic polymorphism at the population level. Here, we have investigated in detail the extent of nucleotide and structural polymorphism in a region harboring Hox1 and several coding genes and presumptive functional elements. Sequencing of several bacterial artificial chromosome inserts representative of this nuclear region uncovered a high level of structural heterogeneity, which is mainly caused by the polymorphic insertion of a diversity of genetic mobile elements. By anchoring this variation through individual genotyping, we demonstrated that sequence diversity could be attributed to the allelic pool of a single population, which was confirmed by detection of extensive recombination within the genomic region studied. The high average level of nucleotide heterozygosity provides clues of selection in both coding and noncoding domains. This pattern stresses how selective processes remarkably cope with intense sequence turnover due to substitutions, mobile element insertions, and recombination to preserve the integrity of functional landscape. These findings suggest that genome polymorphism could provide pivotal information for future functional annotation of genomes.
PMCID: PMC2997562  PMID: 20829282
structural polymorphism; mobile elements; Hox genes; population genomics; Chaetognatha; metazoan evolution
6.  Analysis of Virion Structural Components Reveals Vestiges of the Ancestral Ichnovirus Genome 
PLoS Pathogens  2010;6(5):e1000923.
Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts.
Author Summary
The polydnaviruses (PDVs) are a unique virus type used by an organism (a parasitic wasp) to manipulate the physiology of another organism (a lepidopteran host) in order to ensure successful parasitism. The evolutionary origin of these unusual viruses, found in ∼17,500 braconid wasps (Bracoviruses) and ∼15,000 ichneumonid wasps (Ichnoviruses), has been a major question for the last decade. We thus undertook an exclusive work aiming at investigating this origin via the characterization of genes encoding structural components for both types of PDVs. The present paper constitutes the first report on the identity and genome organisation of the viral machinery producing Ichnovirus virions. Our results strongly suggest that Ichnoviruses originated from a virus belonging to a group as yet uncharacterized that integrated its genome into that of an ichneumonid wasp ancestor. More importantly, our results demonstrate that the ancestor of Ichnoviruses differs from that of Bracoviruses, which originated from a nudivirus. We have now identified, for the two types of PDVs, the non packaged viral genes and their products involved in producing particles injected into the host during oviposition. Together, these data provide an example of convergent evolution where different groups of wasps have independently domesticated viruses to deliver genes into their hosts.
PMCID: PMC2877734  PMID: 20523890
7.  Chætognath transcriptome reveals ancestral and unique features among bilaterians 
Genome Biology  2008;9(6):R94.
The chætognath transcriptome reveals unusual genomic features in the evolution of this protostome and suggests that it could be used as a model organism for bilaterians.
The chætognaths (arrow worms) have puzzled zoologists for years because of their astonishing morphological and developmental characteristics. Despite their deuterostome-like development, phylogenomic studies recently positioned the chætognath phylum in protostomes, most likely in an early branching. This key phylogenetic position and the peculiar characteristics of chætognaths prompted further investigation of their genomic features.
Transcriptomic and genomic data were collected from the chætognath Spadella cephaloptera through the sequencing of expressed sequence tags and genomic bacterial artificial chromosome clones. Transcript comparisons at various taxonomic scales emphasized the conservation of a core gene set and phylogenomic analysis confirmed the basal position of chætognaths among protostomes. A detailed survey of transcript diversity and individual genotyping revealed a past genome duplication event in the chætognath lineage, which was, surprisingly, followed by a high retention rate of duplicated genes. Moreover, striking genetic heterogeneity was detected within the sampled population at the nuclear and mitochondrial levels but cannot be explained by cryptic speciation. Finally, we found evidence for trans-splicing maturation of transcripts through splice-leader addition in the chætognath phylum and we further report that this processing is associated with operonic transcription.
These findings reveal both shared ancestral and unique derived characteristics of the chætognath genome, which suggests that this genome is likely the product of a very original evolutionary history. These features promote chætognaths as a pivotal model for comparative genomics, which could provide new clues for the investigation of the evolution of animal genomes.
PMCID: PMC2481426  PMID: 18533022
8.  A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1 
We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.
PMCID: PMC2290942  PMID: 18319726
Acinetobacter baylyi ADP1; deletion mutants; essential genes; growth phenotype; metabolism; systems biology
9.  Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy 
Journal of Clinical Investigation  2007;117(8):2225-2232.
Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34+ progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.
PMCID: PMC1934585  PMID: 17671652
10.  Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis 
BMC Genomics  2007;8:118.
The western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in X. tropicalis.
A gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers.
These cDNA sequences permit in silico cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study X. tropicalis biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.
PMCID: PMC1890556  PMID: 17506875

Results 1-10 (10)