Identification of Transcribed Sequences (ESTs) in the Trypanosoma cruzi Genome Project

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.

A genome-wide association study of early menopause and the combined impact of identified variants.

Early menopause (EM) affects up to 10% of the female population, reducing reproductive lifespan considerably. Currently, it constitutes the leading cause of infertility in the western world, affecting mainly those women who postpone their first pregnancy beyond the age of 30 years. The genetic aetiology of EM is largely unknown in the majority of cases. We have undertaken a meta-analysis of genome-wide association studies (GWASs) in 3493 EM cases and 13 598 controls from 10 independent studies. No novel genetic variants were discovered, but the 17 variants previously associated with normal age at natural menopause as a quantitative trait (QT) were also associated with EM and primary ovarian insufficiency (POI). Thus, EM has a genetic aetiology which overlaps variation in normal age at menopause and is at least partly explained by the additive effects of the same polygenic variants. The combined effect of the common variants captured by the single nucleotide polymorphism arrays was estimated to account for ∼30% of the variance in EM. The association between the combined 17 variants and the risk of EM was greater than the best validated non-genetic risk factor, smoking.

arrayMap 2014: an updated cancer genome resource.

Somatic copy number aberrations (CNA) represent a mutation type encountered in the majority of cancer genomes. Here, we present the 2014 edition of arrayMap (http://www.arraymap.org), a publicly accessible collection of pre-processed oncogenomic array data sets and CNA profiles, representing a vast range of human malignancies. Since the initial release, we have enhanced this resource both in content and especially with regard to data mining support. The 2014 release of arrayMap contains more than 64,000 genomic array data sets, representing about 250 tumor diagnoses. Data sets included in arrayMap have been assembled from public repositories as well as additional resources, and integrated by applying custom processing pipelines. Online tools have been upgraded for a more flexible array data visualization, including options for processing user provided, non-public data sets. Data integration has been improved by mapping to multiple editions of the human reference genome, with the majority of the data now being available for the UCSC hg18 as well as GRCh37 versions. The large amount of tumor CNA data in arrayMap can be freely downloaded by users to promote data mining projects, and to explore special events such as chromothripsis-like genome patterns.

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

A Study of Cryptosporidium parvum Genotypes and Population Structure

Genetic evidence for the occurrence of two Cryptosporidium parvum subgroups is presented. This evidence is based on restriction fragment length polymorphism analysis of several independent loci. Sequence analysis of the b -tubulin intron revealed additional polymorphism. The stability of the genetic profiles following passage of C. parvum isolates between different hosts was investigated.

Orthogonal higher order structure and confirmatory factor analysis of the French Wechsler Adult Intelligence Scale (WAIS-III).

According to the most widely accepted Cattell-Horn-Carroll (CHC) model of intelligence measurement, each subtest score of the Wechsler Intelligence Scale for Adults (3rd ed.; WAIS-III) should reflect both 1st- and 2nd-order factors (i.e., 4 or 5 broad abilities and 1 general factor). To disentangle the contribution of each factor, we applied a Schmid-Leiman orthogonalization transformation (SLT) to the standardization data published in the French technical manual for the WAIS-III. Results showed that the general factor accounted for 63% of the common variance and that the specific contributions of the 1st-order factors were weak (4.7%-15.9%). We also addressed this issue by using confirmatory factor analysis. Results indicated that the bifactor model (with 1st-order group and general factors) better fit the data than did the traditional higher order structure. Models based on the CHC framework were also tested. Results indicated that a higher order CHC model showed a better fit than did the classical 4-factor model; however, the WAIS bifactor structure was the most adequate. We recommend that users do not discount the Full Scale IQ when interpreting the index scores of the WAIS-III because the general factor accounts for the bulk of the common variance in the French WAIS-III. The 4 index scores cannot be considered to reflect only broad ability because they include a strong contribution of the general factor.

Spatio-temporal variations in reproductive patterns and population structure of Pasiphaea multidentata (Decapoda: Caridea) in the Blanes canyon and adjacent margin, North-western Mediterranean Sea

The bathyal faunal communities of the NW Mediterranean slopes have been studied consistently in the last two decades, with a special focus on population structure, trophic dynamics and benthopelagic coupling of commercial deep-sea decapod crustaceans and fishes (reviewed in Sardà et al. 2004) and associated species (Cartes and Sardà, 1993; Company and Sardà, 1997, 2000; Cartes et al., 2001; Company et al., 2001, 2003, 2004). One of the major topographic features in the North-western Mediterranean slope is the presence of submarine canyons. Canyons play a major role in funnelling energy and organic matter from the shelf to bathyal and abyssal depths (Puig et al., 2000), but the implications of this enhanced organic supply in the deep-sea benthic communities is still mostly unknown. Trophic supply can follow two major pathways – vertical deposition in the water column (Billett et al., 1983; Baldwin et al., 1998; Lampitt et al., 2001) or down-slope advection on the margins (Puig et al., 2001; Bethoux et al., 2002; Canals et al., 2006) – and can be a limiting factor in the deep-sea, being especially important in the oligotrophic Mediterranean Sea (Sardà et al., 2004). Differences in the quantity, quality and timing of organic matter input to the deep seafloor have been used to explain patterns of biomass and abundance in benthic communities (Levin et al., 1994; Gooday & Turley, 1990; Billett et al., 2001; Galéron et al., 2001; Puig et al., 2001; Gage, 2003) as well as other biological process and in particular the existence of seasonal reproduction (Tyler et al., 1994; Company et al., 2004 (MEPS). Reproduction is a highly energetic process tightly linked to food availability and quality.

Genetic Structure of Triatoma sordida (Hemiptera: Reduviidae) Domestic Populations from Bolivia: Application on Control Interventions

The genetic population of Triatoma sordida group 1, a secondary vector of Chagas disease in Bolivia, was studied by multi-locus enzyme electrophoresis. A total of 253 nymphal and adult specimens collected from seven neighbouring localities in the Velasco Province, Department of Santa Cruz, were processed. The relatively low genetic variability was confirmed for this species (rate of polymorphism: 0.20). The absence of genetic disequilibrium detected within the seven localities was demonstrated. A geographical structuration appears between localities with distances greater than 20 km apart. Although T. sordida presents a relatively reduced dispersive capacity, its panmictic unit is wider than compared with T. infestans. Genetic distances between T. sordida populations were correlated with geographic distance. Gene flow between geographic populations of T. sordida provides an efficient framework for effective vigilance and control protocols.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterization and effect of aprotinin on cellular structure

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37ºC. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.