Expressed sequence tags for genes: a review

Expressed sequence tags (ESTs) are partial sequences from the extremities of complementary DNA (CDNA) resulting from a single pass sequencing of clones from cDNA libraries, and different ESTs can be obtained from one gene. Sequence information from ESTs can be used for deciphering the function and the organisation of the genome. From a functional viewpoint, they allow the determination of the expression profiles of genes in any particular tissue, in different conditions or status, and thus the identification of regulated genes. In order to identify genes involved in particular processes one can select a specific group of mRNAs. For such a selection, classical techniques include subtraction or differential screening and new techniques, using polymerase chain reaction (PCR) amplification, are now available. For studies on the organisation of the genome the main use of ESTs is the determination of chromosomal localisation of the corresponding genes using a somatic hybrid cell panel. This chromosomal localisation information is needed to identify genes or quantitative trait loci, according to the ’positional candidate’ approach. ESTs also contribute to comparative genetics and they can help to decipher gene function by comparison between species, even genetically distant ones. Thus, combining sequence, functional and localisation data, ESTs contribute to an integrated approach to the genome.Les « étiquettes » correspondent aux séquences des extrémités des ADN complémentaires, obtenues de manière systématique à partir d’une seule réaction de séquençage. Cependant, à partir d’un seul gène plusieurs étiquettes différentes peuvent être obtenues : celles qui correspondent aux deux extrémités de l’ADN complémentaire, aux ADN complémentaires de tailles différentes synthétisés à partir d’un même ARN messager, et aux différents ARN messagers issus d’une même séquence d’ADN génomique. L’identification des gènes correspondants est faite par comparaison avec les séquences nucléiques ou protéiques contenues dans les bases de données publiques (GenBank ou EMBL, SwissProt), en utilisant des logiciels d’alignement automatique tels que FASTA ou BLAST. Les séquences annotées des étiquettes sont stockées dans une base de données particulière, dbEST, et soumises régulièrement à des tests de comparaison avec les bases de données citées. En raison de la présence d’une longue région non codante à l’extrémité 3’ des ARN messagers, les étiquettes de l’extrémité 3’ sont souvent non informatives. La comparaison des étiquettes entre elles permet d’essayer de regrouper celles qui peuvent appartenir à un même gène et de déterminer ainsi une séquence consensus, plus longue et donc plus informative. Au niveau fonctionnel, les étiquettes permettent d’établir les profils d’expression des gènes d’un tissu donné dans différentes situations physiologiques ou expérimentales et donc d’identifier les gènes qui sont régulés. Ces profils sont établis en utilisant les étiquettes pour mesurer la fréquence des différents ADNc dans une génothèque préparée à partir de ce tissu dans les différentes conditions étudiées. Dans une nouvelle stratégie, la SAGE (Serial Analysis of Gene expression), des étiquettes d’une dizaine de nucléotides sont collectées, mises bout à bout et séquencées en série, ce qui permet d’accélérer l’acquisition de ces profils d’expression. Une autre approche est basée sur l’hybridation d’un grand nombre de clones déposés sur une même membrane en nylon «filtres haute densité », ou, dans un format miniature, sur une lame de verre, « microarrays». Pour identifier les gènes impliqués dans des processus bien définis, différentes stratégies de soustraction ou de comparaison permettent de sélectionner une population particulière d’ARN messagers ; les techniques les plus récentes utilisent l’amplification par PCR. Au niveau de l’organisation du génome, les étiquettes contribuent au développement de la cartographie génique : les gènes correspondants sont localisés en utilisant un panel d’hybrides somatiques, les amorces nécessaires pour amplifier l’ADN des hybrides sont choisies grâce aux informations de séquence fournies par les étiquettes. Cette information de localisation chromosomique est indispensable pour identifier les gènes responsables des caractères étudiés par une stratégie de gène candidat positionnel. L’utilisation d’étiquettes d’une autre espèce peut également permettre d’effectuer ces localisations et donc de développer des cartes comparées entre espèces qui mettent en évidence une certaine conservation de l’organisation des gènes sur les chromosomes. Enfin, la conservation des gènes n’est pas limitée à la séquence et à l’organisation : grâce aux étiquettes, des analogies fonctionnelles de gènes appartenant à des espèces génétiquement éloignées ont été décrites et sont recherchées systématiquement pour identifier la fonction des gènes. Ainsi, en permettant de combiner des données de séquence, d’expression et de localisation chromosomique, les étiquettes participent au développement d’une approche intégrée du génome

Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library

cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH) regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH) and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs

A pig multi-tissue normalised cDNA library: large-scale sequencing, cluster analysis and 9K micro-array resource generation

<p>Abstract</p> <p>Background</p> <p>Domestic animal breeding and product quality improvement require the control of reproduction, nutrition, health and welfare in these animals. It is thus necessary to improve our knowledge of the major physiological functions and their interactions. This would be greatly enhanced by the availability of expressed gene sequences in the databases and by cDNA arrays allowing the transcriptome analysis of any function.</p> <p>The objective within the AGENAE French program was to initiate a high-throughput cDNA sequencing program of a 38-tissue normalised library and generate a diverse microarray for transcriptome analysis in pig species.</p> <p>Results</p> <p>We constructed a multi-tissue cDNA library, which was normalised and subtracted to reduce the redundancy of the clones. Expressed Sequence Tags were produced and 24449 high-quality sequences were released in EMBL database. The assembly of all the public ESTs (available through SIGENAE website) resulted in 40786 contigs and 54653 singletons. At least one Agenae sequence is present in 11969 contigs (12.5%) and in 9291 of the deeper-than-one-contigs (22.8%). Sequence analysis showed that both normalisation and subtraction processes were successful and that the initial tissue complexity was maintained in the final libraries. A 9K nylon cDNA microarray was produced and is available through CRB-GADIE. It will allow high sensitivity transcriptome analyses in pigs.</p> <p>Conclusion</p> <p>In the present work, a pig multi-tissue cDNA library was constructed and a 9K cDNA microarray designed. It contributes to the Expressed Sequence Tags pig data, and offers a valuable tool for transcriptome analysis.</p

The GENETPIG database: a tool for comparative mapping in pig (Sus scrofa)

The GENETPIG database has been established for storing and disseminating the results of the European project: ‘GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpi

VarGoats project : a dataset of 1159 whole-genome sequences to dissect Capra hircus global diversity

Since their domestication 10,500 years ago, goat populations with distinctive genetic backgrounds have adapted to a broad variety of environments and breeding conditions. The VarGoats project is an international 1000-genome resequencing program designed to understand the consequences of domestication and breeding on the genetic diversity of domestic goats and to elucidate how speciation and hybridization have modeled the genomes of a set of species representative of the genus Capra. A dataset comprising 652 sequenced goats and 507 public goat sequences, including 35 animals representing eight wild species, has been collected worldwide. We identified 74,274,427 single nucleotide polymorphisms (SNPs) and 13,607,850 insertion-deletions (InDels) by aligning these sequences to the latest version of the goat reference genome (ARS1). A Neighbor-joining tree based on Reynolds genetic distances showed that goats from Africa, Asia and Europe tend to group into independent clusters. Because goat breeds from Oceania and Caribbean (Creole) all derive from imported animals, they are distributed along the tree according to their ancestral geographic origin. We report on an unprecedented international effort to characterize the genome-wide diversity of domestic goats. This large range of sequenced individuals represents a unique opportunity to ascertain how the demographic and selection processes associated with post-domestication history have shaped the diversity of this species. Data generated for the project will also be extremely useful to identify deleterious mutations and polymorphisms with causal effects on complex traits, and thus will contribute to new knowledge that could be used in genomic prediction and genome-wide association studies

Opposite role of Bax and BCL-2 in the anti-tumoral responses of the immune system

BACKGROUND: The relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood. METHODS: The rat glioma cell line A15A5 was stably transfected with human Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo. RESULTS: In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells. CONCLUSIONS: We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune respons

Measles in Democratic Republic of Congo: an outbreak description from Katanga, 2010--2011

BACKGROUND: The Democratic Republic of Congo experiences regular measles outbreaks. From September 2010, the number of suspected measles cases increased, especially in Katanga province, where Medecins sans Frontieres supported the Ministry of Health in responding to the outbreak by providing free treatment, reinforcing surveillance and implementing non-selective mass vaccination campaigns. Here, we describe the measles outbreak in Katanga province in 2010--2011 and the results of vaccine coverage surveys conducted after the mass campaigns. METHODS: The surveillance system was strengthened in 28 of the 67 health zones of the province and we conducted seven vaccination coverage surveys in 2011. RESULTS: The overall cumulative attack rate was 0.71% and the case fatality ratio was 1.40%.The attack rate was higher in children under 4 and decreased with age. This pattern was consistent across districts and time. The number of cases aged 10 years and older barely increased during the outbreak. CONCLUSIONS: Early investigation of the age distribution of cases is a key to understanding the epidemic, and should guide the vaccination of priority age groups

Distinct Roles of Bcl-2 and Bcl-Xl in the Apoptosis of Human Bone Marrow Mesenchymal Stem Cells during Differentiation

Background: Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells. Principal Findings: Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs. Significance: In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions

La recherche en médecine générale en France : enjeux et perspectives.

Cet article a pour objet de cerner, à partir d'un état des lieux, ce que représente aujourd'hui, en France, la recherche en médecine générale, en insistant sur les enjeux et les perspectives de ce domaine d'activité qui n'a pas pour autant l'évidence d'une "nouvelle discipline". Une analyse détaillée de revues professionnelles a permis de construire un corpus de publications de recherche et de proposer une typologie des recherches, en approchant en même temps le groupe professionnel des "chercheurs en médecine générale", tel qu'il est repérable aujourd'hui en France. L'article présente en premier lieu le contexte institutionnel et les enjeux de la recherche en médecine générale, ensuite la production de recherche, tant par les thèmes traités et les démarches retenues que par les contraintes et les dynamiques observées, est analysée. Une première comparaison avec certains travaux européens permettra, en conclusion, de brosser quelques perspectives. (extrait du texte)