53 research outputs found
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
Similar Biochemical Signatures and Prion Protein Genotypes in Atypical Scrapie and Nor98 Cases, France and Norway
Similarities raise questions regarding the origin of these recently described cases
Effective Gene Therapy in a Mouse Model of Prion Diseases
Classical drug therapies against prion diseases have encountered serious difficulties. It has become urgent to develop radically different therapeutic strategies. Previously, we showed that VSV-G pseudotyped FIV derived vectors carrying dominant negative mutants of the PrP gene are efficient to inhibit prion replication in chronically prion-infected cells. Besides, they can transduce neurons and cells of the lymphoreticular system, highlighting their potential use in gene therapy approaches. Here, we used lentiviral gene transfer to deliver PrPQ167R virions possessing anti-prion properties to analyse their efficiency in vivo. Since treatment for prion diseases is initiated belatedly in human patients, we focused on the development of a curative therapeutic protocol targeting the late stage of the disease, either at 35 or 105 days post-infection (d.p.i.) with prions. We observed a prolongation in the lifespan of the treated mice that prompted us to develop a system of cannula implantation into the brain of prion-infected mice. Chronic injections of PrPQ167R virions were done at 80 and 95 d.p.i. After only two injections, survival of the treated mice was extended by 30 days (20%), accompanied by substantial improvement in behaviour. This delay was correlated with: (i) a strong reduction of spongiosis in the ipsilateral side of the brain by comparison with the contralateral side; and (ii) a remarkable decrease in astrocytic gliosis in the whole brain. These results suggest that chronic injections of dominant negative lentiviral vectors into the brain, may be a promising approach for a curative treatment of prion diseases
Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization
FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts. A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin
Prion protein genotypes of Italian sheep breeds with lysine-171 and phenylalanine-141 detection
Solution Structure and Dynamics of the I214V Mutant of the Rabbit Prion Protein
Background: The conformational conversion of the host-derived cellular prion protein (PrP C) into the disease-associated scrapie isoform (PrP Sc) is responsible for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Various single-point mutations in PrP C s could cause structural changes and thereby distinctly influence the conformational conversion. Elucidation of the differences between the wild-type rabbit PrP C (RaPrP C) and various mutants would be of great help to understand the ability of RaPrP C to be resistant to TSE agents. Methodology/Principal Findings: We determined the solution structure of the I214V mutant of RaPrP C (91–228) and detected the backbone dynamics of its structured C-terminal domain (121–228). The I214V mutant displays a visible shift of surface charge distribution that may have a potential effect on the binding specificity and affinity with other chaperones. The number of hydrogen bonds declines dramatically. Urea-induced transition experiments reveal an obvious decrease in the conformational stability. Furthermore, the NMR dynamics analysis discloses a significant increase in the backbone flexibility on the pico- to nanosecond time scale, indicative of lower energy barrier for structural rearrangement. Conclusions/Significance: Our results suggest that both the surface charge distribution and the intrinsic backbone flexibility greatly contribute to species barriers for the transmission of TSEs, and thereby provide valuable hints fo
Epigenetic dominance of prion conformers
Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation
Absolute and relative quantification of sheep brain prion protein (PrP) allelic variants by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry
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