Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism.

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS(+) candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS(+) candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5' flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogenin minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells. In contrast to the transfected genes, the endogenous chromosomal vitellogenin genes remain silent, demonstrating that in spite of the presence of the hER and the hormone, the conditions necessary for their activation are not fulfilled.

Sociogenetics of fire ants

RÉSUMÉ GRAND PUBLIC La complexité des sociétés d'insectes (telles que les abeilles, les termites ou les fourmis) a depuis longtemps fasciné l'Homme. Depuis le débfit du XIXème siècle, de nombreux travaux observationnels, comportementaux et théoriques leur on été consacrés afin de mieux les décrire et comprendre. L'avènement de la biologie moléculaire à la fin du XXèrne siècle a offert de nouveaux outils scientifiques pour identifier et étudier les gènes et molécules impliqués dans le développement et le comportement des êtres vivants. Alors que la majorité de ces études s'est focalisée sur des organismes de laboratoire tel que la mouche ou les nématodes, l'utilisation de ces outils est restée marginale jusqu'à présent dans l'étude des sociétés d'insectes. Lors de ma thèse, j'ai développé des outils moléculaires permettant de déterminer le niveau d'activité de zo,ooo gènes chez la fourmi de feu, Solenopsis invicta, ainsi qu'une base de données et un portail en ligne regroupant les informations relatives à l'étude génétique des fourmis: Fourmidable. J'ai ensuite utilisé ces outils dans le cadre d'une étude comportementale chez la fourmis S. invicta. Dans les sociétés d'insectes, une hiérarchie peut déterminer le statut reproducteur des individus. Suite à la mort d'un dominant, les subordonnés entrent en compétition en vue d'améliorer leur statut. Un tel phénomène se produit au sein des colonies de S. invicta contenant une unique reine mère, des milliers d'ouvrières et des centaines de reines vierges ailées. A la mort de la reine mère, un grand nombre de reines vierges tentent de la remplacer en arrachant leurs ailes et en activant leurs organes reproducteurs plutôt que de partir en vol nuptial. Ces tentatives sont le plus souvent arrêtées par les ouvrières qui exécutent la plupart de ces reines sur la base de signaux olfactifs produits lors de l'activation des organes reproducteurs. Afin de mieux comprendre les mécanismes moléculaires impliqués, j'ai étudié l'activité de gènes au sein des reines au début de ce processus. J'ai ainsi déterminé que des gènes impliqués dans communication olfactive, le développement des organes reproducteurs et la métabolisation de l'hormone juvénile sont activês à ce moment là. La vitesse à laquelle les reines perdent leurs ailes ainsi que les niveaux d'expression de gènes sont ensuite liés à leur probabilité de survie. ABSTRACT : Honeybees, termites and ants occupy the "pinnacle of social evolution" with societies of a complexity that rivals our own. Humans have long been fascinated by social insects, but studying them has been mostly limited to observational and behavioral experiments. The advent of molecular biology first made it possible to investigate the molecular-genetic basis of development in model systems such as the fruit fly Drosophila melarcogaster or the roundworm Caenorhabditis elegans and subsequently their behavior. Molecular and genomic tools are now becoming available for the study of social insects as well. To permit genomic research on the fire ant, Solenopsis invicta, we developed a cDNA microarray that can simultaneously determine the expression levels of approximately 1oooo genes. These genes were assembled and bioinformatically annotated using custom pipelines. The obtained data formed the cornerstones for Fourmidable, a web portal centralizing sequence, gene annotation and gene expression data as well as laboratory protocols for research on ants. In many animals living in groups the reproductive status of individuals is determined by their social status. In species with social hierarchies, the death of dominant individuals typically upheaves the social hierarchy and provides an opportunity for subordinate individuals to improve their social status. Such a phenomenon occurs in the monogyne form of S. invicta, where colonies typically contain a single wingless reproductive queen, thousands of workers and hundreds of winged non-reproductive virgin queens. Upon the death of the mother queen, many virgin queens shed their wings and initiate reproductive development instead of departing on a mating flight. Workers progressively execute almost all of them over the following weeks. The workers base their collective decision on pheromonal cues associated with the onset of reproductive development of the virgin queens which occurs after orphaning. We used the aforementioned tools to determine that genes putatively involved in processes including olfactory signaling, reproductive development and Juvenile Hormone metabolism are differentially expressed at the onset of competition. Additionally, we found that queens that initiate reproductive development faster and, to a certain extent, shed their wings faster after orphaning are more likely to become replacement queens. These results provide candidate genes that are putatively linked to competition outcome. To determine the extent to which specific genes affect different aspects of life in ant colonies, functional tests such as gene activation and silencing will still be required. We conclude by discussing some of the challenges and opportunities for molecular-genetic research on ants. RÉSUMÉ Les sociétés d'abeilles, de termites et de fourmis sont d'une complexité proche de celle de la nôtre et ont depuis longtemps fasciné l'Homme. Cependant, leur étude était jusqu'à présent limitée aux observations et expériences comportementales. L'avènement de la biologie moléculaire a d'abord rendu possible l'étude moléculaire et génétique du développement d'organismes modèles tels que la mouche Drosophila melanogaster ou le nématode Caenorhabditis elegans, puis dans un second temps de leur comportement. De telles études deviennent désormais possibles pour les insectes sociaux. Nous avons développé une puce à ADN permettant de déterminer simultanément les niveaux d'expression de 1oooo gènes de la fourmi de feu, Solenopsís invicta. Ces gènes ont été séquencés puis assemblés et annotés à l'aide de pipelines que nous avons développés. En se basant sur les informations obtenues, nous avons créé un portail web, Fourmidable. Ce portail vise à centraliser toutes les informations de séquence, d'annotation et d'expression de gènes, ainsi que les protocoles de laboratoire utilisés pour la recherche sur les fourmis. Par la suite, nous avons utilisé les outils développés pour étudier un aspect particulier de S. invicta. Chez les animaux grégaires, une hiérarchie sociale peut déterminer le statut reproducteur des individus. Suite à la mort d'un individu dominant, les individus subordonnés peuvent entrer en compétition en vue d'améliorer leur statut. Un tel phénomène se produit au sein des colonies monogynes de S. invicta, qui contiennent habituellement une unique reine mère, des milliers d'ouvrières et des centaines de reines vierges ailées. Suite à la mort de la reine mère, dominante, un grand nombre de reines vierges, subordonnées, perdent leurs ailes et activent leurs organes reproducteurs au lieu de partir en vol nuptial. Au cours des semaines suivantes, les ouvrières exécutent la plupart de ces reines sur la base de signaux olfactifs produits lors de l'activation des organes reproducteurs. Afin de mieux comprendre les mécanismes moléculaires impliqués, nous avons étudié l'expression de gènes au début de cette compétition. Nous avons identifié 297 gènes différemment exprimés, dont l'annotation indique qu'ils seraient impliqués dans des processus biologiques dont la communication olfactive, le développement des organes reproducteurs et la métabolisation de l'hormone juvénile. Par la suite, nous avons déterminé que la vitesse à laquelle les reines perdent leurs ailes en début de compétition ainsi que les niveaux d'expression de gènes sont corrélés à la probabilité de survie des reines. Nous concluons en discutant des opportunités offertes par la recherche génétique sur les fourmis ainsi que les défis qu'elle devra surmonter.

Prominent use of distal 5' transcription start sites and discovery of a large number of additional exons in ENCODE regions.

This report presents systematic empirical annotation of transcript products from 399 annotated protein-coding loci across the 1% of the human genome targeted by the Encyclopedia of DNA elements (ENCODE) pilot project using a combination of 5' rapid amplification of cDNA ends (RACE) and high-density resolution tiling arrays. We identified previously unannotated and often tissue- or cell-line-specific transcribed fragments (RACEfrags), both 5' distal to the annotated 5' terminus and internal to the annotated gene bounds for the vast majority (81.5%) of the tested genes. Half of the distal RACEfrags span large segments of genomic sequences away from the main portion of the coding transcript and often overlap with the upstream-annotated gene(s). Notably, at least 20% of the resultant novel transcripts have changes in their open reading frames (ORFs), most of them fusing ORFs of adjacent transcripts. A significant fraction of distal RACEfrags show expression levels comparable to those of known exons of the same locus, suggesting that they are not part of very minority splice forms. These results have significant implications concerning (1) our current understanding of the architecture of protein-coding genes; (2) our views on locations of regulatory regions in the genome; and (3) the interpretation of sequence polymorphisms mapping to regions hitherto considered to be "noncoding," ultimately relating to the identification of disease-related sequence alterations.

VEGF is a mediator of retinal pigment epithelium permeability leading to photoreceptor cell death in light-induced retinal degeneration : gene therapy strategy to prevent AMD-disorders

ABSTRACTIn normal tissues, a balance between pro- and anti-angiogenic factors tightly controls angiogenesis. Alterations of this balance may have pathological consequences. For instance, concerning the retina, the vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor, and has been identified has a key player during ocular neovascularization implicated in a variety of retinal diseases. In the exudative form (wet-form) of age-related macular degeneration (AMD), neovascularizations occurring from the choroidal vessels are responsible for a quick and dramatic loss of visual acuity. In diabetic retinopathy and retinopathy of prematurity, sprouting from the retinal vessels leads to vision loss. Furthermore, the aging of the population, the increased- prevalence of diabetes and the better survival rate of premature infants will lead to an increasing rate of these conditions. In this way, anti-VEGF strategy represents an important therapeutic target to treat ocular neovascular disorders.In addition, the administration of Pigmented Epithelial growth factor, a neurotrophic and an anti- angiogenic factor, prevents photoreceptor cell death in a model of retinal degeneration induced by light. Previous results analyzing end point morphology reveal that the light damage (LD) model is used to mimic retinal degenerations arising from environmental insult, as well as aging and genetic disease such as advanced atrophic AMD. Moreover, light has been identified as a co-factor in a number of retinal diseases, speeding up the degeneration process. This protecting effect of PEDF in the LD retina raises the possibility of involvement of the balance between pro- and anti-angiogenic factors not only for angiogenesis, but also in cell survival and maintenance.The aim of the work presented here was to evaluate the importance of this balance in neurodegenerative processes. To this aim, a model of light-induced retinal degeneration was used and characterized, mainly focusing on factors simultaneously controlling neuron survival and angiogenesis, such as PEDF and VEGF.In most species, prolonged intense light exposure can lead to photoreceptor cell damage that can progress to cell death and vision loss. A protocol previously described to induce retinal degeneration in Balb/c mice was used. Retinas were characterized at different time points after light injury through several methods at the functional and molecular levels. Data obtained confirmed that toxic level of light induce PR cell death. Variations were observed in VEGF pathway players in both the neural retina and the eye-cup containing the retinal pigment epithelium (RPE), suggesting a flux of VEGF from the RPE towards the neuroretina. Concomitantly, the integrity of the outer blood-retinal-barrier (BRB) was altered, leading to extravascular albumin leakage from the choroid throughout the photoreceptor layer.To evaluate the importance of VEGF during light-induced retinal degeneration process, a lentiviral vector encoding the cDNA of a single chain antibody directed against all VEGF-A isoforms was developed (LV-V65). The bioactivity of this vector to block VEGF was validated in a mouse model of laser-induced choroidal neovascularization mediated by VEGF upregulation. The vector was then used in the LD model. The administration of the LV-V65 contributed to the maintenance of functional photoreceptors, which was assessed by ERG recording, visual acuity measurement and histological analyses. At the RPE level, the BRB integrity was preserved as shown by the absence of albumin leakage and the maintenance of RPE cell cohesion.These results taken together indicate that the VEGF is a mediator of light induced PR degeneration process and confirm the crucial role of the balance between pro- and anti-angiogenic factors in the PR cell survival. This work also highlights the prime importance of BRB integrity and functional coupling between RPE and PR cells to maintain the PR survival. VEGF dysregulation was already shown to be involved in wet AMD forms and our study suggests that VEGF dysregulation may also occur at early stages of AMD and could thus be a potential therapeutic target for several RPE related diseases.RESUMEDans les différents tissues de l'organisme, l'angiogenèse est strictement contrôlée par une balance entre les facteurs pro- et anti-angiogéniques. Des modifications survenant dans cette balance peuvent engendrer des conséquences pathologiques. Par exemple, concernant la rétine, le facteur de croissance de l'endothélium vasculaire (VEGF) est un facteur pro-angiogénique important. Ce facteur a été identifié comme un acteur majeur dans les néovascularisations oculaires et les processus pathologiques angiogéniques survenant dans l'oeil et responsables d'une grande variété de maladies rétiniennes. Dans la forme humide de la dégénérescence maculaire liée à l'âge (DMLA), la néovascularisation choroïdienne est responsable de la perte rapide et brutale de l'acuité visuelle chez les patients affectés. Dans la rétinopathie diabétique et celle lié à la prématurité, l'émergence de néovaisseaux rétiniens est la cause de la perte de la vision. Les néovascularisations oculaires représentent la principale cause de cécité dans les pays développés. De plus, l'âge croissant de la population, la progression de la prévalence du diabète et la meilleure survie des enfants prématurés mèneront sans doute à l'augmentation de ces pathologies dans les années futures. Dans ces conditions, les thérapies anti- angiogéniques visant à inhiber le VEGF représentent une importante cible thérapeutique pour le traitement de ces pathologies.Plusieurs facteurs anti-angiogéniques ont été identifiés. Parmi eux, le facteur de l'épithélium pigmentaire (PEDF) est à la fois un facteur neuro-trophique et anti-angiogénique, et l'administration de ce facteur au niveau de la rétine dans un modèle de dégénérescence rétinienne induite par la lumière protège les photorécepteurs de la mort cellulaire. Des études antérieures basées sur l'analyse morphologique ont révélé que les modifications survenant lors de la dégénération induite suite à l'exposition à des doses toxiques de lumière représente un remarquable modèle pour l'étude des dégénérations rétiniennes suite à des lésions environnementales, à l'âge ou encore aux maladies génétiques telle que la forme atrophique avancée de la DMLA. De plus, la lumière a été identifiée comme un co-facteur impliqué dans un grand nombre de maladies rétiniennes, accélérant le processus de dégénération. L'effet protecteur du PEDF dans les rétines lésées suite à l'exposition de des doses toxiques de lumière suscite la possibilité que la balance entre les facteurs pro- et anti-angiogéniques soit impliquée non seulement dans les processus angiogéniques, mais également dans le maintient et la survie des cellules.Le but de ce projet consiste donc à évaluer l'implication de cette balance lors des processus neurodégénératifs. Pour cela, un modèle de dégénération induite par la lumière à été utilisé et caractérisé, avec un intérêt particulier pour les facteurs comme le PEDF et le VEGF contrôlant simultanément la survie des neurones et l'angiogenèse.Dans la plupart des espèces, l'exposition prolongée à une lumière intense peut provoquer des dommages au niveau des cellules photoréceptrices de l'oeil, qui peut mener à leur mort, et par conséquent à la perte de la vision. Un protocole préalablement décrit a été utilisé pour induire la dégénération rétinienne dans les souris albinos Balb/c. Les rétines ont été analysées à différents moments après la lésion par différentes techniques, aussi bien au niveau moléculaire que fonctionnel. Les résultats obtenus ont confirmé que des doses toxiques de lumière induisent la mort des photorécepteurs, mais altèrent également la voie de signalisation du VEGF, aussi bien dans la neuro-rétine que dans le reste de l'oeil, contenant l'épithélium pigmentaire (EP), et suggérant un flux de VEGF provenant de ΙΈΡ en direction de la neuro-rétine. Simultanément, il se produit une altération de l'intégrité de la barrière hémato-rétinienne externe, menant à la fuite de protéine telle que l'albumine, provenant de la choroïde et retrouvée dans les compartiments extravasculaires de la rétine, telle que dans la couche des photorécepteurs.Pour déterminer l'importance et le rôle du VEGF, un vecteur lentiviral codant pour un anticorps neutralisant dirigée contre tous les isoformes du VEGF a été développé (LV-V65). La bio-activité de ce vecteur a été testé et validée dans un modèle de laser, connu pour induire des néovascularisations choroïdiennes chez la souris suite à l'augmentation du VEGF. Ce vecteur a ensuite été utilisé dans le modèle de dégénération induite par la lumière. Les résultats des électrorétinogrammes, les mesures de l'acuité visuelle et les analyses histologiques ont montré que l'injection du LV-V65 contribue à la maintenance de photorécepteurs fonctionnels. Au niveau de l'EP, l'absence d'albumine et la maintenance des jonctions cellulaires des cellules de l'EP ont démontré que l'intégrité de la barrière hémato-rétinienne externe est préservée suite au traitement.Par conséquent, tous les résultats obtenus indiquent que le VEGF est un médiateur important impliquée dans le processus de dégénération induit par la lumière et confirme le rôle cruciale de la balance entre les facteurs pro- et anti-angiogéniques dans la survie des photorécepteurs. Cette étude révèle également l'importance de l'intégrité de la barrière hémato-rétinienne et l'importance du lien fonctionnel et structurel entre l'EP et les photorécepteurs, essentiel pour la survie de ces derniers. Par ailleurs, Cette étude suggère que des dérèglements au niveau de l'équilibre du VEGF ne sont pas seulement impliqués dans la forme humide de la DMLA, comme déjà démontré dans des études antérieures, mais pourraient également contribuer et survenir dans des formes précoces de la DMLA, et par conséquent le VEGF représente une cible thérapeutique potentielle pour les maladies associées à des anomalies au niveau de l'EP.

A novel mutation in the sulfate transporter gene SLC26A2 (DTDST) specific to the Finnish population causes de la Chapelle dysplasia.

BACKGROUND: Mutations in the sulfate transporter gene SLC26A2 (DTDST) cause a continuum of skeletal dysplasia phenotypes that includes achondrogenesis type 1B (ACG1B), atelosteogenesis type 2 (AO2), diastrophic dysplasia (DTD), and recessive multiple epiphyseal dysplasia (rMED). In 1972, de la Chapelle et al reported two siblings with a lethal skeletal dysplasia, which was denoted "neonatal osseous dysplasia" and "de la Chapelle dysplasia" (DLCD). It was suggested that DLCD might be part of the SLC26A2 spectrum of phenotypes, both because of the Finnish origin of the original family and of radiographic similarities to ACG1B and AO2. OBJECTIVE: To test the hypothesis whether SLC26A2 mutations are responsible for DLCD. METHODS: We studied the DNA from the original DLCD family and from seven Finnish DTD patients in whom we had identified only one copy of IVS1+2T>C, the common Finnish mutation. A novel SLC26A2 mutation was found in all subjects, inserted by site-directed mutagenesis in a vector harbouring the SLC26A2 cDNA, and expressed in sulfate transport deficient Chinese hamster ovary (CHO) cells to measure sulfate uptake activity. RESULTS: We identified a hitherto undescribed SLC26A2 mutation, T512K, homozygous in the affected subjects and heterozygous in both parents and in the unaffected sister. T512K was then identified as second pathogenic allele in the seven Finnish DTD subjects. Expression studies confirmed pathogenicity. CONCLUSIONS: DLCD is indeed allelic to the other SLC26A2 disorders. T512K is a second rare "Finnish" mutation that results in DLCD at homozygosity and in DTD when compounded with the milder, common Finnish mutation.

Pigs Transgenic For Human Dominant Mutations Of The GUCY2D Gene

Purpose: Studies on large animal models are an important step to test new therapeutical strategies before human application. Considering the importance of cone function for human vision and the paucity of large animal models for cone dystrophies having an enriched cone region, we propose to develop a pig model for cone degeneration. With a lentiviral-directed transgenesis, we obtained pigs transgenic for a cone-dominant mutant gene described in a human cone dystrophy.Methods: Lentiviral vectors encoding the human double mutant GUCY2DE837D/R838S cDNA under the control of a region of the pig arrestin-3 promoter (Arr3) was produced and used for lentiviral-derived transgenesis in pigs. PCR-genotyping and southern blotting determined the genotype of pigs born after injection of the vector at the zygote stage. Retina function analysis was performed by ERG and behavioral tests at 11, 24 and 54 weeks of age. OCT and histological analyses were performed to describe the retina morphology.Results: The ratio of transgenic pigs born after lentiviral-directed transgenesis was close to 50%. Transgenic pigs with 3 to 5 transgene copies per cell clearly present a reduced photopic response from 3 months of age on. Except for one pig, which has 6 integrated transgene copies, no dramatic decrease in general mobility was observed even at 6 months of age. OCT examinations reveal no major changes in the ONL structure of the 6-months old pigs. The retina morphology was well conserved in the 2 pigs sacrificed (3 and 6 months old) except a noticeable displacement of some cone nuclei in the outer segment layer.Conclusions: Lentiviral-directed transgenesis is a rapid and straightforward method to engineer transgenic pigs. Some Arr3-GUCY2DE837D/R838S pigs show signs of retinal dysfunction but further work is needed to describe the progression of the disease in this model.

Generation of a tightly regulated all-cis beta cell-specific tetracycline-inducible vector.

Ability to induce protein expression at will in a cell is a powerful strategy used by scientists to better understand the function of a protein of interest. Various inducible systems have been designed in eukaryotic cells to achieve this goal. Most of them rely on two distinct vectors, one encoding a protein that can regulate transcription by binding a compound X, and one hosting the cDNA encoding the protein of interest placed downstream of promoter sequences that can bind the protein regulated by compound X (e.g., tetracycline, ecdysone). The commercially available systems are not designed to allow cell- or tissue-specific regulated expression. Additionally, although these systems can be used to generate stable clones that can be induced to express a given protein, extensive screening is often required to eliminate the clones that display poor induction or high basal levels. In the present report, we aimed to design a pancreatic beta cell-specific tetracycline-inducible system. Since the classical two-vector based tetracycline-inducible system proved to be unsatisfactory in our hands, a single vector was eventually designed that allowed tight beta cell-specific tetracycline induction in unselected cell populations.

eVOC: a controlled vocabulary for unifying gene expression data.

Expression data contribute significantly to the biological value of the sequenced human genome, providing extensive information about gene structure and the pattern of gene expression. ESTs, together with SAGE libraries and microarray experiment information, provide a broad and rich view of the transcriptome. However, it is difficult to perform large-scale expression mining of the data generated by these diverse experimental approaches. Not only is the data stored in disparate locations, but there is frequent ambiguity in the meaning of terms used to describe the source of the material used in the experiment. Untangling semantic differences between the data provided by different resources is therefore largely reliant on the domain knowledge of a human expert. We present here eVOC, a system which associates labelled target cDNAs for microarray experiments, or cDNA libraries and their associated transcripts with controlled terms in a set of hierarchical vocabularies. eVOC consists of four orthogonal controlled vocabularies suitable for describing the domains of human gene expression data including Anatomical System, Cell Type, Pathology and Developmental Stage. We have curated and annotated 7016 cDNA libraries represented in dbEST, as well as 104 SAGE libraries,with expression information,and provide this as an integrated, public resource that allows the linking of transcripts and libraries with expression terms. Both the vocabularies and the vocabulary-annotated libraries can be retrieved from http://www.sanbi.ac.za/evoc/. Several groups are involved in developing this resource with the aim of unifying transcript expression information.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

RPS4Y gene family evolution in primates

Background: The RPS4 gene codifies for ribosomal protein S4, a very well-conserved protein present in all kingdoms. In primates, RPS4 is codified by two functional genes located on both sex chromosomes: the RPS4X and RPS4Y genes. In humans, RPS4Y is duplicated and the Y chromosome therefore carries a third functional paralog: RPS4Y2, which presents a testis-specific expression pattern. Results: DNA sequence analysis of the intronic and cDNA regions of RPS4Y genes from species covering the entire primate phylogeny showed that the duplication event leading to the second Y-linked copy occurred after the divergence of New World monkeys, about 35 million years ago. Maximum likelihood analyses of the synonymous and non-synonymous substitutions revealed that positive selection was acting on RPS4Y2 gene in the human lineage, which represents the first evidence of positive selection on a ribosomal protein gene. Putative positive amino acid replacements affected the three domains of the protein: one of these changes is located in the KOW protein domain and affects the unique invariable position of this motif, and might thus have a dramatic effect on the protein function.Conclusion: Here, we shed new light on the evolutionary history of RPS4Y gene family, especially on that of RPS4Y2. The results point that the RPS4Y1 gene might be maintained to compensate gene dosage between sexes, while RPS4Y2 might have acquired a new function, at least in the lineage leading to humans.

Thermal evolution of gene expression profiles in Drosophila subobscura

Background: Despite its pervasiveness, the genetic basis of adaptation resulting in variation directly or indirectly related to temperature (climatic) gradients is poorly understood. By using 3-fold replicated laboratory thermal stocks covering much of the physiologically tolerable temperature range for the temperate (i.e., cold tolerant) species Drosophila subobscura we have assessed whole-genome transcriptional responses after three years of thermal adaptation, when the populations had already diverged for inversion frequencies, pre-adult life history components, and morphological traits. Total mRNA from each population was compared to a reference pool mRNA in a standard, highly replicated two-colour competitive hybridization experiment using cDNA microarrays.Results: A total of 306 (6.6%) cDNA clones were identified as 'differentially expressed' (following a false discovery rate correction) after contrasting the two furthest apart thermal selection regimes (i.e., 13°C vs . 22°C), also including four previously reported candidate genes for thermotolerance in Drosophila (Hsp26, Hsp68, Fst, and Treh). On the other hand, correlated patterns of gene expression were similar in cold- and warm-adapted populations. Analysis of functional categories defined by the Gene Ontology project point to an overrepresentation of genes involved in carbohydrate metabolism, nucleic acids metabolism and regulation of transcription among other categories. Although the location of differently expressed genes was approximately at random with respect to chromosomes, a physical mapping of 88 probes to the polytene chromosomes of D. subobscura has shown that a larger than expected number mapped inside inverted chromosomal segments.Conclusion: Our data suggest that a sizeable number of genes appear to be involved in thermal adaptation in Drosophila, with a substantial fraction implicated in metabolism. This apparently illustrates the formidable challenge to understanding the adaptive evolution of complex trait variation. Furthermore, some clustering of genes within inverted chromosomal sections was detected. Disentangling the effects of inversions will be obviously required in any future approach if we want to identify the relevant candidate genes.

Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics

Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.

Short-term oleoyl-estrone treatment affects capacity to manage lipids in rat adipose tissue

Background: Short-term OE (oleoyl-estrone) treatment causes significant decreases in rat weight mainly due to adipose tissue loss. The aim of this work was to determine if OE treatment affects the expression of genes that regulate lipid metabolism in white adipose tissue. Results: Gene expression in adipose tissue from female treated rats (48 hours) was analysed by hybridization to cDNA arrays and levels of specific mRNAs were determined by real-time PCR. Treatment with OE decreased the expression of 232 genes and up-regulated 75 other genes in mesenteric white adipose tissue. The use of real-time PCR validate that, in mesenteric white adipose tissue, mRNA levels for Lipoprotein Lipase (LPL) were decreased by 52%, those of Fatty Acid Synthase (FAS) by 95%, those of Hormone Sensible Lipase (HSL) by 32%, those of Acetyl CoA Carboxylase (ACC) by 92%, those of Carnitine Palmitoyltransferase 1b (CPT1b) by 45%, and those of Fatty Acid Transport Protein 1 (FATP1) and Adipocyte Fatty Acid Binding Protein (FABP4) by 52% and 49%, respectively. Conversely, Tumour Necrosis Factor (TNF¿) values showed overexpression (198%). Conclusion: Short-term treatment with OE affects adipose tissue capacity to extract fatty acids from lipoproteins and to deal with fatty acid transport and metabolism.