Nous montrons l’utilisation de la puce exon d’Affymetrix pour l’analyse simultanée de l’expression des gènes et de la variation d’isoformes. Nous avons utilisé les échantillons d’ARN du cerveau et des tissus de référence qui ont été antérieurement utilisés dans l’étude du consortium MicroArray Quality Control (MAQC). Nous démontrons une forte concordance de la quantification de l’expression des gènes entre trois plateformes d’expression populaires à savoir la puce exon d’Affymetrix, la puce Illumina et la puce U133A d’Affymetrix. Plus intéressant nous montrons que la majorité des discordances entre les trois plateformes résulterait des positions différentes des sondes à travers les plateformes et que les variations d’isoforme exactes ne peuvent être identifiées que par la puce exon. Nous avons détecté avec succès, entre les tissus de référence et ceux du cerveau, une centaine de cas d’évènements d’épissage alternatif. La puce exon est requise dans l’analyse de l’épissage alternatif associé aux pathologies telles que les cancers et les troubles neurologiques. Comme application de cette technologie, nous avons analysé les variations d’épissage dans la métastase du cancer de sein développé dans le model de la souris. Nous avons utilisé une gamme bien définie de trois lignées de tumeur mammaire ayant différents potentiels métastatiques. Par des analyses statistiques, nous avons répertorié 2623 transcripts présentant des variations d’expression et d’isoformes entre les types de tumeur. Une analyse du réseau de gènes montre qu’environ la moitié d’entre eux est impliquée dans plusieurs activités cellulaires, ainsi que dans nombreux cancers et désordres génétiques. / We demonstrate how the Affymetrix Exon Array, can be used to simultaneously profile gene expression level, and detect variations at the isoform level. We use a well studied set of brain and reference RNA samples previously used by the MicroArray Quality Control (MAQC) consortium study. We demonstrate a high concordance of gene expression measurements among three popular expression platforms – Affymetrix Exon Array, Illumina, and Affymetrix 3’ targeted array (U133A). More interestingly, we show that in many cases of discordant results, the effect can be explained by differential probe placements across platforms, and that the exact isoform change can only be captured by the Exon Array. Finally, we are able to detect hundreds of cases of splicing, transcript initiation, and termination differences between the brain and reference tissue samples. We propose that the Exon Array is a highly effective tool for transcript isoform profiling, and that it should be used in a variety of systems where such changes are known to be associated with diseases, such as neurological disorders and cancer. As application, we used the Affymetrix Exon Array to identify metastatis-specific alternative splicing in mouse model of breast cancer at the whole genome level. We utilize a well characterized series of three mouse mammary tumor lines exhibiting varying levels of metastatic potential. We catalogued 2623 transcripts which exhibit splicing aberrations during the progression of cancer. A genetic pathway analysis shows the half of them implicated in several cell activities, cancers and genetic disorders.

La thrombasthénie de Glanzmann (TG) est une maladie caractérisée par un défaut d’agrégation plaquettaire. C’est une maladie génétique autosomale récessive causée par une anomalie du récepteur plaquettaire pour le fibrinogène. Ce récepteur est une intégrine localisée à la surface plasmatique qui est formée par un complexe composé des sous‐unités αIIb et β3. Nous avons identifié un cheval démontrant les caractéristiques clinicopathologiques de la TG. Des études par cytométrie de flux ont révélé une déficience au niveau de la portion αIIb du récepteur. Ces résultats suggèrent une ou plusieurs mutations au niveau du gène codant pour cette portion αIIb du récepteur. L’objectif de notre étude était de caractériser l’ADNc et l’ADN génomique codant pour les gènes ITGA2B et ITGB3 codant respectivement pour les deux sous‐unités αIIb et β3 chez un cheval atteint de la TG. L’ADNc a été synthétisé par RT‐PCR en utilisant l’ARN total récolté à partir des plaquettes. L’ADN génomique a été extrait à partir des globules blancs. Des amorces spécifiques ont été utilisées pour l’amplification par PCR d’ITGA2B et d’ITGB3. Les séquences d’ADNc et d’ADN génomique de notre patient ont été caractérisées par séquençage et comparées par l’analyse BLAST (GenBank). Une substitution d’une guanine par une cytosine a été mise en évidence au niveau de l’exon 2 d’ITGA2B amenant à la substitution d’une arginine (Arg72) par une proline (Pro72). Ce changement d’acide aminé pourrait résulter en une conformation structurelle anormale qui amènerait à une sous‐unité αIIb inactive. L’analyse de l’ADN génomique a démontré que ce cheval était homozygote pour cette mutation. Le séquençage de l’ADN génomique des parents et de la grand‐mère du patient a démontré que ces individus étaient hétérozygotes pour cette mutation. Le séquençage d’ITGB3 n’a démontré aucune anomalie. / Glanzmann thrombasthenia (GT) is characterized by a defect of platelet aggregation. This autosomal recessive genetic disorder is caused by an abnormality of the platelet receptor for fibrinogen. This receptor is an integrin located on the plasma membrane formed by a complex of αIIb‐β3 subunits. Recently, we identified a horse with clinical and pathological features of GT. Flow cytometry studies revealed a deficiency of the αIIb subunit suggesting the presence of one or several mutations in the gene encoding for the αIIb subunit. The aim of this study was to describe this case of GT at the molecular level by characterizing the cDNAs encoding for the β3 (ITGB3) and αIIb (ITGA2B) subunits. Total RNA was extracted from platelets and converted into cDNA by reverse transcription using a poly‐dT primer. Genomic DNA was extracted from white blood cells. Specific primers for αIIb and β3 sequences were used to amplify by PCR the corresponding cDNA or genomic regions that were further characterized by sequencing and compared by BLAST analysis (GenBank). A point mutation from G to C in exon 2 of ITGA2B was identified, causing a substitution of the expected amino acid arginine 72 (Arg72) by a proline (Pro72). This amino acid change may result in abnormal structural conformations that yield an inactive αIIb subunit. The analysis of genomic DNA showed that this horse was homozygous for the missense mutation. The genomic DNA sequences encoding exon 2 of the dam, grand dam and the sire were heterozygous for this nucleic acid change and were clinically normal. The analysis of ITGB3 was unremarkable.

No description available.

Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.

<p>The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo / genease&rdquo / activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001 / van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.</p> <p><br /> Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding /intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons.</p> <p><br /> Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.</p>

<p>The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.</p> / Dissertation

O sistema complemento participa da imunidade natural e da imunidade adquirida. Este sistema pode ser ativado por três diferentes vias: a via clássica, a via alternativa e a via das lectinas, desencadeando uma cascata proteolítica que irá resultar na possível eliminação de microorganismos e, por sua vez, no restabelecimento da homeostasia do indivíduo. A proteína C3 possui um papel central participando das três vias deste sistema. Sua clivagem gera fragmentos que estão associados a várias funções biológicas como opsonização, quimiotaxia, anafilatoxinas, além da formação da C3-convertases, que amplificam a via de ativação efetora contra patógenos. A completa deficiência de C3 é rara, está comumente associada a repetidas infecções, ao desenvolvimento de doenças mediadas por imunocomplexos e glomerulonefrite. Neste trabalho nós investigamos as bases moleculares desta deficiência em uma paciente com histórico de consanguinidade. Nosso estudo identificou uma mutação genética responsável pela completa deleção do éxon 27, implicando na perda de 99 nucleotídeos (região 3450 até 3549 correspondente ao cDNA do C3). O sequenciamento do gene C3 da região 6690313 até 6690961 mostrou uma troca de nucleotídeos T por um C (T &rarr; C) na posição 6690626, causando a exclusão do o éxon 27 e deficiência da proteína C3 do complemento humano nessa paciente. Também confirmamos o padrão de herança autossômica recessiva desta deficiência. Verificamos ainda que os fibloblastos da paciente estimulados com LPS por 24 h foram incapazes de secretar a proteína C3, o que nos leva a suspeitar que esta proteína mutante seja rapidamente degradada ainda dentro da célula, impedindo sua liberação para o meio extracelular. / The complement system participates in the natural immunity and acquired immunity. This system can be activated by three different pathways: the classical pathway, the alternative pathway and the lectin pathway, triggering a proteolytic cascade that will possibly result in the elimination of microorganisms and, in turn, in restoring the homeostasis of the individual. The C3 protein has a central role in the activation of all pathways. Its cleavage produces fragments that are associated with various biological functions such as opsonization, chemotaxis, and production of anaphylatoxins. In addition, the C3 participates in formation of C3 convertase of the alternative pathway which amplifies the activation of effector against pathogens. The complete C3 deficiency is rare and commonly associated with recurrent infections, development of diseases mediated by immune complexes and glomerulonephritis. In this work, we investigated the molecular basis of this deficiency in a patient family with a history of consanguinity. Our study has identified a genetic mutation responsible for the complete deletion of exon 27, resulting in the loss of 99 nucleotides (region 3450 to 3549 of the cDNA corresponding to C3). The sequencing of the C3 region 6690313 to 6690961 showed a T nucleotide exchange to C (T &rarr; C) at position 6690626 causing deletion of exon 27 leading to deficiency of the human complement protein C3 in this patient. We also confirmed the autosomal recessive patters of this patient deficiency in the family. We observed that patient fibroblasts stimulated with LPS for 24 h were unable to secrete C3, which leads us to suspect that this protein is degraded within the cell preventing its release into the extracellular.

<p>Alternativ splicing är en process som ger upphov till att olika mRNA-sekvenser bildas från en enda gen, vilket bidrar till en ökad proteindiversitet hos organismen. Olika mRNA-sekvenser kan uppstå eftersom att det förekommer olika varianter av alternativ splicing som även kan kombineras på flera olika sätt: cassette exon (inkludering/exkludering av exon), intron retention (intronet behålls), alternative 5´splice-site choice (olika 5´ splice sites kan väljas) och slutligen alternative 3´ splice-site choice (andra 3´ splice sites kan väljas). För att alternativ splicing ska äga rum i olika pre-mRNA måste den regleras av cis-reglerande element. De cis-reglerande elementen utgörs av fyra grupper: exonic splicing enhancers (ESE), exonic splicing silencers (ESS), intronic splicing enhancers (ISE) samt intronic splicing silencers (ISS). Som namnen förtäljer finns de antingen i exoner eller introner, där de interagerar med transagerande faktorer, SR-proteiner (aktiverare) eller hnRNPs (hämmare). Alternativ splicing förekommer både i djur och i växter. Hos <em>Homo sapiens </em>genomgår över 74 % av de 25,000 gener som finns hos organismen, alternativ splicing. Däremot i växten <em>Arabidopsis thaliana</em>, genomgår endast 22 %, av den totala mängden på cirka 26,000 gener, alternativ splicing. Eftersom att processen bidrar till en ökad proteindiversitet, kommer det medföra att olika processer i organismerna påverkas, exempelvis celltillväxt, celldöd samt utvecklingen av olika sjukdomar, såsom Parkinson och cystisk fibros. Många studier har gjorts som bekräftar dess betydelse för organismerna men på grund av processens komplexitet är det fortfarande ett ämne som ständigt måste utforskas.</p><p> </p>

Alternativ splicing är en process som ger upphov till att olika mRNA-sekvenser bildas från en enda gen, vilket bidrar till en ökad proteindiversitet hos organismen. Olika mRNA-sekvenser kan uppstå eftersom att det förekommer olika varianter av alternativ splicing som även kan kombineras på flera olika sätt: cassette exon (inkludering/exkludering av exon), intron retention (intronet behålls), alternative 5´splice-site choice (olika 5´ splice sites kan väljas) och slutligen alternative 3´ splice-site choice (andra 3´ splice sites kan väljas). För att alternativ splicing ska äga rum i olika pre-mRNA måste den regleras av cis-reglerande element. De cis-reglerande elementen utgörs av fyra grupper: exonic splicing enhancers (ESE), exonic splicing silencers (ESS), intronic splicing enhancers (ISE) samt intronic splicing silencers (ISS). Som namnen förtäljer finns de antingen i exoner eller introner, där de interagerar med transagerande faktorer, SR-proteiner (aktiverare) eller hnRNPs (hämmare). Alternativ splicing förekommer både i djur och i växter. Hos Homo sapiens genomgår över 74 % av de 25,000 gener som finns hos organismen, alternativ splicing. Däremot i växten Arabidopsis thaliana, genomgår endast 22 %, av den totala mängden på cirka 26,000 gener, alternativ splicing. Eftersom att processen bidrar till en ökad proteindiversitet, kommer det medföra att olika processer i organismerna påverkas, exempelvis celltillväxt, celldöd samt utvecklingen av olika sjukdomar, såsom Parkinson och cystisk fibros. Många studier har gjorts som bekräftar dess betydelse för organismerna men på grund av processens komplexitet är det fortfarande ett ämne som ständigt måste utforskas.

Exons are expression DNA sequences. A DNA sequence which includes gene has exons and introns. During transcription and translation, introns will be removed, and exons will remain to become protein. Many researchers need exon primers for PCR experiments. However, it is a difficult to find that many exon primers satisfy all primer design constraints at the same time. Here, we proposed an efficient exon primer design algorithm. The algorithm applies multiobjective genetic algorithm (MGA) instead of the single objective algorithm which can easily lend to unsuitable solutions. And a hash-index algorithm is applied to make specificity checking in a reasonable time. The algorithm has tested by a variety of mRNA sequences. These dry dock experiments show that our proposed algorithm can find primers which satisfy all exon primer design constraints.