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.

<p>\abstract</p><p>Transcription and translation describe the flow of genetic information from DNA to mRNA to protein. Recent studies show that at a single cell level, these processes are stochastic, which results in the variation of the number of mRNA and proteins even under identical environmental conditions. Because the number of mRNA and protein in each single cell are actually very small, these variations can be crucial for cellular function in diverse contexts, such as development, stress response, immunological and nervous system function. Most studies examine the origin and effects of stochastic gene expression using computer simulations. My goal is to develop a theoretical framework to study activity-dependent gene expression using simplified models that capture essential features. </p><p>I have examined the dynamics of stochastic gene regulation in three contexts. First, I examine how fluctuations in promoter accessibility lead to "bursty" transcription, during which genes are turned "on" or "off" stochastically. I describe a mathematical formalism to represent bursty gene expression in a coarse-grained manner as a Markov process and derive a master equation for the time evolution of the probability distribution of the number of mRNA molecules. This allows us to examine how transcript number responds to time varying stimuli. This model forms a basic building block for understanding the signal transmission and noise of the transcription process to time varying inputs as would be sensed by cells in dynamic environments. In addition to synthesis, gene expression is subject to additional modes of regulation. One such mechanism that controls transcript numbers is by microRNAs (miRNAs), which pair with target mRNAs to repress protein production following transcription. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. I explore the functional roles of feedback regulation by miRNAs and show that protein fluctuations strongly depend on the mode of miRNA-mediated repression. I discuss the functional implications of protein fluctuations arising from miRNA-mediated repression on gene regulatory networks. Finally, I examine the impact of fluctuations on alternative splicing, which is a major source for proteomic complexity in higher eukaryotes. Although the proteins regulating alternative splicing have been extensively studied, little is known about how noise arising from the stochastic nature of alternative splicing contributes to the entire gene expression process. I explore the functional roles and noise properties of alternative splicing, focusing on the case of exon skipping and intron retention. I show that while the overall counts of the mRNAs of the two isoforms are independent and Poisson distributed, diffusion and binding of the splicing factors contributes to the variance in the abundance of the isoforms. </p><p>Noise in gene expression may be of particular relevance in the nervous system. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify neuronal excitability and connectivity, ultimately influencing higher order brain function. Finally, I examine the implications of our studies for activity dependent gene expression in the nervous system.</p> / Dissertation

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease which currently lacks rapid and definitive diagnostic tests. Recently identified neuron specific splice variant molecules, Per28 and NFL-60, have been shown to contain unique epitopes and to have altered levels of expression in ALS patients. It is believed that these factors make Per28 and NFL-60 excellent candidate biomarkers for the ALS disease state. In this study, we attempted to develop ELISA assays directed against Per28 and NFL-60, as well as a generalized guideline for splice variant ELISA development, which could be used in a clinical setting. Limitations in currently identified antibodies to the splice variants allowed only for the completion of a Per28 ELISA, which lacked the sensitivity for clinical relevance. This assay creation process, however, did produce a guideline for similar ELISA development, which should allow for the more expeditious creation future ELISA.

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Supplementary material to figures contained on DVD only available with manuscript. / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences, 2005. / Bibliography: p. 221-244. / Introduction -- Materials and methods -- Isolation of the hex1 gene from Trichoderma reesei and Ophiostoma floccosum -- Expression of DsRed under the cbh1 promoter and the hex1 promoter with random integration -- Modified expression vectors containing a fusion to a portion of hex1 gene sequence -- Expression of DsRed from the hex1 locus and the phenotypic characteristics of a hex1 deletion mutant -- Summary and concluding discussion. / For Trichoderma reesei to be developed as an effiecient producer of a large variety of proteins, the expression system requires diversification. In particular, the choice of promoters available needs to be broadened to include promoters which are active in conditions other than those conducive to induction of cellulase expression. Using proteomics, the HEX1 protein was identified as an abundant protein of the cell envelope of T. reesei when grown on a range of carbon sources, suggesting that a strong constitutive promoter drives the expression of this physiologically important protein. This thesis is an exploration into the hex1 gene promoter and the role of hex1 in the maintenance of mycelium integrity in T. reesei with consideration for the application of this gene in the further development of filamentous fungi as protein expression systems. -- The single copy hex1 gene and flanking regions were isolated from T. reesei and another biotechnologically important fungus, Ophiostoma floccosum. The fluorescent reporter protein DsRed1-E5 was expressed under the T. reesei hex1 promoter and promoter activity was monitored by fluorescence CLSM and RNA analysis. During the rapid growth phase of a culture, the hex1 promoter was active in a range of carbon sources and three transcipt types with alternative tsp and splicing sites were discovered for the hex1 gene. The distribution of fluorescence throughout the mycelium suggested spatial regulation of the hex1 promoter as well as temporal regulation. The promoter was continually active in the absence of a functional hex1 gene product suggesting that the hex1 promoter is regulated in part, by negative feedback from the endogenous gene product. Interruption of the hex1 gene produced hyphae that leaked excessive volumes of cytoplasm when physically damaged which may be advantageous for the externalisation of selected protein products. The results indicate that the regulation of the hex1 hene promoter is complex and that the hex1 gene is integral to the maintenance of the integrity of the fungal mycelium. / Mode of access: World Wide Web. / xv, 244 p. ill

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 215-225). Also available on the Internet.

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

Due to global warming, our planet will experience more extreme weather conditions. Plants can protect themselves against these abiotic stress conditions with their stress response, which includes alternative splicing of certain genes. Alternative splicing is a post-transcriptional process where a single gene gives rise to different mRNAs, which in turn produces different proteins. In plants, this is usually done by intron retention. One type of protein that may be involved in this stress response are the non-specific lipid transfer proteins (LTPs). Indeed, evidence of intron retention has been found in the LTP genes in the liverwort Marchantia polymorpha, called MpLTPd. To investigate whether this alternative splicing is caused by abiotic stress or not, I subjected the moss to two different types of stress trials, drought and cold, and compared the general expression of the intron in MpLTPd2 and MpLTPd3 from the stressed samples to samples from a moss grown under normal conditions. I found that the expression of the intron did change in the stressed moss, but none of the differences were significant. This suggests that alterative splicing in MpLTPd2 and MpLTPd3 is not caused by cold and drought and that the intron-containing protein plays no role in the protection of M. polymorpha against abiotic stress.

RNA splicing is a process where introns are removed and exons are joined together. Human adenovirus type 2 pre-mRNAs undergoes intensive alternative splicing and produce more than 40 differently spliced mRNAs.  This thesis work is focused on the identification of new splice sites in adenovirus. By virtue of Illumina mRNA sequencing technology we have identified 255 splice sites. Splice site analysis of the introns revealed the presence of three types of splice sites GT-AG (61.2%), GC-AG (25.9%) and AT-AC (12.9%). Among 255 splice sites, 224 were new. Significantly, more than 50% of the new splice sites were located in the major late transcription unit on the positive strand of adenovirus DNA. Three new splice sites; 17452-29489 (GC-AG) located on the negative strand of adenovirus DNA in the E2 region, 9668-20346 (AT-AC) and 9699-30505 (GC-AG) on the positive strand of adenovirus DNA in the major late transcription unit were further confirmed by PCR analysis. / Adenovirus replication and transcriptome

Le virus de la maladie de Marek (MDV) est un virus oncogène responsable des lymphomes T chez les poulets. L´infection par ce virus est divisée en une phase lytique dépendante de l´expression du gène très précoce ICP4 et une phase latente, caractérisée par l´expression de l’ARN long non codant LAT localisé en antisens. Nous avons montré que l’expression différentielle des miARN du cluster mdv1-miR-M8-M10 était directement corrélée à l’épissage alternatif de l’intron 1 du LAT et plus particulièrement à la biogenèse par le splicéosome du premier mirtron viral. La présence du mirtron mdv1-miR-M6 au milieu du cluster est associée à une cinétique d’expression des miARN. En parallèle, nous avons identifié deux promoteurs alternatifs de type Sp1, quatre signaux poly-A et trois exons associés à la régulation de la transcription du transcrit ICP4. Nous avons prédits cinq isoformes potentielles pour la protéine ICP4 et avons pu observer par immunodétection que la protéine était exprimée principalement dans le cytoplasme des cellules infectées en phase lytique ou de réactivation. / The Marek disease virus (MDV) is an oncogenic herpesvirus responsible of T-cell lymphoma in chicken. MDV infections are divided into a lytic phase, depending on the expression of immediate early gene like ICP4, and a latent phase characterized by the expression of the long non-coding RNA LAT localized in antisense. In this study, we have shown the differential expression of the cluster of miRNA mdv1-miR-M8-M10 was directly correlated with the alternative splicing of LAT’s intron 1 and more specifically with the first viral mirtron biogenesis by the spliceosome. The location of the mirtron mdv1-miR-M6 inside of the cluster is associated with a two-step biogenesis of the miARN of the cluster. On the other hand, we have identified a dual promoter that responded to Sp1, four poly-A signals and three exons that are responsible of transcriptional regulation of ICP4 transcript. We also have predicted five potential isoproteines for ICP4 and were able to observe by immunodetection that ICP4 was mainly expressed in the cytoplasm of infected cells during the lytic phase or the reactivation one.