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Study of abd-A regulation by the iab-8 noncoding RNA using recombineering techniques

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Denomination Master en Biologie orientation Génétique, Développement et Evolution
Defense Maîtrise : Univ. Genève, 2014
Abstract The homeotic genes (or Hox genes) are responsible for the determination of the anteriorposterior axis during embryogenesis in Metazoan animals. In Drosophila, there are eight homeotic genes, clustered into two groups: the Antennapedia Complex (AP-C) and the Bithorax Complex (BX-C). In the BX-C we find three Hox genes: Ultrabithorax, abdominal-A and Abdominal-B. The BX-C also includes a long non-coding RNA of 92kb, the iab-8 ncRNA, which represses the expression of abdominal-A in the central nervous system of parasegment 13 in the embryo. This repression is exerted by a microRNA formed from this non-coding RNA, and by a cis-acting mechanism that seems to be transcriptional interference. The iab-8 ncRNA seems to have a high biological importance, as the disruption of its function causes sterility. During this project we have tested the conservation of the iab-8 non-coding RNA between three species of the family Drosophilidae (Drosophila melanogaster, Drosophila pseudoobscura and Drosophila virilis). In order to do this, we have performed RNA in situ hybridizations against embryos of these species, using RNA probes with similar sequences to the iab-8 ncRNA sequence in D. melanogaster. We have proven that the iab-8 ncRNA is expressed in a similar way in these three species, and that its transcriptional unit seems to have a similar length. We also wanted to provide evidence to the hypothesis of transcriptional interference being a regulatory mechanism of abd-A. To fulfill this objective, we have designed a genetically engineered reporter system, which should reproduce the interaction between the iab-8 ncRNA and abd-A. This system is based on the construction of two different reporter Drosophila lines, that can be later combined using Flp/FRT recombineering, recreating a mini-iab-8 noncoding RNA expressed upstream of a tagged version of abdominal-A. In addition, each construct can be used independently as a reporter for studying further the role of these two genes.
Keywords Homeotic genesBithoraxIab-8 ncRNAAbdominal-ATranscriptional interferenceMicroRNADrosophila melanogasterDrosophila pseudoobscuraDrosophila virilisRecombineeringGap repair
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CASTRO ALVAREZ, Javier. Study of abd-A regulation by the iab-8 noncoding RNA using recombineering techniques. Université de Genève. Maîtrise, 2014. https://archive-ouverte.unige.ch/unige:42311

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Deposited on : 2014-12-01

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