Genetics - Theses
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ItemExpression and function of five ligand-gated chloride channel genes of Drosophila melanogaster( 2012)Annotation of the genome of the vinegar fly, Drosophila melanogaster, has identified 12 ligand-gated chloride ion channel (LCCH) subunit genes. The ligands for the receptors produced from these LCCH subunit genes are the neurotransmitters GABA (γ amino butyric acid), glutamate, histamine and glycine. The ligands of some subunits are yet to be discovered. The GABA-gated LCCH subunits have sequence homology with vertebrate GABAA receptors that have vital functions in nervous system development and neurotransmission. The Rdl gene of D. melanogaster encodes a LCCH subunit. This gene came to prominence when it was found that a naturally occurring allele, in D. melanogaster and many insect pest species, conferred high level target site resistance to the insecticide dieldrin. Subsequently the Rdl (Resistance to DieLdrin) gene and its product (RDL) have been intensively investigated. The Rdl gene has been associated with sleep, learning and memory, epileptic seizure and phenotypes due to defects in Drosophila Fragile X mental retardation gene function. There are four other D. melanogaster genes that been reported to encode subunits that assemble into GABA-gated cation channels (Lcch3 and Grd) or have the GABA-binding consensus sequences found in vertebrate GABA receptor subunits (CG8916 and CG12344). These other genes are also likely to play important functional roles in neurotransmission and/or development but, to date, there has been a striking lack of research focused on elucidating their functions. In this thesis two approaches are combined to examine the function of these genes. In the first of these, the expression of the Rdl, Lcch3, Grd, CG8916 and CG12344 genes is examined using in situ hybridization. Further, the cloned endogenous promoters for the five genes are used to drive the expression of the green fluorescent protein (GFP) using the GAL4/UAS system (Chapter 2). The spatial and temporal expression patterns for these genes provide some clues to their function. The second approach uses RNA interference to knockdown gene expression to generate mutant phenotypes indicative of gene function (Chapter 3). A number of phenotypes are described here – defects in larval forward locomotion and wing inflation, necrotic tissues and lethality. The links between these phenotypes and the function of the five genes are carefully considered. While it is not the major focus of this thesis, the applications of the research described are considered. The RDL protein has been targeted with a range of insecticides. This thesis identifies two other GABA-gated cation channels (GRD and LCCH3) as potential insecticide targets. Given the similarities between the five GABA-gated LCCH subunits and their human counterparts, some fruitful lines of research that may positively impact the further study of human biology and health are identified. Most importantly, the research reported here provides a foundation of knowledge and reagents that will be of great value in an ongoing systematic analysis of the role that these five GABA-gated LCCH subunits play in insect development and neurotransmission.