Drosophila Genes Associated With Mushroom Body Enhancers

Mounsey, Andrew (1997) Drosophila Genes Associated With Mushroom Body Enhancers. PhD thesis, University of Glasgow.

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The mushroom bodies of Drosophila melanogaster are discrete substructures of the fly brain that have been implicated in mediating the processes that underlie learning and memory. Specific chemical ablation of the mushroom bodies greatly reduces flies' performance in associative learning tasks. The two best characterised mutants deficient in learning and memory are dunce and rutabaga. Both have preferential expression in the mushroom bodies, and have helped elucidate the biochemical mechanisms underpinning learning in Drosophila. Identification of other genes with mushroom body preferential, or specific, expression will be necessary to understand other aspects of these biochemical mechanisms. Enhancer-traps are engineered P-element transposons that carry a reporter gene under the control of a minimal promoter. Genomic insertion of the enhancer-trap in the vicinity of an enhancer allows the expression of the reporter gene, and the identification of tissue specific expression patterns. Genes flanking the enhancer-trap may have a similar tissue specific expression pattern. Twenty-four fly lines, with enhancer-trap expression in the mushroom bodies, were isolated from a screen of 1400 fly lines containing a single copy of a P1GAL4J enhancer-trap (Yang, 1995, Armstrong, 1995). These twenty-four fly lines were made available to me for further study. Initially, genomic DNA flanking the enhancer-traps was acquired by plasmid-rescue, and analysed by reverse Northern, using probes copied from fly head or body derived mRNA. Many of the rescued-plasmids were shown to contain transcriptional units, some of which clearly had head elevated expression. Two fly lines, 201Y and 43Y, were selected for further detailed study. The genes adjacent to the 201Y and 43Y enhancer-traps were identified by screening cDNA libraries with probes derived from rescued-plasmids or genomic DNA clones, and subjecting isolated cDNA clones to sequencing analysis. The genes flanking the 201Y enhancer-trap are the previously uncharacterised Drosophila homologue of 12kDa FK506 binding proteins (FKBP12), and a new gene (known as A421) with no significant homology to other genes in the DNA sequence databases. Northern analysis revealed that the FKBP12 gene is expressed at all developmental stages, but it was not determined if it has elevated expression in the mushroom bodies. Conceptual translation of the largest A421 ORF gives a protein of 510 aa, that has a glutamine-rich region, a potential leucine-zipper (of novel structure), and a new zinc-finger-like motif of the form WXCX2-4CX3N6CX2CX3-4R/K. This previously undescribed zinc-finger-like motif was identified in twenty other proteins of diverse functionality, and may play a role in mediating protein-protein interactions. Northern analysis indicates that the A421 gene produces four transcripts, two of which clearly have elevated expression in fly heads. Furthermore, in situ hybridisation of DIG labelled cDNA probes to fly head sections, suggests that the A421 gene is expressed in neurons of the fly brain, but not with elevated or specific expression in the mushroom bodies. The genes flanking the 43Y enhancer-trap are ultraspiracle and alpha-actinin. Both of these genes have been previously cloned and characterised in Drosophila. The ultraspiracle gene product has homology to the retinoid X receptor class of proteins, and interacts with the ecdysone receptor to produce a DNA binding heterodimer that mediates ecdysteroid-induced gene expression (Yao et al., 1993). Immuno- histochemical analysis indicates that the ultraspiracle gene product is present in all neurons of the fly CNS, with no noticeable concentration in the mushroom bodies, alpha-Actinin is an actin cross-linking protein commonly associated with the Z-discs of muscle fibres (Blanchard et al., 1989). However, immunohistochemistry, using an antibody to waterbug alpha-actinin, suggests that alpha-actinin is preferentially expressed in the mushroom bodies. alpha-Actinin is an integral constituent of the the neuronal cytoskeleton, and may play a role in the mushroom bodies by mediating neuronal plasticity, alpha-Actinin is thought to link NMDA receptors and integrins to the neuronal cytoskeleton and may function in mushroom bodies by regulating the activity of these molecules.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75890
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:39
Last Modified: 19 Nov 2019 17:39
URI: http://theses.gla.ac.uk/id/eprint/75890

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