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Title: A reverse genetics approach to Drosophila learning and memory
Author: Goodwin, Stephen F.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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A reverse genetics approach termed "site-selected" transposon mutagenesis (SSM) has been developed in Drosophila, which allows the detection and isolation of a P-element transposon into or near a cloned gene of interest (Kaiser and Goodwin, 1990; Kaiser, 1990). This approach exploits the mutagenic properties of strains carrying multiple P-elements (Robertson, 1988) and the polymerase chain reaction (PCR; Saiki et al., 1988). In a model system, SSM has been used successfully to isolate three independent P-element insertions in the singed gene, a known 'hot-spot' for P-element insertion, and six independent P-element insertions in the ductin gene, encoding the 16 kDa proteolipid subunit of the vacuolar H+-ATPase. Furthermore, SSM has been used to identify two lines (RI715 and RI11D4) containing P-element insertions in the regulatory (RI) subunit of Drosophila cAMP-dependent protein kinase (PKA; Kalderon and Rubin, 1988). Both lines have a defective P-element insertion within a 26 by region containing multiple transcriptional start sites. Both lines were homozygous viable, presenting no obvious phenotype. Although the two mutant lines (RI7I5 and RI11D4) were generated independently, they both have insertions identical in size, insertion site, and orientation and I consider them to be functionally equivalent. Northern analysis of mRNA produced by these lines reveals that RI transcription is disrupted relative to wild-type. A number of single-gene defects have been isolated that perturb associative and nonassociative learning processes in Drosophila. The two most studied mutants are dunce (dnc), which encodes the cAMP-specific phosphodiesterase II and rutabaga (rut), which encodes a Ca2+/Calmodulin-activated adenylate cyclase. These findings implicated the cAMP signal transduction pathway in the neuromodulatory mechanisms underlying Drosophila learning and memory. In order to test the part played by PKA in learning and memory, both lines were isogenised with a high learning index wild-type strain and tested in a classical olfactory conditioning paradigm (Tully and Quinn, 1985). They displayed a significant initial learning decrement with respect to wild-type. Preliminary experiments in a nonassociative learning paradigm, habituation of the jump reflex to olfactory cues (McKenna et al., 1989), suggested that waning of the jump reflex was normal. However, spontaneous recovery was higher than normal wild-type controls. In situ hybridisation to sections of wild-type adult heads showed the RI gene to be expressed throughout the CNS, but to be prominent in the mushroom bodies, supporting the theory that mushroom bodies are the chief sites mediating olfactory learning and memory (Heisenberg, 1985). Although a previous study involving expression of a peptide inhibitor of PKA was the first direct role for PKA in Drosophila learning (Drain et al., 1991), mutational studies provide the only way to investigate the relative roles of different isoforms of the regulatory and catalytic subunits.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available