Functional DTH expression from undriven UAS-DTHg. Emma J. Garren, Karol Cichewicz, Jay Hirsh. Biology, University of Virginia, Charlottesville, VA.

   Dopamine (DA) biosynthesis is regulated by the rate limiting enzyme tyrosine hydroxylase (DTH, ple). Our lab has created a Drosophila genetic background lacking DA in the CNS. The approach uses a large genomic BAC clone containing a DTH gene modified to selectively express in the hypoderm, rescuing ple² lethality but not CNS DA (see Cichewicz et al. poster). This background allows for further genetic manipulations using the Gal4-UAS binary expression system to study the role of DA modulation in circadian behavior. The intended approach relies on cell-specific rescue of DA using selective Gal4 drivers to express UAS-DTHg, genomic DTH. Unexpectedly, undriven UAS-DTHg is sufficient for DTH expression in a pattern similar to that observed with TH-Gal4. Immunostaining revealed DTH expression at a level much lower than expression driven by TH-Gal4 or in wild type. However, HPLC measurement of DA in whole brain extracts shows normal levels of DA, indicating that post-translational regulation of DTH can compensate for reduced DTH protein expression. Furthermore, circadian period phenotypes of the undriven UAS-DTHg in the DA-deficient background are the same as TH-Gal4-driven (see Cichewicz et al. poster). We are testing DTH derivatives lacking putative regulatory elements to allow our initial approach to succeed.