The cis-acting sequences controlling developmental stage and tissue specific expression of the Drosophila dopa decarboxylase (Ddc) gene will be studied by in vitro mutagenesis of cloned copies of the gene coupled with in vivo analysis of the resulting phenotypes after reintegration of the genes into the Drosophila genome. The enzyme encoded by this locus is required for the proper hardening of the exoskeleton at various reproducible stages during development and for neurotransmitter biosynthesis. Previous work has demonstrated that clone copies of Ddc are correctly regulated when reintegrated into the Drosophila genome and in vitro mutagenesis experiments have uncovered some of the cis-acting control elements essential for proper regulation. The large amount of genetic and molecular information already known about Ddc make it a model system for studying the mechanisms by which higher eukaryotic genes are regulated. The proposed experiments will further define the properties of the individual elements and probe just how those elements interact in regulating the gene. We will investigate how the two known central nervous systems specific elements interact with the other controls; our data suggest that they do so in conjunction with previously unknown control elements as well as sequences previously thought to be involved only in hypodermal (exoskeleton) expression. We will use similar techniques to probe the spatial and functional interactions between the two major hypodermal elements and to determine whether or not the Ddc controls act as transcriptional enhancer. These experiments will help us elucidate the regulatory circuitry that controls this gene. The regulation of gene expression in eukaryotic cells is complex. Some factors have been discovered and investigated but it is clear that for genes which must be expressed at different times in development and whose product is needed for different functions, the regulatory circuitry can be complex. Drosophila is one of the most powerful experimental organisms available for dissecting this circuitry.
