The overall goal of our proposed work is to understand the genetic and epigenetic strategies as well as the molecular mechanisms used to regulate expression of neurotransmitter specific genes. Appropriate expression of neurotransmitter specific genes determines the phenotypic properties of a neuron as well as its functional state and thus underlies the formation and operation of specific neuronal circuits. Inappropriate expression will result in defective neurotransmission and may contribute to the etiology or pathology of neurotransmitter related diseases. it will be essential to have precise gene regulatory information to properly evaluate the contribution of inappropriate expression to disease states and also to design effective gene therapeutic approaches to treat neurotransmitter related diseases. Our recent work has provided the tools, uncovered some of the strategies and begun to address the mechanisms regulating expression of choline acetyltransferase (ChAT; EC2,3,1,6), the biosynthetic enzyme for acetylcholine production. The ChAT gene (Cha) has recently been discovered to be part of a more complex genetic locus also coding for the vesicular acetylcholine transporter (VAChT) which packages transmitter into synaptic vesicles. The genomic organization of these two distinct but related genetic functions is unique and has been conserved from Drosophila to humans. our proposed studies will attempt to define the significance of this unique genomic organization for the cholinergic locus using Drosophila as a model system. We plan continued identification and analysis of transcriptional control elements and cognate transcription factors regulating expression of the cholinergic locus using P-element """"""""rescue"""""""" of mutant phenotypes and biochemical experiments. We will confirm the importance of pdm1/dPOU19 as a key transcriptional regulator of the cholinergic locus, analyze peripheral nervous system regulatory motifs and attempt to define the combination of DNA regulatory elements which are both necessary and sufficient for cholinergic expression. Our preliminary results indicate that cholinergic specificity may be determined by an accessory factor and we thus plan to identify and clone the putative cholinergic co-activator of pdm1/dPOU19. We will also determine the extent and nature of co-ordinate regulation between ChAT and VAChT using immunocytochemistry, in situ hybridization and quantitative methods on wild-type and mutant Drosophila. New aspects of cholinergic locus regulation will be investigated by analyzing suppressor of Cha15 mutations by genetic, transgenic and biochemical methods. We also plan to investigate post-transcriptional regulatory mechanisms for ChAT and VAChT by focusing on translational control mechanisms. Our final goal is to determine the consequences of altered cholinergic gene expression on higher ordered visual behaviors and learning and memory tasks using P-element transformants with abnormal ChAT mini-gene expression patterns.
| Munoz-Maines, V J; Slemmon, J R; Panicker, M M et al. (1988) Production of polyclonal antisera to choline acetyltransferase using a fusion protein produced by a cDNA clone. J Neurochem 50:167-75 |
