The proposed research is a continuation of our longstanding studies on the structure and function of the cholinesterases. Since the primary and secondary structures of the enzyme were delineated and organization of the gene described, studies will now be directed to the assignment of functional residues or domains important for catalysis, inhibitor binding and governing the cellular localization of the enzyme. In addition, we plan to examine regions within the gene critical for regulating transcription, mRNA stability and alternative mRNA splicing patterns specific to particular tissues. As in the past, our experimental strategy will emphasize structural considerations so that regulatory phenomena can be related to gene and protein structure. To these ends, we will complete our analysis of the Torpedo genome and from the genomic sequence, recombinant constructs will be developed to study transcriptal and regulation in vitro. In addition, mutagenesis studies will be undertaken on both genomic and cDNA clones in order to assign residues critical for catalysis, peripheral and active center inhibitor binding, the cellular localization of the enzyme and differential expression of mRNA species. Studies will then progress from Torpedo to mouse where we will complete our cloning of the mouse acetyl- and butyrylcholinesterase genes. In particular, we hope to characterize the 5' noncoding and other regulatory regions of the gene to define cis elements and trans acting factors which control gene expression. Such studies will involve transfection into mouse cell lines to define a molecular basis for expression of cholinergic proteins and the factors which control alternative mRNA processing of the gene. Quite apart from the importance of acetylcholinesterase in somatic motor, central cholinergic and autonomic neurotransmission, the control of expression of protein in the cholinergic nervous system carries important implications in degenerative neurologic diseases such as Alzheimer's disease.
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