E. Hare and C. M. Loer.* Dept. of Biology, University of San Diego, CA 92110.
We are attempting to identify mechanisms by which neurons choose a neurotransmitter during development. We are currently characterizing genes used by the serotonergic neurons of the nematode C. elegans to learn how they are regulated. Worms with mutations in the bas-1 gene (biogenic amine synthesis abnormal) are serotonin- and dopamine-deficient. Wildtype serotonin can be restored by the application of exogenous serotonin, but not its immediate precursor 5-HTP; this phenotype is consistent with the loss of serotonin-synthetic AAADC activity. We have previously shown that wildtype serotonin immunofluroescence can be rescued in bas-1 mutants by injection of a 15.1 kb genomic clone (C05D2XN) containing two adjacent AAADC homologous sequences. The genomic structure of the region suggests the two AAADC sequences (called C05D2.4 and C05D2.3) will be transcribed together as an operon (e.g., the two coding regions are less than 400 bp apart). The pair of sequences must be from an ancient gene duplication since the predicted amino acid sequences are only 66% identical. Interestingly, the second sequence (C05D2.3) is missing a highly conserved segment of protein, suggesting it may not be able to function as an AAADC. The existence of a partial cDNA for C05D2.3, however, does demonstrate the gene is expressed. We are currently attempting to determine which of the two predicted sequences is necessary for serotonin synthesis and how the AAADC gene is expressed and regulated. Two bas-1 mutant alleles sequenced to date contain point mutations in C05D2.4 coding sequence resulting in premature stop codons. Furthermore, a construct in which C05D2.4 is mutated does not rescue bas-1 mutants whereas a construct mutated in C05D2.3 does rescue bas-1 mutants. Our preliminary results strongly suggest that C05D2.4 constitutes the bas-1 gene; we are still attempting to determine what role C05D2.3 might play. This work supported by the Fletcher Jones Foundation and an NIH (AREA) Grant.