C. M. Loer, S. DePaul & E. Hare. Dept. of Biology, University of San Diego, CA 92110.
We are characterizing genes used by serotonergic neurons in the nematode C. elegans to learn how they are regulated; among these are the bas-1 and cat-4 genes. Worms with mutations in bas-1 and cat-4 genes lack serotonin and dopamine. We have shown that the bas-1 gene encodes a serotonin- and dopamine-synthetic aromatic amino acid decarboxylase (AAADC). The cat-4 gene appears to encode GTP Cyclohydrolase I (GCH), an enzyme necessary for synthesis of biopterin. A biopterin cofactor is used by both tryptophan and tyrosine hydroxylase, enzymes catalyzing the first step in serotonin and dopamine synthesis, respectively. We have identified the mutations in four bas-1 mutant alleles; a deletion allele was generated by the C. elegans Gene Knockout Consortium. Although two AAADC-homologous sequences are found in the region, our analyses show that disruption of only one sequence (C05D2.4) causes the Bas-1 phenotype. We rescued bas-1 mutants by injection of wildtype C05D2.4 DNA; an introduced mutation in this sequence blocks rescue. Bas-1 cDNAs include at least two alternately spliced forms; one has an extra 27 bp exon after exon 2. We have sequenced the original cat-4 mutant allele and found a mutation in the GCH homolog coding sequence. Mutants with a GCH gene deletion have a similar phenotype to the original cat-4 mutant. These results are consistent with the cat-4 gene encoding GCH. Finally, the recent completion of C. briggsae genomic sequence will allow us to make cross-species comparisons of regulatory regions with C. elegans to discover conserved regulatory elements in genes used by serotonergic neurons. Supported by: Fletcher Jones Foundation & NIGMS.