A PUTATIVE SEROTONIN-SYNTHETIC AROMATIC AMINO ACID HYDROXYLASE FROM THE NEMATODE C. ELEGANS
C. M. Loer* and J. McKerrow. Dept. of Pathology, University of California, San Francisco, CA 94121 and *Dept. of Biology, Lafayette College, Easton, PA 18042
Editorial Note: Brian Davidson, an undergraduate research student, made a significant contribution to the work that was presented at the 1994 meeting. Because he did not begin working in the lab until after the abstract submission deadline, his name could not appear on the abstract.
We are seeking to identify genes that regulate the expression of the neurotransmitter phenotype of serotonere using two approaches: 1) isolation and characterization of serotonin-deficient mutants, some of which should identify regulatory genes, and 2) identification of marker genes used by serotonergic neurons, the genes controlled by the regulators of neurotransmitter type.
The first step in serotonin synthesis is catalyzed by an aromatic amino acid hydroxylase (AAAH). In vertebrates, this enzyme is named tryptophan hydroxylase (TrpH); it is one of three related AAAH's, including phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TH). Recent evidence suggests that in the fruit fly Drosophila, only two such enzymes exist, with the TrpH and PheH functions carried out by a single Phe/Trp hydroxylase with broader substrate specificity (Neckameyer and White, J. Biol. Chem. 267: 4199-4206, 1992). We have previously cloned an AAAH gene from C. elegans using PCR with degenerate oligonucleotides The enzyme encoded by the longest cDNA sequenced is most similar to the Phe/TrpH from Drosophila(C. Loer and C. Kenyon, unpublished). In order to determine whether the protein is a serotonin-synthetic enzyme, we have cloned the cDNA into an fusion-protein expression vector (pMAL), expressed the protein in bacteria, and tested the substrate specificity of the resulting enzyme. Preliminary evidence using crude lysates of bacteria expressing the protein suggests that this enzyme is also capable of hydroxylating both Phe and Trp.
Editorial Note #2: The gene identified here is now called bas-2 and is likely the phenylalanine hydroxylase of C. elegans. Three AAAHs, all on LG2, have now been identified in the worm, likely corresponding in function to those found in the vertebrates. The likely tryptophan hydroxylase is found on cosmid ZK1290; the likely tyrosine hydroxylase on B0432 (and probably identifies the cat-2 gene).