Worm Breeder's Gazette 11(3): 18
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Specification of neurotransmitter phenotype is an important requirement for generating a functional nervous system. A specific neurotransmitter phenotype requires the coordinate expression of biosynthetic enzymes, a degradation or re-uptake system, and perhaps a specific packaging apparatus. The Drosophila dopa decarboxylase gene ( Ddc), the last enzyme required for serotonin (5-HT) and dopamine (DA) synthesis is differentially regulated and spliced in the nervous system and the epidermis, where is required for cuticle synthesis ( Morgan et al., 1986, EMBO J. 5: 3335; Bray et al., 1988, EMBO J. 7:177). A number of neuron-specific enhancers in the 5' region of Ddc have been identified, including one that binds a POU-type homeodomain protein. Mutation in this enhancer region eliminate DA expression in a subset of dopaminergic CNS neurons (Johnson and Hirsh, 1990, Nature 343: 467). The biogenic amine neurotransmitters 5-HT and DA are used by a number of neurons in C. elegans, including the HSN, NSM, and some CPs (5-HT) and ADE, PDE, CEPs, and Ray neurons 5A, 7A, and 9A (DA) ( Sulston et al., 1975, J. Comp. Neurol., 163: 215; Horvitz et al., 1982, Science 216:1012). 5-HT and DA are derived by two enzymatic steps from the aromatic amino acids tryptophan and tyrosine, respectively (see below). In vertebrates and flies, specific enzymes catalyze the first step in the reaction (i.e., tryptophan hydroxylase and tyrosine hydroxylase), whereas the decarboxylation step for both 5- HT and DA is catalyzed by a single enzyme with broader specificity (i. e., dopa decarboxylase). [See Figure 1] As a first step towards analyzing the control of neurotransmitter phenotype, we are seeking to clone genes encoding biosynthetic enzymes for these neurotransmitters. We used the polymerase chain reaction ( PCR) with degenerate oligonucleotides encoding highly conserved regions of aromatic amino acid hydroxylases to amplify a genomic fragment encoding a C. elegans AAA Hydroxylase. Of 5 clones sequenced, 3 were garbage and 2 encoded the AA sequence shown below. As indicated, the sequence included a typical small worm intron with good consensus splice donor and acceptor sequences at an evolutionarily conserved splice site. Typical C. elegans codon usage was also apparent in the coding region sequenced. [See Figure 2] We used this 185 bp fragment (TH1,2) to isolate a lambda genomic clone and also to probe a genomic southern blot. At high stringency, the TH1,2 fragment showed a single band in all lanes except for a SacI digest (TH1,2 contains a SacI site); thus, the gene seems to be found in a single copy in the genome. We are using the lambda genomic clone to isolate cDNAs for sequencing. We plan to use in situ hybridization to define sites of expression of the gene. Since we may not be able to determine what kind of AAA hydroxlase this gene encodes by sequence alone, such information may clearly indicate the probable specificity of the enzyme (e.g., if it is found only in dopaminergic cells, it should be a tyrosine hydroxylase!). We also plan to fuse -gal or another reporter gene into the AAAH gene to use for identifying mutations that alter its expression and to examine expression in certain known mutants. Such a screen should yield genes that are required for the regulation of neurotransmitter synthesis (the cat mutations may be alleles of such genes) as well as genes that determine which neurons produce a specific neurotransmitter.
