Characterization of the Xenopus tryptophan hydroxylase gene

Purpose: Tryptophan hydroxylase is the rate limiting enzyme in serotonin production. In Xenopus laevis retina, it is expressed in photoreceptors and is transcriptionally regulated by a photoreceptor circadian clock (Green, Cahill and Besharse, Vis. Neuro. 12, 663-670, 1995; Green and Besharse, J. Neurochem. 62, 2420-2428, 1994). In this capacity, it plays a role in the rhythmic production of melatonin (Cahill and Besharse, J. Neurochem. 54: 716-719, 1990). As a prelude to studies of its transcriptional regulation, we report here characterization of the Xenopus tryptophan hydroxylase gene. Methods: Clone TPHG11, corresponding to the tryptophan hydroxylase gene, was isolated from a λFIXII Xenopus genomic library purchased from Stratagene. This clone was mapped using restriction enzymes and exon/intron boundaries were characterized by Southern blotting and DNA sequencing. The transcription start site was identified by primer extension. Results: The locations of the exon/intron boundaries for clone TPHG11 were found to be conserved when compared to the human tryptophan hydroxylase gene (Boularand et.al. J. Biochem. 270, 3748-3756, 1995). The transcription initiation site is preceded by a TATA box at -30bp. This clone contains approximately two kilobases of sequence upstream of the transcription start site. Conclusion: We have isolated and characterized the genomic clone TPHG11, which contains the Xenopus tryptophan hydroxylase gene. This clone contains two kilobases of upstream sequence which is currently being analyzed for additional promoter elements. Promoter studies of the tryptophan hydroxylase gene will provide insight both on what confers tissue specificity and on mechanisms by which the circadian clock controls its rhythmic expression.