Dimorphic expression of tryptophan hydroxylase in the brain of XX and XY Nile tilapia during early development.
ABSTRACT Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.
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ABSTRACT: A full-length cDNA for tryptophan hydroxylase was cloned from rabbit pineal body by screening an expression library with antibody against rat phenylalanine hydroxylase, which crossreacts with rabbit tryptophan hydroxylase. Clones producing immunoreactive material contain sequences homologous to, yet distinct from, phenylalanine hydroxylase. The rabbit cDNA hybridizes to mRNA in pineal body and brainstem but not in liver. Comparison of the rabbit tryptophan hydroxylase sequence with the sequences of phenylalanine hydroxylase and tyrosine hydroxylase demonstrates that these three biopterin-dependent aromatic amino acid hydroxylases are highly homologous, reflecting a common evolutionary origin from a single primordial genetic locus. The pattern of sequence homology supports the hypothesis that the carboxyl-terminal two-thirds of the molecules constitute the enzymatic activity cores, and the amino-terminal thirds of the molecules constitute domains for substrate specificity.Proceedings of the National Academy of Sciences 09/1987; 84(16):5530-4. · 9.74 Impact Factor
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ABSTRACT: The mouse tryptophan hydroxylase gene was isolated and its intron/exon boundaries and putative regulatory sequences identified. To isolate the gene a mouse mastocytoma cDNA clone encoding tryptophan hydroxylase was used to identify and isolate ten overlapping DNA fragments from a mouse genomic library. Restriction mapping and sequence analysis of the clones revealed that the gene contains 11 exons and covers a region of DNA of approximately 21 kb. The transcription initiation site was mapped and the major site of initiation yields an untranslated leader sequence of 124 nucleotides. A minor initiation site is located 9 nucleotides 3' of the major site. The 5' untranslated sequence is interrupted by the first intron. Analysis of the sequence upstream of the initiation site showed the presence of several putative promoter and regulatory sequences. Nine of the ten intron/exon boundaries of tryptophan hydroxylase are conserved with tyrosine hydroxylase and phenylalanine hydroxylase, further delineating the evolutionary relationship of these three genes.Journal of Neuroscience Research 05/1991; 28(4):457-65. · 2.97 Impact Factor
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ABSTRACT: Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.Journal of Biological Chemistry 10/2006; 281(38):28105-12. · 4.65 Impact Factor