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Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine.

ABSTRACT: Octopamine is likely to be an important neuroactive molecule in invertebrates. Here we report the molecular cloning of the Drosophila melanogaster gene, which encodes tyramine beta-hydroxylase (TBH), the enzyme that catalyzes the last step in octopamine biosynthesis. The deduced amino acid sequence of the encoded protein exhibits 39% identity to the evolutionarily related mammalian dopamine beta-hydroxylase enzyme. We generated a polyclonal antibody against the protein product of T beta h gene, and we demonstrate that the TBH expression pattern is remarkably similar to the previously described octopamine immunoreactivity in Drosophila. We further report the creation of null mutations at the T beta h locus, which result in complete absence of TBH protein and blockage of the octopamine biosynthesis. T beta h-null flies are octopamine-less but survive to adulthood. They are normal in external morphology, but the females are sterile, because although they mate, they retain fully developed eggs. Finally, we demonstrate that this defect in egg laying is associated with the octopamine deficit, because females that have retained eggs initiate egg laying when transferred onto octopamine-supplemented food.

SUBMITTER: Monastirioti M

PROVIDER: S-EPMC6578608 | biostudies-literature | 1996 Jun

REPOSITORIES: biostudies-literature

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Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine.

Monastirioti M M Linn C E CE White K K

The Journal of neuroscience : the official journal of the Society for Neuroscience 19960601 12

Octopamine is likely to be an important neuroactive molecule in invertebrates. Here we report the molecular cloning of the Drosophila melanogaster gene, which encodes tyramine beta-hydroxylase (TBH), the enzyme that catalyzes the last step in octopamine biosynthesis. The deduced amino acid sequence of the encoded protein exhibits 39% identity to the evolutionarily related mammalian dopamine beta-hydroxylase enzyme. We generated a polyclonal antibody against the protein product of T beta h gene, ...[more]

PMID: 8656284

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Project description:In insects, the biogenic amines octopamine (OA) and tyramine (TA) are involved in controlling several physiological and behavioural processes. OA and TA act as neurotransmitters, neuromodulators or neurohormones, performing their functions by binding to specific receptors belonging to the G protein-coupled receptor (GPCR) superfamily. OA and TA along with their receptors are involved in reproduction, smell perception, metabolism, and homeostasis. Moreover, OA and TA receptors are targets for insecticides and antiparasitic agents, such as the formamidine Amitraz. In the dengue and yellow fever vector, Aedes aegypti, limited research has been reported on their OA or TA receptors. Here, we identify and molecularly characterize the OA and TA receptors in A. aegypti. Bioinformatic tools were used to identify four OA and three TA receptors in the genome of A. aegypti. The seven receptors are expressed in all developmental stages of A. aegypti; however, their highest transcript abundance is observed in the adult. Among several adult A. aegypti tissues examined, including the central nervous system, antennae and rostrum, midgut, Malpighian tubules, ovaries, and testes, the type 2 TA receptor (TAR2) transcript is most abundant in the ovaries and the type 3 TA receptor (TAR3) is enriched in the Malpighian tubules, leading us to propose putative roles for these receptors in reproduction and diuresis, respectively. Furthermore, a blood meal influenced OA and TA receptor transcript expression patterns in adult female tissues at several time points post blood meal, suggesting these receptors may play key physiological roles associated with feeding. To better understand OA and TA signalling in A. aegypti, the transcript expression profiles of key enzymes in their biosynthetic pathway, namely tyrosine decarboxylase (Tdc) and tyramine β-hydroxylase (Tβh), were examined in developmental stages, adult tissues, and brains from blood-fed females. These findings provide information for better understanding the physiological roles of OA, TA, and their receptors in A. aegypti, and additionally, may help in the development of novel strategies for the control of these human disease vectors.

Project description:BACKGROUND:Norepinephrine/noradrenaline is a neurotransmitter implicated in arousal and other aspects of vertebrate behavior and physiology. In invertebrates, adrenergic signaling is considered absent and analogous functions are performed by the biogenic amines octopamine and its precursor tyramine. These chemically similar transmitters signal by related families of G-protein-coupled receptors in vertebrates and invertebrates, suggesting that octopamine/tyramine are the invertebrate equivalents of vertebrate norepinephrine. However, the evolutionary relationships and origin of these transmitter systems remain unclear. RESULTS:Using phylogenetic analysis and receptor pharmacology, here we have established that norepinephrine, octopamine, and tyramine receptors coexist in some marine invertebrates. In the protostomes Platynereis dumerilii (an annelid) and Priapulus caudatus (a priapulid), we have identified and pharmacologically characterized adrenergic ?1 and ?2 receptors that coexist with octopamine ?, octopamine ?, tyramine type 1, and tyramine type 2 receptors. These receptors represent the first examples of adrenergic receptors in protostomes. In the deuterostome Saccoglossus kowalevskii (a hemichordate), we have identified and characterized octopamine ?, octopamine ?, tyramine type 1, and tyramine type 2 receptors, representing the first examples of these receptors in deuterostomes. S. kowalevskii also has adrenergic ?1 and ?2 receptors, indicating that all three signaling systems coexist in this animal. In phylogenetic analysis, we have also identified adrenergic and tyramine receptor orthologs in xenacoelomorphs. CONCLUSIONS:Our results clarify the history of monoamine signaling in bilaterians. Given that all six receptor families (two each for octopamine, tyramine, and norepinephrine) can be found in representatives of the two major clades of Bilateria, the protostomes and the deuterostomes, all six receptors must have coexisted in the last common ancestor of the protostomes and deuterostomes. Adrenergic receptors were lost from most insects and nematodes, and tyramine and octopamine receptors were lost from most deuterostomes. This complex scenario of differential losses cautions that octopamine signaling in protostomes is not a good model for adrenergic signaling in deuterostomes, and that studies of marine animals where all three transmitter systems coexist will be needed for a better understanding of the origin and ancestral functions of these transmitters.

Dataset Information

Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine.

Publications

Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine.

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