11 research outputs found
Drosophila tan Encodes a Novel Hydrolase Required in Pigmentation and Vision
Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5Ⲡuntranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function
Histaminkreislauf im visuellen System von
Von den Photorezeptorzellen im Auge der Taufliege wird der Neurotransmitter Histamin ausgeschßttet. Dieser durchläuft nach der derzeitigen Modellvorstellung einen Kreislauf aus Ausschßttung, Inaktivierung und Reaktivierung. Die Identifizierung des Gens sowie Untersuchungen zur Funktionalität und Expression der beta-Alanyl-Amin Hydrolase Tan werden beschrieben. Mit elektrophysiologischen Mitteln wird ein direkter Einfluss des Expressionsgrades auf die Qualität der Signaltransmission im optischen System nachgewiesen.
Die Herstellung eines spezifischen Antiserums gegen beta-Alanyl-Histamin erlaubte eine Verfolgung der inaktiven Form des Transmitters an Gewebeschnitten
Tan Expression and Function in the Eye and Optic Lobes
<div><p>(A) RNA in situ hybridization with an antisense <i>tan</i> cDNA probe to wild-type adult head sections. Horizontal section cut through the eye and the optic lobes.</p><p>(B) Sagittal section through the ommatidial array of the eye.</p><p>(C) Control sense probe does not show significant staining (horizontal section).</p><p>(DâG) ERG phenotypes of wild-type flies (D) and <i>tan</i> mutants (EâG). (D) <i>Canton<sup>S</sup></i> (wild-type) ERG showing normal âonâ and âoffâ transients. Upper trace in each recording indicates duration of light input stimulus (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010063#s4" target="_blank">Materials and Methods</a>). (E) <i>tan<sup>2</sup></i> mutant showing complete absence of âonâ and âoffâ transients. (F) <i>P{d07784}</i> exhibits a normal âonâ and âoffâ transient even though it has a <i>tan</i>-like pigmentation phenotype. (G) <i>P{g1557}</i> lacks âonâ and âoffâ transients.</p><p>(H and I) Normalized âonâ (H) and âoffâ (I) transients for wild-type, <i>tan<sup>2</sup> (t[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010063#pgen-0010063-b002" target="_blank">2</a>])</i>, <i>tan</i>-like <i>P</i> excision lines, and ârevertantâ <i>P</i> excision lines (see text) expressed as a percentage of the respective sustained negative potential. Insets: Correlation between head histamine content and the normalized magnitude of ERG âonâ and âoffâ transients (see text). Error bars: Âą 1 standard error in both dimensions.</p><p>(J) Head histamine contents for <i>tan</i>-like and revertant <i>P</i> excision lines (see text), relative to control <i>w<sup>1118</sup></i> and <i>tan<sup>1</sup>w<sup>1118</sup></i> flies. Head histamine increased in all flies that drank 0.5% carcinine in 4% glucose (black bars), relative to controls that drank only 4% glucose (open bars). Revertant and <i>tan</i>-like excision lines are each arranged in rank order. Note that excision line flies that drank only 4% glucose have histamine contents too small to show above the abscissa. Error bars represent Âą 1 standard error.</p><p>La, lamina; Me, medulla; Re, retina. Scale bars = 50 Îźm.</p></div
<i>Drosophila CG12120</i> Is Conserved across Insects and Is Related to Fungal IAT
<div><p>(A) Alignment of <i>D. melanogaster, D. pseudoobscura,</i> and <i>A. gambiae</i> CG12120 orthologs and <i>P. chrysogenum</i> IAT protein. Residues in blue are identical (*) across all four species, and residues in green are functionally similar, showing strong (:) or weak (.) similarity. Residue highlighted â1â indicates conserved arginine residue at position 217 that is mutated to proline in <i>tan<sup>1</sup></i> and <i>tan<sup>4</sup></i> mutants. Residue highlighted â2â indicates methionine residue at position 256 that is mutated to isoleucine in <i>tan<sup>5</sup></i> mutant. Cyan rectangle indicates auto-processing site of <i>P. chrysogenum</i> at which pro-IAT is cleaved between glycine 102 and cysteine 103.</p><p>(B) Percent sequence identity (blue in upper right) and sequence similarity (red in lower left) among <i>Drosophila</i> spp., <i>A. gambiae,</i> and <i>P. chrysogenum</i> proteins.</p><p>(C) Percent sequence identity (blue in upper right) and sequence similarity (red in lower left) of N-terminal <i>Drosophila</i> spp., <i>A. gambiae,</i> and <i>P. chrysogenum</i> proteins with presumptive <i>B. mori</i> CG12120 ortholog (see text).</p></div
Tan Functions in Diverse Developmental and Metabolic Pathways
<div><p>(A) In developing adult epidermal cells, Tan catalyzes the production of dopamine from NBAD during pigment development. This is one of four parallel pathways by which dopa or dopamine derivatives are secreted into the developing cuticle as precursors for distinct pigments. aaNAT, arylalkylamine-N-acetyl transferase; DDC, dopa decarboxylase; NADA, N-acetyl dopamine; PO, phenol oxidase; TH, tyrosine hydroxylase.</p><p>(B) In the photoreceptor, Tan catalyzes the hydrolysis of N-β-alanyl histamine (carcinine) to histamine for re-uptake by the presynaptic photoreceptor cell (R). CA, carcinine; EG, epithelial glial cell; HA, histamine.</p></div
<i>P</i>-Element Insertions in <i>CG12120</i> Cause <i>tan</i>-Like Pigmentation Phenotypes and Ectopic Expression of <i>CG12120</i> Rescues the <i>tan</i> Phenotype and Causes Ectopic Melanin Formation
<div><p>(A) The <i>tan</i> region of the <i>D. melanogaster</i> X chromosome. Known and predicted genes with directions of transcription are depicted below the line. Cytological divisions and flanking <i>P</i>-element insertions, as well as meiotic mapping data (see text), are indicated above the line.</p><p>(B) Transcription unit of predicted gene <i>CG12120</i>. Boxes indicate exons. Black indicates coding region. Transcription start site, inferred from EST data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010063#pgen-0010063-b015" target="_blank">15</a>], is at the position designated +1. Start codon is at position +250. Below are indicated positions of <i>P{d07784}</i> and <i>P{g1557}</i> insertions, just 3Ⲡto the transcription initiation site (+1) of <i>CG12120</i>. Transcription unit is indicated in bold.</p><p>(CâF) Adult (3â5 d) female body coloration of (C) <i>Canton<sup>S</sup></i> (wild-type), (D) <i>tan<sup>5</sup>,</i> (E) <i>w P{d07784}</i>, and (F) <i>w P{g1557}</i>.</p><p>(G and H) <i>w tan<sup>1</sup>/w tan<sup>1</sup>; CyO/+; P{w<sup>+</sup> C765-GAL4}/+</i> control female (G) and <i>w tan<sup>1</sup>/w tan<sup>1</sup>; P{w<sup>+</sup> UAS-CG12120}/+; P{ w<sup>+</sup> C765-GAL4}/+</i> female (H) showing rescue of abdomen phenotype (arrows).</p><p>(I and J) <i>w/w; CyO/+; P{w<sup>+</sup> pnr-GAL4}/+</i> control female (I) and <i>w/w; P{w<sup>+</sup> UAS-CG12120}/+; P{w<sup>+</sup> pnr-GAL4}/+</i> female (J) showing ectopic melanin pattern along dorsal midline (arrowheads).</p><p>All flies depicted were 3â5 d of age.</p></div