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 5′ untranslated 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

Evolution of ventricular myocyte electrophysiology

The relative importance of regulatory versus structural evolution for the evolution of different biological systems is a subject of controversy. The primacy of regulatory evolution in the diversification of morphological traits has been promoted by many evolutionary developmental biologists. For physiological traits, however, the role of regulatory evolution has received less attention or has been considered to be relatively unimportant. To address this issue for electrophysiological systems, we examined the importance of regulatory and structural evolution in the evolution of the electrophysiological function of cardiac myocytes in mammals. In particular, two related phenomena were studied: the change in action potential morphology in small mammals and the scaling of action potential duration across mammalian phylogeny. In general, the functional properties of the ion channels involved in ventricular action potential repolarization were found to be relatively invariant. In contrast, there were large changes in the expression levels of multiple ion channel and transporter genes. For the Kv2.1 and Kv4.2 potassium channel genes, which are primary determinants of the action potential morphology in small mammals, the functional properties of the proximal promoter regions were found to vary in concordance with species-dependent differences in mRNA expression, suggesting that evolution of cis-regulatory elements is the primary determinant of this trait. Scaling of action potential duration was found to be a complex phenomenon, involving changes in the expression of a large number of channels and transporters. In this case, it is concluded that regulatory evolution is the predominant mechanism by which the scaling is achieved

Comparative Genomics of Salmonella enterica Serovar Typhi Strains Ty2 and CT18

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics

The <i>CG12120</i> Gene Product Possesses NBAD Hydrolase and N-β-Alanyl Histamine (Carcinine) Hydrolase Activities

<div><p>HPLC-ED chromatograms from activity assays.</p><p>(A) Lysate from control (recombinant AGT) transfected <i>Sf9</i> cells exhibited no activity. Oxidation product (OP) peak denotes uncharacterized presumptive oxidation product of NBAD present in NBAD substrate in all experiments.</p><p>(B) NBAD incubated with CG12120 protein purified from <i>Sf9</i> cells expressing <i>CG12120</i> baculovirus construct after 20 min incubation with NBAD. Dopamine (DA) peak is evident.</p><p>(C) Same experiment as in (B) but after 50 min incubation, showing increased accumulation of dopamine and depletion of NBAD.</p><p>(D) Control HPLC chromatogram containing dopamine and NBAD standards.</p><p>(E) Extract from control (recombinant AGT) transfected <i>Sf9</i> cells exhibits no production of β-alanine and histamine when incubated with carcinine (CA). Unknown contaminant (UC) peak denotes an unknown contaminant in carcinine. β-mercaptoethanol (BME) peak denotes β-mercaptoethanol present in the reaction solution.</p><p>(F) Extract from <i>Sf9</i> cells transfected with <i>CG12120</i> baculovirus incubated with carcinine demonstrates production of histamine (HA) and β-alanine (BA). Under the applied conditions, essentially all carcinine was hydrolyzed to histamine and β-alanine.</p><p>(G) Control HPLC chromatogram containing β-mercaptoethanol, carcinine, histamine, and β-alanine standards. β-alanine and histamine detection were enabled by OPT conjugation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010063#s4" target="_blank">Materials and Methods</a>).</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