GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.

We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

The biosynthesis of structural and signaling molecules depends on intracellular concentrations of essential amino acids, which are maintained by a specific system of plasma membrane transporters. We identify a unique population of nutrient amino acid transporters (NATs) within the sodium-neurotransmitter symporter family and have characterized a member of the NAT subfamily from the larval midgut of the Yellow Fever vector mosquito, Aedes aegypti (aeAAT1, AAR08269), which primarily supplies phenylalanine, an essential substrate for the synthesis of neuronal and cuticular catecholamines. Further analysis suggests that NATs constitute a comprehensive transport metabolon for the epithelial uptake and redistribution of essential amino acids including precursors of several neurotransmitters. In contrast to the highly conserved subfamily of orthologous neurotransmitter transporters, lineagespecific, paralogous NATs undergo rapid gene multiplication͞ substitution that enables a high degree of evolutionary plasticity of nutrient amino acid uptake mechanisms and facilitates environmental and nutrient adaptations of organisms. These findings provide a unique model for understanding the molecular mechanisms, physiology, and evolution of amino acid and neurotransmitter transport systems and imply that monoamine and GABA transporters evolved by selection and conservation of earlier neuronal NATs.

On the basis of anatomy and larval behavior, the apical sensory organ (ASO) of gastropod veliger larvae has been implicated as the site of perception of cues for settlement and metamorphosis. Until now, there have been no experimental data to support this hypothesis. In this study, cells in the ASO of veliger larvae of the tropical nudibranch Phestilla sibogae were stained with the styryl vital dye DASPEI and then irradiated with a narrow excitatory light beam on a fluorescence microscope. When its ASO cells were bleached by irradiation for 20 min or longer, an otherwise healthy larva was no longer able to respond to the usual metamorphic cue, a soluble metabolite from a coral prey of the adult nudibranch. The irradiated cells absorbed the dye acridine orange, suggesting that they were dying. When larvae not stained with DASPEI were similarly irradiated, or when stained larvae were irradiated with the light beam focused on other parts of the body, there was no loss of ability to metamorphose. Together these data provide strong support for the hypothesis. Potassium and cesium ions, known to induce metamorphosis in larvae of many marine-invertebrate phyla, continue to induce metamorphosis in larvae that have lost the ability to respond to the coral inducer due to staining and irradiation. These results demonstrate that (1) the ASO-ablated larvae have not lost the ability to metamorphose and (2) the ions do not act only on the metamorphic-signal receptor cells, but at other sites downstream in the metamorphic signal transduction pathway.

The recently discovered prokaryotic signal transducer HemAT, which has been described in both Archaea and Bacteria, mediates aerotactic responses. The N-terminal regions of HemAT from the archaeon Halobacterium salinarum (HemAT-Hs) and from the Gram-positive bacterium Bacillus subtilis (HemAT-Bs) contain a myoglobin-like motif, display characteristic heme-protein absorption spectra, and bind oxygen reversibly. Recombinant HemAT-Hs and HemAT-Bs shorter than 195 and 176 residues, respectively, do not bind heme effectively. Sequence homology comparisons and three-dimensional modeling predict that His-123 is the proximal heme-binding residue in HemAT from both species. The work described here used site-specific mutagenesis and spectroscopy to confirm this prediction, thereby providing direct evidence for a functional domain of prokaryotic signal transducers that bind heme in a globin fold. We postulate that this domain is part of a globin-coupled sensor (GCS) motif that exists as a two-domain transducer having no similarity to the PER-ARNT-SIM (PAS)-domain superfamily transducers. Using the GCS motif, we have identified several two-domain sensors in a variety of prokaryotes. We have cloned, expressed, and purified two potential globin-coupled sensors and performed spectral analysis on them. Both bind heme and show myoglobin-like spectra. This observation suggests that the general function of GCS-type transducers is to bind diatomic oxygen and perhaps other gaseous ligands, and to transmit a conformational signal through a linked signaling domain.proximal histidine ͉ transducer G lobins are heme-containing proteins that are involved in binding and͞or transport of diatomic oxygen. Presently, more than 700 globin sequences are known (1). It has been proposed that all globins have evolved from an ancestral redox protein of about 17 kDa that displayed the globin fold, which is characterized by the presence of eight helices, designated A through H (2). The residues absolutely conserved among all globins are the proximal histidine in the F helix (F8) and phenylalanine in the CD region (CD1) (3, 4). Highly conserved residues include the distal histidine in the E helix (E7), phenylalanine in the CD4 region, and proline at the beginning of the C helix (C2).We recently discovered heme-containing transducers in the archaeon Halobacterium salinarum (HemAT-Hs) and the Grampositive bacterium Bacillus subtilis (HemAT-Bs). These proteins bind diatomic oxygen and mediate an aerotactic response (5). The N termini of these transducers resemble myoglobin, and their C termini are homologous to the cytoplasmic signaling domain of bacterial chemoreceptors. We have also described three-dimensional homology models of the putative oxygensensing domain of HemATs (6). In these models the overall globin topology, including the orientation of the heme prosthetic group, is preserved, as is the hydrophobic core of the hemebinding pocket and the electrostatic stabilization of the CD region. Therefore, an experimental determination of the organizatio...

SUMMARY Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1)uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA,but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B0 type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes(Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned,nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

The CG3252 gene product, DmNAT1, represents the first Nutrient Amino acid Transporter cloned from Drosophila. It absorbs a broader set of neutral amino acids versus earlier characterized insect NATs and mammalian NATs-B 0 system transporters from the Sodium Neurotransmitter symporter Family (SNF, a.k.a. solute carrier family 6, SLC6). In addition to B 0 -specific L-substrates, DmNAT1 equally or more effectively transports D-amino acids with sub millimolar affinities and 1:1 sodium:amino acid transport stoichiometry. DmNAT1 is strongly transcribed in the absorptive and secretory regions of the larval alimentary canal and larval brain, revealing its roles in the primary absorption and redistribution of large neutral L-amino acids as well as corresponding D-isomers. The absorption of D-amino acids via DmNAT1 may benefit the acquisition of fermented and symbiotic products, and may support the unique capacity of fruit fly larvae to utilize a diet with substitution of essential amino acids by D-isomers. It also suggests a remarkable adaptive plasticity of NAT-SLC6 mechanisms via alterations of a few identifiable sites in the substrate-binding pocket. The strong transcription in the brain suggests roles for DmNAT1 in neuronal nutrition and clearance of L-neutral amino acids from the fly brain. In addition, neuronal DmNAT1 may absorb synaptic D-serine and modulate NMDA receptor-coupled signal transduction. The characterization of the first invertebrate B 0 -like transporter extends the biological roles of the SLC6 family, revealing adaptations for the absorption of D-isomers of the essential amino acids. These findings suggest that some members of the NAT-SLC6 subfamily evolving specific properties which contribute to nutrient symbiotic relationships and neuronal functions.