Cloning and characterization of an odorant receptor in five Pacific salmon.

The olfactory system of fish is extremely important as it is able to recognize and distinguish a vast of odorous molecules involved in wide ranges of behaviors including reproduction, homing, kin recognition, feeding and predator avoidance; all of which are paramount for their survival. We cloned and characterized one type olfactory receptors (ORs) from five congeneric salmonids: lacustrine sockeye salmon (Oncorhynchus nerka), pink salmon (O. gorbuscha), chum salmon (O. keta), masu salmon (O. masou) and rainbow trout (O. mykiss). Lacustrine sockeye salmon olfactory receptor 1 (LSSOR1) showed high sequence homology to the OR subfamily, and was expressed only in the olfactory epithelium (as indicated by PCR amplified genomic DNA and cDNA). OR genes from the five salmonids examined all showed strong homology (96-99%) to each other. Hypervariable regions, believed to be ligand-binding pockets, showed homologous completely matched amino acid sequences except for one amino acid in pink salmon olfactory receptor 1 (PSOR1), revealing that these ORs may be well conserved among salmon species. These results suggest that the isolated 5 salmonid ORs might play an important role in salmon life cycles.

Gaseous neuromodulator-related genes expressed in the brain of honeybee Apis mellifera.

Nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO) are thought to act as gaseous neuromodulators in the brain across species. For example, in the brain of honeybee Apis mellifera, NO plays important roles in olfactory learning and discrimination, but the existence of H2S- and CO-mediated signaling pathways remains unknown. In the present study, we identified the genes of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), cystathionine beta-synthase (CBS), and heme oxygenase (HO) from the honeybee brain. The honeybee brain contains at least one gene for each of NOS, CBS, and HO. The deduced proteins for NOS, CBS, and HO are thought to contain domains to generate NO, H2S, and CO, respectively, and to contain putative Ca2+/calmodulin-binding domains. On the other hand, the honeybee brain contains three subunits of sGC: sGCalpha1, sGCbeta1, and sGCbeta3. Phylogenetic analysis of sGC revealed that Apis sGCalpha1 and sGCbeta1 are closely related to NO- and CO-sensitive sGC subunits, whereas Apis sGCbeta3 is closely related to insect O2-sensitive sGC subunits. In addition, we performed in situ hybridization for Apis NOS mRNA and NADPH-diaphorase histochemistry in the honeybee brain. The NOS gene was strongly expressed in the optic lobes and in the Kenyon cells of the mushroom bodies. NOS activity was detected in the optic lobes, the mushroom bodies, the central body complex, the lateral protocerebral lobes, and the antennal lobes. These findings suggest that NO is involved in various brain functions and that H2S and CO can be endogenously produced in the honeybee brain.

Amphipathic alpha-helix mediates the heterodimerization of soluble guanylyl cyclase.

Soluble guanylyl cyclase (soluble GC) is an enzyme consisting of alpha and beta subunits and catalyzes the conversion of GTP to cGMP. The formation of the heterodimer is essential for the activity of soluble GC. Each subunit of soluble GC has been shown to comprize three functionally different parts: a C-terminal catalytic domain, a central dimerization domain, and an N-terminal regulatory domain. The central dimerization domain of the beta(1) subunit, which contains an N-terminal binding site (NBS) and a C-terminal binding site (CBS), has been postulated to be responsible for the formation of alpha/ beta heterodimer. In this study, we analyzed heterodimerization by the pull-down assay using the affinity between a histidine tag and Ni(2+) Sepharose after co-expression of various N- and C-terminally truncated FLAG-tagged mutants of the alpha(1) subunit and the histidine-tagged wild type of the beta(1) subunit in the vaculovirus/Sf9 system, and demonstrated that the CBS-like sequence of the alpha(1) subunit is critical for the formation of the heterodimer with the beta(1) subunit and the NBS-like sequence of the alpha(1) subunit is essential for the formation of the enzymatically active heterodimer, although this particular sequence was not involved in heterodimerization. The analysis of the secondary structure of the alpha(1) subunit predicted the existence of an amphipathic alpha-helix in residues 431-464. Experiments with site-directed alpha(1) subunit mutant proteins demonstrated that the amphipathicity of the alpha-helix is important for the formation of the heterodimer, and Leu(463) in the alpha-helix region plays a critical role in the formation of a properly arranged active center in the dimer.

Expression of guanylyl cyclase genes in medaka hybrids (Oryzias curvinotus x Oryzias latipes).

The Hong Kong-originated medaka fish Oryzias curvinotus expresses nine genes (OcGC1 approximately OcGC8 and OcGC-R2) for membrane guanylyl cyclases (membrane GCs) and three genes (OcGCS-alpha(1), OcGCS-alpha(2), and OcGCS-beta(1)) for soluble GC subunits. The deduced amino acid sequences of membrane GCs expressed in O. curvinotus were quite similar to those expressed in the Japanese medaka Oryzias latipes, including a novel membrane GC gene, OlGC8, first isolated and characterized in O. latipes. O. curvinotus was able to produce hybrids with O. latipes, irrespective of the direction of crossing, and the resulting hybrids expressed both maternal and paternal soluble GC subunit genes, suggesting the possibility of the formation of a chimeric heterodimer in the hybrids. However, in early embryogenesis of hybrids, the maternal soluble GC subunit genes were expressed earlier than the paternal soluble GC subunit genes, suggesting that the maternal soluble GC subunit genes interact more effectively with maternal effector molecules such as transcription factors than with those of paternal origin.