Haruhiko Toyohara

Identification of receptors of main sex-pheromone components of three Lepidopteran species

Male moths discriminate conspecific female‐emitted sex pheromones. Although the chemical components of sex pheromones have been identified in more than 500 moth species, only three components in Bombyx mori and Heliothis virescens have had their receptors identified. Here we report the identification of receptors for the main sex‐pheromone components in three moth species, Plutella xylostella, Mythimna separata and Diaphania indica. We cloned putative sex‐pheromone receptor genes PxOR1, MsOR1 and DiOR1 from P. xylostella, M. separata and D. indica, respectively. Each of the three genes was exclusively expressed with an Or83b orthologous gene in male olfactory receptor neurons (ORNs) that are surrounded by supporting cells expressing pheromone‐binding‐protein (PBP) genes. By two‐electrode voltage‐clamp recording, we tested the ligand specificity of Xenopus oocytes co‐expressing PxOR1, MsOR1 or DiOR1 with an OR83b family protein. Among the seven sex‐pheromone components of the three moth species, the oocytes dose‐dependently responded only to the main sex‐pheromone component of the corresponding moth species. In our study, PBPs were not essential for ligand specificity of the receptors. On the phylogenetic tree of insect olfactory receptors, the six sex‐pheromone receptors identified in the present and previous studies are grouped in the same subfamily but have no relation with the taxonomy of moths. It is most likely that sex‐pheromone receptors have randomly evolved from ancestral sex‐pheromone receptors before the speciation of moths and that their ligand specificity was modified by mutations of local amino acid sequences after speciation.

Fish glucocorticoid receptor with splicing variants in the DNA binding domain

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid‐responsive mouse mammary tumor virus promoter to comparable levels.

A hyperosmotic stress-induced mRNA of carp cell encodes Na+- and Cl−-dependent high affinity taurine transporter

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.

Cellulose digestion by common Japanese freshwater clam Corbicula japonica

Cellulose digestion by Corbicula japonica was investigated according to the hypothesis that without any symbiotic aid, this organism can utilize cellulose as a carbon source. Enzymatic studies revealed the complete cellulase activity of this species, and molecular cloning resulted in the isolation of cDNA with an ORF encoding a 596-amino-acid protein that shares significant homology with abalone and termite cellulases with an amino acid identity of 52.2% and 50.5%, respectively. The isolated cellulase had a carbohydrate-binding module at the N-terminal region that was also reportedly present in abalone cellulase, and its mRNA were specifically expressed in the digestive gland. These findings strongly support the assumption that C. japonica has an endogenous cellulose, as well as abalones and termites. It is further believed that C. japonica plays an important roll in decomposing cellulose, and consequently contributes to the carbon-cycle in the aquatic environment, as termites do in terrestrial forests.

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.

Sequence and expression of a cDNA encoding the red seabream androgen receptor

The cDNA of the androgen receptor (AR) has been isolated from the ovary of red seabream, Pagrus major, and sequenced. The amino acid sequence of red seabream AR (rsAR) shows about 45% identity with those of Xenopus, rat, mouse, and human ARS. It is shown that rsAR has the ability to trans-activate the responsive gene depending on the presence of androgen.

The Diversity of Shell Matrix Proteins: Genome-Wide Investigation of the Pearl Oyster, Pinctada fucata

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 (http://marinegenomics.oist.jp/pinctada_fucata). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.

A stable line of transgenic medaka (Oryzias latipes) carrying the CAT gene

Abstract A stable homozygous line of transgenic medaka (Oryzias latipes) was produced by injecting plasmid DNA containing rainbow trout metallothionein A promoter region followed by bacterial acetyltransferase gene (rtMT-A-CAT), into the cytoplasm of 111 fertilized eggs. The line transmitted active CAT gene to all of the offsprings until sixth generation in mendelian fashion. The Southern blot analysis and the crossing experiments indicated that the DNA was integrated into the chromosome. These results reveal that the medaka is a good model for basic studies of the production of transgenic fish.

Food sources for the bivalve Corbicula japonica in the foremost fishing lakes estimated from stable isotope analysis

Carbon and nitrogen stable isotope ratios in tissue of the bivalve corbicula Corbicula japonica and particulate organic matter (POM) were measured in Lake Jusan, Lake Ogawara and Lake Shinji, which are the foremost fishing grounds for the corbicula in Japan, to determine their food sources. The bivalves in Lake Ogawara and Lake Shinji showed enriched isotope composition, while those in Lake Jusan were depleted. In addition, the difference in the isotope ratios between the sampling sites was remarkable in Lake Jusan. Chlorophyll concentrations were significantly higher in Lake Ogawara and Lake Shinji than those in the inflow rivers, although that in Lake Jusan was equivalent to that in the river. Residence time of river water was estimated at 1 day, 455 days and 88 days in Lake Ogawara and Lake Shinji assimilate autochthonous phytoplankton, while those in Lake Jusan assimilate terrestrial matter in the upper reaches and marine phytoplankton in the lower reaches because of low production in the lake.