Hiroyuki Okamoto

A second generation genetic linkage map of Japanese flounder ( Paralichthys olivaceus )

BackgroundJapanese flounder (Paralichthys olivaceus) is one of the most economically important marine species in Northeast Asia. Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. Commercial production of Japanese flounder could be increased by developing disease-resistant fish and improving commercially important traits. Previous maps have been constructed with AFLP markers and a limited number of microsatellite markers. In this study, improved genetic linkage maps are presented. In contrast with previous studies, these maps were built mainly with a large number of codominant markers so they can potentially be used to analyze different families and populations.ResultsSex-specific genetic linkage maps were constructed for the Japanese flounder including a total of 1,375 markers [1,268 microsatellites, 105 single nucleotide polymorphisms (SNPs) and two genes]; 1,167 markers are linked to the male map and 1,067 markers are linked to the female map. The lengths of the male and female maps are 1,147.7 cM and 833.8 cM, respectively. Based on estimations of map lengths, the female and male maps covered 79 and 82% of the genome, respectively. Recombination ratio in the new maps revealed F:M of 1:0.7. All linkage groups in the maps presented large differences in the location of sex-specific recombination hot-spots.ConclusionsThe improved genetic linkage maps are very useful for QTL analyses and marker-assisted selection (MAS) breeding programs for economically important traits in Japanese flounder. In addition, SNP flanking sequences were blasted against Tetraodon nigroviridis (puffer fish) and Danio rerio (zebrafish), and synteny analysis has been carried out. The ability to detect synteny among species or genera based on homology analysis of SNP flanking sequences may provide opportunities to complement initial QTL experiments with candidate gene approaches from homologous chromosomal locations identified in related model organisms.

M2 and M3 muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild‐type mice and mice genetically lacking M2 or M3 muscarinic receptors, in order to characterize the mechanisms involved in M2 and M3 receptor‐mediated contractile responses. Single applications of carbachol (0.1–100u2003μM) produced concentration‐dependent contractions in preparations from M2‐knockout (KO) and M3‐KO mice, mediated via M3 and M2 receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M2‐preferring antagonist methoctramine (300u2003nM) or the M3‐preferring antagonist 4‐DAMP (30u2003nM). The M2‐mediated contractions were mimicked in shape by submaximal stimulation with high K+ concentrations (up to 35u2003mM), almost abolished by voltage‐dependent Ca2+ channel (VDCC) antagonists or depolarization with 140u2003mM K+ medium, and greatly reduced by pertussis toxin (PTX) treatment. The M3‐mediated contractions were only partially inhibited by VDCC antagonists or 140u2003mM K+‐depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K+ depolarization consisted of different components, either sensitive or insensitive to extracellular Ca2+. The carbachol contractions observed with wild‐type preparations consisted of PTX‐sensitive and ‐insensitive components. The PTX‐sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M2 receptor, through PTX‐sensitive mechanisms, induces ileal contractions that depend on voltage‐dependent Ca2+ entry, especially associated with action potential discharge, and that the M3 receptor, through PTX‐insensitive mechanisms, induces contractions that depend on voltage‐dependent and ‐independent Ca2+ entry and intracellular Ca2+ release. In intact tissues coexpressing M2 and M3 receptors, M2 receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Effects of G-protein-specific antibodies and Gβγ subunits on the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells

The effects on the whole‐cell carbachol‐induced muscarinic cationic current (mIcat) of antibodies against the α‐subunits of various G proteins, as well as the effect of a Gβγ subunit, were studied in single guinea‐pig ileal smooth muscle cells voltage‐clamped at −50 mV. Ionized intracellular calcium concentration, [Ca2+]i, was clamped at 100 nM using a 1,2‐bis(2‐aminophenoxyl‐ethane‐N,N,N′,N′‐tetraacetic acid)/Ca2+ mixture. Application of ascending concentrations of carbachol (1–300 μM) activated mIcat (mean amplitude 0.83 nA at 300 μM carbachol; EC50 8 μM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of Gi3/Go or Go antibodies resulted in about a 70% depression of the maximum response without change in the EC50 value. In contrast, antibodies against α‐subunits of Gi1, Gi1/Gi2, Gi3, Gq/G11 or Gs protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gβ or infusion of the Gβγ subunit itself had no effect on mIcat. If cells were exposed briefly to carbachol (50 or 100 μM) at early times (<3 min) after infusion of antibodies to Gαi3/Gαo or to Gαo had begun, carbachol responses remained unchanged even after 20–60 min; that is, the depression of mIcat by these antibodies was prevented. These data show that Gαo protein couples the muscarinic receptor to the cationic channel in guinea‐pig ileal longitudinal smooth muscle and that Gβγ is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long‐lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.

Muscarinic agonist potencies at three different effector systems linked to the M2 or M3 receptor in longitudinal smooth muscle of guinea-pig small intestine

The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, McN‐A343, methacholine, pilocarpine) to inhibit isoprenaline‐induced cyclic AMP production in chopped fragments (via M2 receptors), and to evoke cationic current (Icat) (via M2 receptors) or calcium store release (via M3 receptors) in enzyme‐dispersed, single voltage‐clamped cells from longitudinal smooth muscle of the guinea‐pig small intestine were examined. All muscarinic agonists (1u2003–u2003300u2003μM) examined inhibited isoprenaline (1u2003μM)‐induced accumulation of cyclic AMP, the IC50 varying from 52 to 248u2003μM. However, their relative potencies to evoke this M2 effect were not significantly correlated with their ability to evoke Icat, also a M2 effect, whether or not calcium stores were depleted; pilocarpine and McN‐A343 inhibited the Icat response to carbachol. Muscarinic agonists (concentration 300 or 1000u2003μM), except pilocarpine and McN‐A343 which were ineffective, evoked Ca2+‐activated K+ current (IK‐Ca) resulting from Ca2+ store release (M3 effect). Their effectiveness was tested by estimating residual stored calcium by subsequent application of caffeine (10u2003mM). The relative potencies to evoke Ca2+ store release (M3) and for Icat activation (M2) were closely correlated (P<0.001). These data might be explained if M2‐mediated adenylyl cyclase inhibition and Icat activation involve different G proteins, or involve different populations of M2 receptors. The observed correlation of agonist potency between Icat activation and Ca2+ store release supports the proposal ( Zholos & Bolton, 1997 ) that M3 activation can potentiate M2‐cationic channel coupling through Ca2+‐independent mechanisms.

Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle

In guinea‐pig ileal longitudinal muscle, muscarinic partial agonists, 4‐(N‐[3‐chlorophenyl]‐carbomoyloxy)‐2‐butynyl‐trimethylammonium (McN‐A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca2+ concentration ([Ca2+]c) with a higher EC50 than that of the full agonist carbachol. The maximum response of [Ca2+]c or tension was not much different among the three agonists. The Ca2+ channel blocker nicardipine markedly inhibited the effects of all three agonists The contractile response to any agonist was antagonized in a competitive manner by M2 receptor selective antagonists (N,N′‐bis[6‐[[(2‐methoyphenyl)methyl]amino]hexyl]‐1,8‐octanediamine tetrahydrochloride and 11‐[[2‐[(diethlamino)methyl]‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4] benzodiazepine‐6‐one), and the apparent order of M2 antagonist sensitivity was McN‐A343>pilocarpine>carbachol. M3 receptor selective antagonists, 1,1‐dimethyl‐4‐diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN‐A343, while darifenacin had a similar action in the case of pilocarpine. Both M3 antagonists behaved in a competitive manner in the case of the carbachol response. McN‐A343 failed to release Ca2+ from the intracellular stores, and the Ca2+‐releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca2+ sensitivity of the contractile proteins. McN‐A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. The results suggest that muscarinic agonist‐evoked contractions result primarily from the integration of Ca2+ entry associated with the increased spike discharge and myofilaments Ca2+ sensitization, and that Ca2+ store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M2 and M3 receptors plays a crucial role in mediating the contraction response.

Cloning of cDNA encoding vitellogenin and its expression in red sea urchin, Pseudocentrotus depressus

Abstract Both male and female red sea urchins, Pseudocentrotus depressus, accumulate a large quantity of the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before the initiation of game-togenesis. To examine the accumulation mechanism of this protein in the gonad, we cloned full-length cDNA encoding vitellogenin (Vg; the MYP precursor in the coelomic fluid), and investigated its expression in various tissues of immature adults. The nucleotide sequence of Vg contains an open reading frame of 4050 bp encoding 1349 amino acids. The deduced amino acid sequence near the N-terminal showed 25% homology to the vertebrate transferrin family. Vitellogenin mRNA was detected in the ovary, testis, stomach, intestine and rectum by Northern blot analysis, with the highest level of mRNA expression in the gonad. Weak expression was also detected in the esophagus and coelomocytes by RT-PCR. In situ hybridization demonstrated that nutritive phagocytes, which exclusively fill the lumina of the immature gonad, contained Vg mRNA. These results suggested that the MYP stored in the immature gonads is synthesized and accumulated mainly within the nutritive phagocytes.

Analysis of haploid development based on expression patterns of developmental genes in the medaka Oryzias latipes.

The abnormalities of haploid medaka embryos were characterized by comparative analysis of histologic sections and expression patterns of some developmental marker genes between haploids and diploids to clarify whether medaka haploids are useful for identifying mutants. During gastrulation, an obvious defect was first observed as a delay of epiboly and involution. This delay was shown to be caused not by the perturbation of mesoderm induction, but by widespread cell death and disorganization of cell arrangement in the blastoderm. This disorganization of cell arrangement was also detected in various organs, such as the brain, somite and notochord, at a late developmental stage. Ten days after fertilization, a small head and a short body axis were formed; these changes were also observed in haploid embryos in other species, but their cause is unknown. Based on the expression patterns of HNF3β and goosecoid, it was demonstrated that a short and impotent prechordal plate induced near the marginal zone in haploid embryos was responsible for this defect. However, in these experiments it was also demonstrated that many major organs in haploids, such as the somite and notochord, differentiated incompletely but were present. Therefore, it was concluded that haploid screening is suitable for identifying mutations revealed by an obvious phenotype, such as dorsoventral polarity.

Molecular basis of mucopolysaccharidosis type VII: replacement of Ala619 in β-glucuronidase with Val

We have identified a mutation causing beta-glucuronidase (beta Gl) deficiency in a 6-year-old girl with mucopolysaccharidosis type VII. Enzyme assay of lysates of a girls lymphocytes or cultured fibroblasts showed little residual activity and a normal beta Gl-specific mRNA level, as revealed by Northern-blot analysis. Sequencing of the full-length mutated cDNA revealed a C----T transition, an event causing a single Ala619----Val change (we designated this variant beta GGifu). This change is detected by loss of the cleavage site for the enzyme Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient was shown to be a homozygote with the beta GGifu mutation and her parents and brother were heterozygotes. This mutation disrupts a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gls, and it reduces the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA.

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis

BackgroundIn fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions INGenomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported.ResultsIn this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297xa0kb, which is similar to values calculated for zebrafish and medaka TILLING libraries.ConclusionsThese results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Homozygous and heterozygous GH transgenesis alters fatty acid composition and content in the liver of Amago salmon (Oncorhynchus masou ishikawae)

Summary Growth hormone (GH) transgenic Amago (Oncorhynchus masou ishikawae), containing the sockeye GH1 gene fused with metallothionein-B promoter from the same species, were generated and the physiological condition through lipid metabolism compared among homozygous (Tg/Tg) and heterozygous GH transgenic (Tg/+) Amago and the wild type control (+/+). Previously, we have reported that the adipose tissue was generally smaller in GH transgenic fish compared to the control, and that the &Dgr;-6 fatty acyl desaturase gene was down-regulated in the Tg/+ fish. However, fatty acid (FA) compositions have not been measured previously in these fish. In this study we compared the FAs composition and content in the liver using gas chromatography. Eleven kinds of FA were detected. The composition of saturated and monounsaturated fatty acids (SFA and MUFA) such as myristic acid (14:0), palmitoleic acid (16:1n-7), and cis-vaccenic acid (cis-18:1n-7) was significantly (P<0.05) decreased in GH transgenic Amago. On the other hand, the composition of polyunsaturated fatty acids (PUFAs) such as linoleic acid (18:2n-6), arachidonic acid (20:4n-6), and docosapentaenoic acid (22:5n-3) was significantly (P<0.05) increased. Levels of serum glucose and triacylglycerol were significantly (P<0.05) decreased in the GH transgenics compared with +/+ fish. Furthermore, 3′-tag digital gene expression profiling was performed using liver tissues from Tg/Tg and +/+ fish, and showed that Mid1 interacting protein 1 (Mid1ip1), which is an important factor to activate Acetyl-CoA carboxylase (ACC), was down-regulated in Tg/Tg fish, while genes involved in FA catabolism were up-regulated, including long-chain-fatty-acid–CoA ligase 1 (ACSL1) and acyl-coenzyme A oxidase 3 (ACOX3). These data suggest that liver tissue from GH transgenic Amago showed starvation by alteration in glucose and lipid metabolism due to GH overexpression. The decrease of serum glucose suppressed Mid1ip1, and caused a decrease of de novo FA synthesis, resulting in a decrease of SFA and MUFA. This induced expression of ACSL1 and ACOX3 to produce energy through &bgr;-oxidation in the GH transgenic Amago.