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Torafugu myosin heavy chain gene, MYH M2528-1 , is specifically expressed in neonatal slow and fast muscle fibers, suggesting its functional role in indeterminate muscle growth in fish. However, the transcriptional regulatory mechanisms of MYH M2528-1 involved in indeterminate muscle growth in fish remained unknown. We previously isolated a 2100 bp 5'- flanking sequence of torafugu MYH M2528-1 that showed sufficient promoter activity to allow specific gene expression in neonatal muscle fibers of zebrafish. Here, we examined the cis-regulatory mechanism of 2100 bp 5'-flanking region of torafugu MYH M2528-1 using deletion-mutation analysis in zebrafish embryo. We discovered that myoblast determining factor (MyoD) binding elements play a key role and participate in the transcriptional regulation of MYH M2528-1 expression in zebrafish embryos. We further discovered that paired box protein (Pax3) are required for promoting MYH M2528-1 expression and myocyte enhancer factor-2 (MEF2) binding sites participate in the transcriptional regulation of MYH M2528-1 expression in slow/fast skeletal muscles. Our study also confirmed that the nuclear factor of activated T-cell (NFAT) binding sites take part in the transcriptional regulation of MYH M2528-1 expression in slow and fast muscles fiber in relation to indeterminate muscle growth. These results obviously confirmed that multiple cis-elements in the 5'-flanking region of MYH M2528-1 function in the transcriptional regulation of its expression., (Copyright © 2018. Published by Elsevier B.V.)

The Ofunato Bay in Iwate Prefecture, Japan is a deep coastal bay located at the center of the Sanriku Rias Coast and considered an economically and environmentally important asset. Here, we describe the first whole genome sequencing (WGS) study on the microbial community of the bay, where surface water samples were collected from three stations along its length to cover the entire bay; we preliminarily sequenced a 0.2 μm filter fraction among sequentially size-fractionated samples of 20.0, 5.0, 0.8 and 0.2 μm filters, targeting the free-living fraction only. From the 0.27-0.34 Gb WGS library, 0.9 × 10 6 -1.2 × 10 6 reads from three sampling stations revealed 29 bacterial phyla (~80% of assigned reads), 3 archaeal phyla (~4%) and 59 eukaryotic phyla (~15%). Microbial diversity obtained from the WGS approach was compared with 16S rRNA gene results by mining WGS metagenomes, and we found similar estimates. The most frequently recovered bacterial sequences were Proteobacteria, predominantly comprised of 18.0-19.6% Planktomarina (Family Rhodobacteraceae) and 13.7-17.5% Candidatus Pelagibacter (Family Pelagibacterales). Other dominant bacterial genera, including Polaribacter (3.5-6.1%), Flavobacterium (1.8-2.6%), Sphingobacterium (1.4-1.6%) and Cellulophaga (1.4-2.0%), were members of Bacteroidetes and likely associated with the degradation and turnover of organic matter. The Marine Group I Archaea Nitrosopumilus was also detected. Remarkably, eukaryotic green alga Bathycoccus, Ostreococcus and Micromonas accounted for 8.8-15.2%, 3.6-4.9% and 2.1-3.1% of total read counts, respectively, highlighting their potential roles in the phytoplankton bloom after winter mixing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio Currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The Ofunato Bay in the northeastern Pacific Ocean area of Japan possesses the highest biodiversity of marine organisms in the world and has attracted much attention due to its economic and environmental importance. We report here a shotgun metagenomic analysis of the year-round variation in free-living bacterioplankton collected across the entire length of the bay. Phylogenetic differences among spring, summer, autumn and winter bacterioplankton suggested that members of Proteobacteria tended to decrease at high water temperatures and increase at low temperatures. It was revealed that Candidatus Pelagibacter varied seasonally, reaching as much as 60% of all sequences at the genus level in the surface waters during winter. This increase was more evident in the deeper waters, where they reached up to 75%. The relative abundance of Planktomarina also rose during winter and fell during summer. A significant component of the winter bacterioplankton community was Archaea (mainly represented by Nitrosopumilus), as their relative abundance was very low during spring and summer but high during winter. In contrast, Actinobacteria and Cyanobacteria appeared to be higher in abundance during high-temperature periods. It was also revealed that Bacteroidetes constituted a significant component of the summer bacterioplankton community, being the second largest bacterial phylum detected in the Ofunato Bay. Its members, notably Polaribacter and Flavobacterium, were found to be high in abundance during spring and summer, particularly in the surface waters. Principal component analysis and hierarchal clustering analyses showed that the bacterial communities in the Ofunato Bay changed seasonally, likely caused by the levels of organic matter, which would be deeply mixed with surface runoff in the winter., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ofunato Bay, in Japan, is the home of buoy-and-rope-type oyster aquaculture activities. Since the oysters filter suspended materials and excrete organic matters into the seawater, bacterial communities residing in its vicinity may show dynamic changes depending on the oyster culture activities. We employed a shotgun metagenomic technique to study bacterial communities near oyster aquaculture facilities at the center of the bay (KSt. 2) and compared the results with those of two other localities far from the station, one to the northeast (innermost bay, KSt. 1) and the other to the southwest (bay entrance, KSt. 3). Seawater samples were collected every month from January to December 2015 from the surface (1 m) and deeper (8 or 10 m) layers of the three locations, and the sequentially filtered fraction on 0.2-μm membranes was sequenced on an Illumina MiSeq system. The acquired reads were uploaded to MG-RAST for KEGG functional abundance analysis, while taxonomic analyses at the phylum and genus levels were performed using MEGAN after parsing the BLAST output. Discrimination analyses were then performed using the ROC-AUC value of the cross validation, targeting the depth (shallow or deep), locality [(KSt. 1 + KSt. 2) vs. KSt 3; (KSt. 1 + KSt. 3) vs. KSt. 2 or the (KSt. 2 + KSt. 3) vs. KSt. 1] and seasonality (12 months). The matrix discrimination analysis on the adjacent 2 continuous seasons by ROC-AUC, which was based on the datasets that originated from different depths, localities and months, showed the strongest discrimination signal on the taxonomy matrix at the phylum level for the datasets from July to August compared with those from September to June, while the KEGG matrix showed the strongest signal for the datasets from March to June compared with those from July to February. Then, the locality combination was subjected to the same ROC-AUC discrimination analysis, resulting in significant differences between KSt. 2 and KSt. 1 + KSt. 3 on the KEGG matrix. These results suggest that aquaculture activities markedly affect bacterial functions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42-49%, 35-49% and 13-17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A high-temperature selected (HT) strain of rainbow trout was established from the Donaldson (DS) strain by traditional selective breeding in Japan. The aim of this study is to investigate genes related to upper temperature tolerance in this strain utilizing next generation sequencer (NGS), and to establish comprehensive and comparable datasets in brain, liver, muscle, heart and gill tissues between the HT and DS strains. After assembling, clustering and filtering, 242,530 contigs were obtained. Among them, 7624 transcripts had at least 10 counts in expression analysis in all tissues and used as references. BLASTX homology search showed that 7329 transcripts matched with known genes. Compared to the DS strain, the HT strain expressed 90, 775, 349, 188 and 194 genes 2 folds or more in brain, liver, muscle, heart, and gill, respectively in the case of fish before heat-exposure treatment. Meanwhile, the HT strain expressed 292, 363, 433, 322 and 211 genes 2 folds or more in brain, liver, muscle, heart, and gill, respectively in case of fish after heat-exposure treatment. Many of heat shock protein family genes and transcription factor AP-1 related genes were highly expressed in all tissues of the HT strain compared with the DS strain. These results suggest that these genes play key roles in upper temperature tolerance. These comprehensive and comparable datasets will offer broad visions for upper temperature tolerance in fish species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pax3 and Pax7 are the regulators and markers of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. However, information regarding them in vertebrate genome model, torafugu Takifugu rubripes, has remained unknown. Therefore, as an initial step, here we characterized Pax3 and Pax7 from torafugu and investigated their expression patterns during different developmental stages by RT-PCR. In silico analysis with the Fugu genome database (ver. 4.0) yielded two distinct genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b). The 75th amino acid, glutamine (Gln75), from the N-terminus was replaced by proline in the paired box domain (PD) of Pax3a. One single cDNA clone encoding Pax3a had deletion of Gln75 in PD, suggesting the presence of alternatively spliced variants (Q+/Q-). This was further supported by identification of two adjacent alternative 3' splice acceptor sites which produce Pax3b Q+ (aagCAGGGA) and Q- (aagcagGGA) variants. Interestingly, torafugu Pax7a, but not Pax7b, had an insert encoding five amino acid residues (SGEAS) in a C-terminal region of PD in two out of three cDNA clones. Genomic analysis showed two alternate splice donor sites at exon 4 of Pax7a. In synteny analysis, torafugu Pax3a showed syntenic relationship with the corresponding regions in other teleosts only, whereas Pax3b and Pax7b showed high syntenic relationship with the corresponding regions of both mammals and other teleosts. RT-PCR revealed that expression of Pax3a and Pax3b transcripts was restricted to embryonic stages only, whereas those of Pax7a and Pax7b was continued to be expressed in larvae and importantly those of Pax7a were found in adult skeletal muscles. Therefore, Pax3 appears to be most important for primary myogenesis and Pax7 for secondary myogenesis and growth by hyperplasia in fish. In this regard, the transcripts of torafugu Pax3 and Pax7 genes might be used for further investigation as a marker for identification of muscle precursor cells during different phases of growth, and this ambiguity is the next target of our research., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In vertebrates, the development-dependent and tissue-specific expression of myosin heavy chain (MYH) genes (MYHs) contributes to the formation of diverged muscle fiber types. The expression patterns of developmentally regulated MYHs have been investigated in certain species of fish. However, the expression profiles of MYHs during torafugu Takifugu rubripes development, although extensively studied in adult tissues, have not been sufficiently studied, and also the expression orders of MYHs during development have remained unclear. In the present study, we comprehensively cloned four MYHs (MYH(M743-2), MYH(M86-2), MYH(M5) and MYH(M2126-1)) from embryos, and two MYHs (MYH(M2528-1) and MYH(M1034)) from larvae, and characterized their expression pattern in relation to developmental stages of torafugu by reverse transcription (RT)-PCR and in situ hybridization. The expression of MYHs from torafugu embryos and larvae appeared sequentially and varied largely in relation to the developmental stage-dependent and fibers-type-specific manners. The transcripts of MYH(M743-2) appeared first in embryos at 3 days post fertilization (dpf) and were localized in the epaxial and hypaxial domains of fast muscle fibers of larval myotome, whereas those of MYH(M5) and MYH(M86-2) in 3 dpf and 4 dpf, respectively, and both were localized in superficial slow and horizontal myoseptum regions. The expression of MYH(M1034) and MYH(M2126-1) was quite low and mostly undetectable. Different MYHs from torafugu embryos and larvae have also been found to be expressed differentially in pectoral fin and craniofacial muscles. Interestingly, the transcripts of MYH(M2528-1) first appeared at 6 dpf and were distinctly expressed at the dorsal and ventral extremes of larval myotome, suggesting its involvement in stratified hyperplasia. The novel involvement of MYH(M2528-1) in mosaic hyperplasia was further confirmed in juvenile torafugu, where the transcripts were expressed in fast fibers with small diameters as well as the inner part of superficial slow fibers., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The myosin heavy chain gene, MYH(M743-2), is highly expressed in fast muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional regulatory mechanism involved in its expression remained unknown. Here, we analyzed the 2075bp 5'-flanking region of torafugu MYH(M743-2) to examine the spatial and temporal regulation by using transgenic and transient expression techniques in zebrafish embryos. Combining both transient and transgenic analyses, we demonstrated that the 2075bp 5'-flanking sequences was sufficient for its expression in skeletal, craniofacial and pectoral fin muscles. The immunohistochemical observation revealed that the zebrafish larvae from the stable transgenic line consistently expressed enhanced green fluorescent protein (EGFP) in fast muscle fibers. Promoter deletion analyses demonstrated that the minimum 468bp promoter region could direct MYH(M743-2) expression in zebrafish larvae. We discovered that the serum response factor (SRF)-like binding sites are required for promoting MYH(M743-2) expression and myoblast determining factor (MyoD) and myocyte enhancer factor-2 (MEF2) binding sites participate in the transcriptional control of MYH(M743-2) expression in fast skeletal muscles. We further discovered that MyoD binding sites, but not MEF2, participate in the transcriptional regulation of MYH(M743-2) expression in pectoral fin and craniofacial muscles. These results clearly demonstrated that multiple cis-elements in the 5'-flanking region of MYH(M743-2) function in the transcriptional control of its expression., (2011 Elsevier B.V. All rights reserved.)

Although energy metabolism in mammals is critically regulated by adipokines from adipocytes, it is unclear whether this is the case in fish as well. In this study, over 30,000 expressed sequence tags (ESTs) were obtained from adipose tissue in rainbow trout Oncorhynchus mykiss peritoneal cavity and searched for genes possibly related to lipid metabolism. Large numbers of ESTs encoded digestive enzymes and hormones usually found in the pancreas in higher vertebrates, consistent with the fact that pancreatic cells are dispersed in the adipose tissue. Many ESTs encoded apolipoprotein C-I, fatty acid-binding proteins and lymphocyte G0/G1 switch protein 2, which function in lipid transport, fatty acid accumulation and adipocyte differentiation, respectively. None of the ESTs encoded adipokines. We therefore obtained a cDNA encoding adiponectin, an adipokine that regulates oxidation of glucose and lipids in peripheral tissues, using rainbow trout ESTs in the public database. Real-time RT-PCR analyses revealed that its transcript levels were high in muscle and quite low in adipose tissue. These results strongly suggest that adipocytes of rainbow trout and possibly other fish species, unlike those of mammals, are not involved in the production of adipokines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The draft genome data of Medaka Oryzias latipes shows that it has distinct intraspecific genetic variation. To survey the genetic variations contributing to environmental adaptation, we focused on the mitochondrial DNA (mtDNA). The complete mtDNA sequences of Medaka were compared among 8 local population stocks and 4 inbred strains established from genetically divergent groups. Inbred strain HSOK, derived from the Eastern Korean group of Medaka, has a mitochondrial gene order that was distinct from other Medaka groups. Phylogenetic trees based on the mitochondrial genome sequences indicated that the mitogenome from the Shanghai stock (China) and HSOK strain were highly diverged from Japanese Medaka, and that the Japanese Medaka mitogenome was diverged into two groups; this result was fully consistent with those of the previous study using mtDNA-encode gene sequences. Among tRNA genes, the most divergent was the tRNA(Thr) gene as reported in humans previously. The number of tandemly repeated 11 nucleotide units in the Medaka mtDNA control region (CR) varied greatly among local populations. The number of repeats was more variable in the Northern Japanese group (10-34) than in the Southern group (7-12), while two other Oryzias species, inhabiting tropical regions, had no repeats. A comprehensive comparison between the number of repeat units and meteorological data indicated that the number of repeats correlated to the index data of a cold environment and seasonal climatic change. In cold (5 degrees C) acclimated fish, the mRNA levels varied among mitochondria coding genes. mRNA of the cytochrome oxidase subunit I gene in some local stocks was induced by cold temperature and seemed to be correlated with the number of repeated sequences in the CR. This study revealed that the repeated sequences in the mtDNA CR might function for mtDNA gene expression and that the number of tandem repeats in Medaka mtDNA is likely related to adaptation to a harsh habitat., (Copyright 2010 Elsevier B.V. All rights reserved.)

Silver carp Hypopthalmichthys molitrix is eurythermal temperate fish, whose muscle is considered to express several types of myosin heavy chain (MYH) genes at different stages of its growth and to adjust to the environmental temperature. In this study, MYH genes expressed in the muscles of juvenile and adult silver carp were investigated. Five types of MYH cDNA clone were isolated from silver carp (H. molitrix) by RACE strategy using a set of fast-type MYH specific primers, and termed scMYH(F1), scMYH(F2), scMYH(F3), scMYH(F4) and scMYH(F5) in the order of their abundance in cDNA libraries constructed from fast skeletal muscles of adult silver carp. scMYH(F1), scMYH(F3) and scMYH(F5) showed high nucleotide sequence identities of 96, 98 and 96% to gcMYH(F30), gcMYH(F10) and gcMYH(FI), respectively, that encode MYHs predominantly expressed in fast skeletal muscle of grass carp (Ctenopharyngodon idella) acclimated to 30, 10 and 20 degrees C, respectively. scMYH(F2) and scMYH(F4) showed a high identity to A4-type MYH from rock cod (Notothenia coriiceps) slow skeletal muscle. Phylogenetic analysis demonstrated that scMYH(F1) and scMYH(F5) were monophyletic with fish adult fast-type MYHs, whereas scMYH(F2) and scMYH(F4) formed a cluster with fish slow-like fast-type MYH, and scMYH(F3) did with fish embryonic fast-type MYHs. Interestingly, juvenile silver carp predominantly expressed scMYH(F3) irrespective of acclimation temperatures at 10, 18 or 26 degrees C. The comparison among scMYH(F1), scMYH(F2) and scMYH(F3) in the deduced amino acid sequence revealed that the putative binding sites for ATP, actin, and essential and regulatory light chains in myosin subfragment-1 (S1) have high identities with each other (81-100%). However, their loop-1 and loop-2 regions in S1 were highly variable, suggesting their different functions. The deduced amino acid sequences of myosin subfragment-2 and L-meromyosin showed high identities of 90-91% and 86-90%, respectively, among the above three scMYHs.

We characterized the promoter activity of fast skeletal myosin heavy chain genes (MYHs) from medaka Oryzias latipes. The 5'-flanking region of approximately 6 kb in medaka MYHs, mMYH10 and mMYH30, predominantly expressed in medaka acclimated to 10 degrees C and 30 degrees C, respectively, contained various cis-elements that are supposed to bind to transcriptional regulatory factors such as MyoD and myocyte enhancer factor 2 (MEF2) family members and nuclear factor of activated T cells. To localize functional regions responsible for the mMYH expression in a temperature-dependent manner, a series of deletion and site mutation constructs prepared from the 5'-flanking regions were fused to the luciferase gene in a commercially available plasmid and directly injected into the dorsal fast muscle of medaka acclimated to 10 degrees C and 30 degrees C. The truncation of MEF2 binding site located at -966 to -957 in the 5'-flanking region of mMYH10 resulted in distinct gene expression at 10 degrees C. The activation effect by the removal of this binding site was further confirmed by the mutation construct. One of the E box sites, to which MyoD family members are supposed to bind, was located at -613 to -607 of mMYH10, and found to be responsible for the transcriptional activity. In contrast, the MEF2 binding site located at -960 to -951 of mMYH30 was involved in the activation at 30 degrees C. Thus, these transient transfection assays demonstrated that the MEF2 binding site is crucial for a temperature-dependent expression of mMYHs.

Two types of the fast skeletal myosin heavy chain (MYH) genes were cloned from a genomic DNA library of carp (Cyprinus carpio L.) and named MYH10 and MYH30, which showed the sequence similarity to the MYH cDNAs predominantly expressed in carp acclimated to 10 and 30 degrees C, respectively. The 5'-flanking region of about 3 kbp in size each from MYH10 and MYH30 contained various cis-elements to bind to transcriptional regulatory factors such as MyoD family and myocyte enhancer factor 2 (MEF2) family members. To localize functional regions responsible for the MYH gene expression in a temperature-dependent manner, a series of deletion constructs were prepared from the 5'-flanking region, inserted upstream the luciferase gene in a commercially available plasmid, and injected into the dorsal fast muscle of carp acclimated to 10 and 30 degrees C. The sequence of -1004 to -995 bp with the transcriptional activity in MYH30 was identified as an MEF2 binding site. While the activity given by a sequence of -921 to -824 bp in MYH10 contained only a GATA box, that of the activity of the -1 kbp construct from MYH10 was markedly higher in carp reared at 10 degrees C than fish reared at 30 degrees C. On the other hand, no temperature-dependent expressional regulation was observed for MYH30 even with the full-length construct of -3 kbp. The DNA fragment of -921 to -824 bp in MYH10 and MEF2 binding site in MYH30 interacted with nuclear proteins extracted from carp fast skeletal muscle as revealed by electrophoretic mobility shift assay. The signal intensity of a complex formed between the DNA fragment of MYH10 and nuclear extracts from the 10 degrees C-acclimated carp were higher than those with extracts from the 30 degrees C-acclimated fish. Although MEF2-binding site in MYH30 could form complex with nuclear extracts from the 30 degrees C-acclimated carp, the same or stronger signals were detected in complex formed with extracts from the 10 degrees C-acclimated fish.

We have determined the complete DNA nucleotide sequence of the carp Cyprinus carpio fast skeletal myosin heavy chain (MYH) gene. Introns and exons were predicted by comparison with the corresponding carp MYH cDNAs previously reported. The gene encoded the entire mRNA transcript and contained 5958 nucleotides (nt) including 77 nt 5'-untranslated region, 5796 nt coding region for 1931 amino acids, and 85 nt 3'-untranslated region. The coding region was split by 38 introns and the complete gene contained 11,385 nt. This integration of the carp fast skeletal MYH gene was comparable to those of the rat and chicken embryonic MYH genes, which have 41 and 40 exons, respectively. However, the entire gene size of carp MYH was about half those of rat and chicken due to much smaller size of carp introns. We have also demonstrated that this carp MYH gene belonged to so-called intermediate type in a multigene family of carp fast skeletal muscle MYH in comparison of its nucleotide and deduced amino acid sequences to those of carp MYH cDNAs reported previously.

We have characterized the apolipoprotein multigene family of the pufferfish Takifugu rubripes. The pufferfish mainly contains 28-kDa, 27-kDa, and 14-kDa apolipoproteins in its plasma and was designated apo-28 kDa, apo-27 kDa, and apo-14 kDa, respectively. N-terminal amino acid sequencing revealed that pufferfish apo-28 kDa and apo-27 kDa have an identical amino acid sequence except an additional propeptide in the former; and both are homologues of apoA-I from other animals. The sequence of pufferfish apo-14 kDa is homologous to that of eel apo-14 kDa previously reported, both being apparently specific to fish. In silico screening, using the publicly available Fugu genome database confirmed the pufferfish apoA-I and apo-14 kDa genes. The database further contained the genes encoding four types of apoA-IV, one apoC-II and two types of apoE. Thus, pufferfish contains nine genes encoding apolipoprotein multigene family. Two apoA-IV and one apoE genes were tandemly arrayed and located on one scaffold. Thus two sets of these genes formed two gene clusters. The apoC-II and apo-14 kDa genes are also located on a single scaffold. apoA-I and apo-14 kDa gene transcripts were mainly expressed in liver and less abundantly in brain. The transcripts of the former gene were also observed in intestine. In contrast, the transcripts encoding four apoA-IVs, one apoC-II, and two apoEs were mainly expressed in intestine. These structural details of pufferfish apolipoproteins and tissue distribution of their gene transcripts provide a novel evidence for better understanding of evolutionary relationships of apolipoprotein multigene family.

The Japanese pufferfish, torafugu (Fugu rubripes), has a haploid genome of about 400 Mb in size, which has been sequenced to approximately 90% coverage. Here we identified six Fugu tropomyosin (TPM) gene sequences by using the BLASTN program and the sequence of the white croaker TPM1 gene in our collection against the draft assembly of the Fugu genomic sequence database. TPM2, TPM3 and TPM4 genes were identified together with a set of two potentially duplicated genes of TPM1 (TPM1-1 and TPM1-2) as described in our previous report and TPM4 (TPM4-1 and TPM4-2) newly found in this study. The expression patterns of these Fugu TPM genes were determined by reverse transcription polymerase chain reaction (RT-PCR). A phylogenetic tree was constructed using the deduced amino acid sequences, which were encoded by the exons common to all vertebrate TPM genes. This indicated that the Fugu TPM1 and TPM4 genes had resulted from a gene duplication in the fish evolutionary lineage.