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We cloned and sequenced the complete mitochondrial DNA (mtDNA) of Cirrhinus mrigala (♀) × Labeo rohita (♂) by PCR-based method. The total of the mtDNA was 16,595 bp. It consisted of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and one putative control region (D-loop region). The overall base composition on strand was as follows A: 32.09%, G: 15.51%, C: 27.95%, T: 24.45%, and the A + T content 56.54%. All the protein-coding genes initiated by typical ATG codon, and ten genes ended with the complete stop codon TAA or TAG, while COII2, ND4 and Cytb genes terminated with GCC, TAT and GCT. The control region contains a thiolases active site, EGF-like domain signature and two anaphylatoxin domain signature. This mitogenome sequence data would play an important role in population genetics and phylogenetics of the hybrid fish in the Cyprinidae family.

In this study, we determined the complete mitochondrial DNA sequence of the hybrid of Ctenopharyngodon idellus (♀) × Elopichthys bambusa (♂). The total length of the mitochondrial genome was 16,609 bp (accession number KM401549), with the base composition of 31.89% A, 26.33% C, 15.68% G, 26.11% T. The genome contained 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes, and a major non-coding control region (D-loop region). Except for ND6 and 8 tRNAs, all other genes were encoded on the heavy strand. The arrangements of these genes were similar to that found in the other teleosts. The complete mitogenome may provide important date set for the study of genetic mechanism in the hybrid fish of Cyprinidae.

The Chinese perch (Siniperca chuatsi) is one of the most commercially important carnivorous fish species in aquaculture with its large-scale culture in China. Increasing evidence suggests that microRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of the identity of myogenic miRNAs and the effect of nutrient status on miRNA expression in teleost remains limited. In the present study, among the 21 miRNAs identified with high abundance in the fast muscle of adult Chinese perch, 19 miRNAs were differentially expressed in the adults and juveniles. The postprandial changes in the transcript abundance were determined for the 21 miRNAs following a single satiating meal in the juveniles after fasting for 1 week. The results showed that the seven miRNAs (miR-10c, miR-107a, miR-133a-3p, miR-140-3p, miR-181a-5p, miR-206, and miR-214) were sharply upregulated or downregulated within 1 h after refeeding. These miRNAs may be the promising candidate miRNAs involved in a fast-response signaling system that regulates fish skeletal muscle growth. Target prediction and expressional analysis suggested that four miRNAs (miR-10c, miR-107a, miR-140-3p, and miR-181a-5p) might play a role in regulating the translation of target gene transcripts such as myostatin following acute anabolic stimuli.

Myogenin plays a crucial role in myoblast differentiation and maturation. In the present study, the myogenin gene structure and expressional patterns in Siniperca chuatsi were characterized. Sequence analysis indicated that the myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Sequence alignment and phylogenetic analysis showed that Siniperca chuatsi myogenin shared homologous with Epinephelus coioides, Sparus aurata, Takifugu rubripes and Salmo salar. Whole-mount in situ hybridization revealed that myogenin expression was first detected in the gastrula stage embryos and high levels of expression at the 24 somite stage. After the 24 somite stage, myogenin expression began to decrease in the anterior somites where somatic cells were differentiated. Further, the muscle structural gene, MyHC, and myogenin, are concomitantly expressed during S. chuatsi embryonic development as assayed with whole-mount in situ hybridization. In the adult fish, the myogenin showed the highest levels of expression in the brain compared with the kidney, spleen, liver, heart and white muscles. Our work on the myogenin gene from the mandarin fish has provided useful information for fish molecular biology and fish genomics.

The fast and slow skeletal muscle fibers in fish are separated to a much greater degree than in mammals. MyoD is required for the maintenance of normal fiber type balance in muscles. So far, the upstream regulatory factors of MyoD in terms of controlling muscle phenotype are poorly understood. In the present study, we used Siniperca chuatsi as a model system and demonstrated that miR-143 expression was negatively correlated with MyoD expression in the fast and slow muscles of S. chuatsi. The luciferase reporter assay further verified the direct interaction between the miR-143 and MyoD. The miR-143 suppression also led to the significant increase in MyoD and fast myosin heavy chain gene expression in vivo. Taken together, our studies indicated that miRNA participates in controlling the performance of different muscle fiber types in vertebrates.

In this study, we cloned and sequenced the complete mitochondrial DNA of ricefield eel populations from four different areas (Guizhou province, Guangxi province, Hunan province, and Guangdong province) in China. The mitochondrial genome was 16,622 bp in length and deposited in the GenBank with accession numbers KP779622-KP779625. The organizations of the complete mitogenomes of the four ricefield eel were similar to those of other teleost species which contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, as well as a putative control region (CR). Most of these genes were encoded on the H-strand, except for the ND6 and eight tRNA genes, all other genes were encoded on the H-strand. Phylogenetic analyses using N-J computational algorithms showed that the ricefield eel was clustered with Mastacembelus armatus (KJ184553) and they belong to Synbranchiformes. The four ricefield eel populations could be divided into two groups: Guangxi province and the other population cluster together.

Sinipercids are a group of freshwater perciform fish endemic in East Asia with the majority of species recorded in China. In the present study, we analyzed the organizations of complete mitogenomes of the six sinipercid fish populations from the Yangtze River and the Pearl River systems in China. The proportion of G was relatively low just at 16.2%-16.5% among the fish populations analyzed. The similarity and divergence analysis showed that the populations from different drainages have more sequence similarities than those from different species in the same drainages. The rate of point mutation was 0.39% and a total of 422 variable sites were identified in the 12 protein-coding genes (excluding the ND6) among the four S. scherzeri populations. The analysis of evolutionary relationship showed that the four S. scherzeri populations can be divided into two groups of Yangtze River system and the Pearl River. Overall the data provide significant information for the genetic and evolutionary status of sinipercids upon adapting to different environments, and for genetic conservation and selective breeding of these fish species in fishery and aquaculture.

MicroRNAs (miRNAs) participate in the regulation of myogenesis and muscle physiological function. Most skeletal muscles in vertebrates contain a mixture of fibertypes. So far, the regulatory mechanism of the miRNA in terms of controlling muscle phenotype is poorly understood. In the present study, we use Siniperca chuatsi as a model system and demonstrate that miRNAs are involved in regulating the physiological processes and metabolism of different muscle fibers in vertebrates. The miRNA transcriptomes of the white muscle, red muscle, and five other tissues from Siniperca chuatsi were profiled using Solexa deep sequencing. We characterized 186 conserved miRNAs and 3 novel miRNAs from the two small RNA libraries of white and red muscles. Among the 155 miRNAs overlapped between the two libraries, we identified 60 significantly expressed miRNAs between the two types of muscle fibers. Using integrative miRNA target-prediction and network-analysis approaches, an interaction network of differentially expressed and muscle-related miRNAs and their putative targets were constructed. Sch-miR-181a-5p that could act to control the performance of the different muscle fiber types by targeting the myostatin gene was identified.

The extant freshwater sinipercids represent a group of 12 species and they are endemic to East Asia. In this study, we cloned and sequenced the complete mitochondrial DNA of Siniperca obscura from the Lijiang River. The size of the complete mitochondrial genome is 16,492 bp. The organization of the mitochondrial contained 37 genes (13 protein-coding genes, 2 ribosomal RNA and 22 transfer RNAs) and a major non-coding control region as well as those reported sinipercid fishes. Among the 13 protein-coding genes, three reading-frame overlaps were found: ATP8 and ATP6 overlap by 10 nucleotides and ND4 and ND4L overlap by 7 nucleotides and ND5 and ND6 overlap by 5 nucleotides. Phylogenetic analyses using N-J and maximum parsimony (MP) computational algorithms showed that S. chuatsi and S. kneri are sister species, next joined by S. Obscura, based on combined 12 protein-coding genes (excluding DN6).

The sinipercids are a group of 12 species of freshwater percoid fish endemic to East Asia and their phylogenetic placements have perplexed generations of taxonomists. We cloned and sequenced the complete mitochondrial DNA (mtDNA) of three sinipercid fishes (Siniperca chuatsi, S. kneri, and S. scherzeri) to characterize and compare their mitochondrial genomes. The mitochondrial genomes of S. chuatsi, S. kneri, and S. scherzeri were 16,496, 17,002, and 16,585 bp in length, respectively. The organization of the three mitochondrial genomes is similar to those reported from other fish mitochondrial genomes, which contains 37 genes (13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs) and a major non-coding control region. Among the 13 protein-coding genes of all the three sinipercid fishes, three reading-frame overlaps were found on the same strand. There is an 81-bp tandem repeat cluster at the end of CSB-3 in the S. scherzeri control region. The complete mitochondrial genomes of the three sinipercids should be useful for the evolutionary studies of sinipercids and other vertebrate species.

Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.

The objective of this study was to determine developmental changes in mRNA and protein levels for N-acetylglutamate synthase (NAGS; a key enzyme in synthesis of citrulline and arginine from glutamine/glutamate and proline) in the small intestine of suckling piglets. The porcine NAGS gene was cloned using the real-time polymerase-chain reaction (RT-PCR) method. The porcine NAGS gene encoded 368 amino acid residues and had a high degree of sequence similarity to the “conserved domain” of human and mouse NAGS genes. The porcine NAGS gene was expressed in E. coli BL21 and a polyclonal antibody against the porcine NAGS protein was developed. Real-time RT-PCR and western-blot analyses were performed to quantify NAGS mRNA and protein, respectively, in the jejunum and ileum of 1- to 28-day-old pigs. Results indicated that intestinal NAGS mRNA levels were lower in 7- to 28-day-old than in 1-day-old pigs. Immunochemical analysis revealed that NAGS protein was localized in enterocytes of the gut. Notably, intestinal NAGS protein abundance declined progressively during the 28-day suckling period. The postnatal decrease in NAGS protein levels was consistent with the previous report of reduced NAGS enzymatic activity as well as reduced synthesis of citrulline and arginine in the small intestine of 7- to 28-day-old pigs. Collectively, these results suggest that intestinal NAGS expression is regulated primarily at the post-transcriptional level. The findings also provide a new molecular basis to explain that endogenous synthesis of arginine is impaired in sow-reared piglets and arginine is a nutritionally essential amino acid for the neonates.

To identify muscle-related protein isoforms expressed in the white muscle of the mandarin fish Siniperca chuatsi, we analyzed 5,063 high-quality expressed sequence tags (ESTs) from white muscle cDNA library and predicted the integrity of the clusters annotated to these genes and the physiochemical properties of the putative polypeptides with full length. Up to about 33% of total ESTs were annotated to muscle-related proteins: myosin, actin, tropomyosin/troponin complex, parvalbumin, and Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCa). Thirty-two isoforms were identified and more than one isoform existed in each of these proteins. Among these isoforms, 14 putative polypeptides were with full length. In addition, about 2% of total ESTs were significantly homologous to “glue” molecules such as alpha-actinins, myosin-binding proteins, myomesin, tropomodulin, cofilin, profilin, twinfilins, coronin-1, and nebulin, which were required for the integrity and maintenance of the muscle sarcomere. The results demonstrated that multiple isoforms of major muscle-related proteins were expressed in S. chuatsi white muscle. The analysis on these isoforms and other proteins sequences will greatly aid our systematic understanding of the high flexibility of mandarin fish white muscle at molecular level and expand the utility of fish systems as models for the muscle genetic control and function.

The class B scavenger receptor CD36 binds multiple ligands, including oxidized and native lipoprotein species. CD36 and the related receptor SR-B1 have been localized to caveolae, domains that participate in cell signaling, transcytosis, and regulation of cellular cholesterol homeostasis. Previous work has indicated that the ligand preference of CD36 may depend on the cell type in which it is expressed. To determine if the presence or absence of caveolae is the determining factor for lipoprotein preference, we treated CHO-CD36 and C32 cells with filipin. Filipin treatment rapidly increased the binding capacity of CD36 for the native lipoproteins HDL and LDL, but did not affect the binding capacity of CD36 for oxidized LDL. Filipin treatment affected the distribution of caveolin and CD36 suggesting that the presence caveolae may modulate the ligand preference of CD36. However, its molecular mechanism how CD36 and caveolin interaction in regulating lipoprotein transport remains to be further studied.

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus which belongs to the genus Avibirnavirus of the family Birnavirideae. In this study, we determined the complete nucleotide sequences of a reassortment IBDV strain TL2004 with segments A and B derived from attenuated and very virulent strains of IBDV. This strain is pathogenic to SPF-embryonated eggs and chickens, although it is not as virulent as very virulent strain. Genomic sequence in GenBank analysis showed that both types of natural genetic reassortment of infectious bursal disease virus emerged in China. Our findings, which strongly suggest genetic exchange between attenuated and very virulent strains of IBDV, emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines.

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals. During infection and induction of the host immune response, outer membrane proteins of bacteria play an important role. In this study, an outer membrane protein gene (ompW) was cloned from V. alginolyticus and expressed in Escherichia coli. The 645 bp open reading frame (ORF) encodes a protein of 214 amino acid residues with a predicted molecular weight of 23.3 kDa. The amino acid sequence showed a high identity with that of Photobacterium damselae (96.2%) and Vibrio parahaemolyticus (94.4%). The alignment analysis indicated that OmpW was highly conserved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the gene was over-expressed in E. coli BL21(DE3). Western blot analysis revealed that the expressed protein had immunoreactivity. The recombinant protein was purified by affinity chromatography on Ni-NTA Superflow resin. Large yellow croaker vaccinated with the purified OmpW showed significantly increased antibody to OmpW, which could resist the infection by V. alginolyticus. A specific antibody was detected by enzyme-linked immunosorbent assay. This study suggested that the conserved OmpW could be an effective vaccine candidate against infection by V. alginolyticus.

Recently, we cloned the adult alpha-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these alpha-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid alpha- and beta-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked alpha- and beta-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid alpha- and beta-globin genes.

In this study, we cloned and sequenced the complete mitochondrial DNAs of Chinese mandarin fish, Siniperca scherzeri populations from four different areas of the Yangtze River and the Pearl River systems in China. In the Yangtze River system, fish samples were taken from three regions, Dongting Lake (DT), Poyang Lake (PL) and Xiangjiang River (XR). In the Pearl River system, fish samples were collected from the Lijiang River (LR). The sizes of their complete mitochondrial genome were 16,502 bp (LR), 16,508 bp (PL), 16,502 bp (XR) and 16,508 bp (DT), respectively. The organization of the S. scherzeri mitochondrial genomes was similar to those reported from other fish mitochondrial genomes. Phylogenetic analyses using N-J computational algorithms showed that the Sinipercidaes are monophyletic and can be divided into two genera, Siniperca and Coreoperca. The S. scherzeri could be divided into two groups along the Yangtze River system and the Pearl River.

12 páginas, 7 figuras, Background Smyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos. Methodology/Principal Findings To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1. Conclusion/Significance Together, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1., This research was supported by research grant No IS-8713-08 from the Israel Binational Agricultural Research and Development Fund, the United States (BARD), and an intercenter collaboration grant (Du-Fang) from University of Maryland Biotechnology Institute. (http://www.bard-isus.com/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.