Molecular characterization of myostatin in black seabream, Acanthopagrus schlegelii.

Myostatin is a negative regulator of skeletal muscle growth and has a potential application in aquaculture. The black seabream myostatin gene was cloned and sequenced. It had three exons encoding a protein of 382 amino acids. A 90 bp 5'-untranslated region (UTR) and a 536 bp 3'-UTR were obtained by RACE. Four microsatellite sequences, a (CAG)9, a (TC)12, a (CA)16 repeat and an "imperfect" (CA)25 microsatellite, were found in the myostatin. Two introns were 329 and 742 bp in length, respectively. The deduced amino acid sequence of the myostatin had a putative amino terminal signal sequence, a TGF-beta propeptide domain, a RXXR proteolytic processing site, a TGF-beta domain, and 12 conserved cysteine residues. The myostatin gene was expressed in four of the examined ten tissues and organs. The expression of myostatin was the strongest in the skeletal muscle and brain, intermediate in the eye, and low in the heart.     

Molecular Cloning and Expression Analysis of the Myostatin Gene in Sea Perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart. It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further, it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures.     

MiRNA-19a and miRNA-19b regulate proliferation of antler cells by targeting TGFBR2

Transforming growth factor (TGF-β) plays a pivotal role in angiogenesis. The purpose of this study was to explore the microRNA-mediated regulation of TGF-β receptor-II (TGFBR2) expression during rapid antler growth and proliferation of antler cells in sika deer. Deep sequencing–based expression analysis of miRNAs on the antler tip tissue was performed. Then, two bioinformatics software were used to analyze TGFBR2 3′-UTR sequence for predicting the matched and differentially expressed miRNAs in different tissues of the antler. The results indicated that miRNA-19a and miRNA-19b exhibited the highest upregulation among differentially expressed miRNAs. We also found that the TGFBR2 3′-UTR contains a binding site for miRNA-19a and miRNA-19b by transfection of wild-type and mutant dual-luciferase reporter vectors into antler cartilage cells. Meanwhile, overexpression of miRNA-19a and miRNA-19b significantly inhibited the proliferation of cartilage cells in vitro, and decreased the expression level of TGFBR2 protein. Furthermore, the expression levels of insulin-like growth factor 1 (IGF-1) and TGF-β2, which were associated with TGFBR2, reduced after transfection of cartilage cells with miRNA-19a and miRNA-19b. Our results indicate the significant roles of miRNA-19a and miRNA-19b in proliferation of antler cells and its potential application.     

Molecular analysis of the QM gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression on the different ovarian stages of development

In present study, a QM gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn QM (PmQM) cDNA contained a 5′-UTR of 41 bp, an ORF of 663 bp encoding a polypeptide of 220 amino acids with molecular weight 25.5 kDa, and a 3′-UTR of 54 bp. Homology analysis of the deduced amino acid sequence of the PmQM with other known QM sequences by MatGAT software revealed that the PmQM was high homology with other invertebrates. A conserved signature sequence of the QM family was found in the PmQM deduced amino acid sequence. Analysis of the tissue expression pattern of the PmQM gene showed that the PmQM mRNA was expressed in all tissues tested, with highest levels in ovary. Furthermore, the PmQM expression was found to be different in three important ovarian stages of development. The results indicated PmQM might play an important role in ovarian development.     

Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by β-strands 6 and 7 of the crystal structures of bovine RIα and rat RIIβ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–[PV]–R–[ANQV]–A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects α-helix B′ and β strand 7. Received: 2 November 2000/Accepted: 14 June 2001     

Cloning and characterization of squalene synthase gene from <Emphasis Type="Italic">Fusarium fujikuroi</Emphasis> (Saw.) Wr.

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome. Accession numbers AF015784 and AF015785 at the GenBank.     

Two Highly Homologous Promoters of a Squash Aspartic Protease Inhibitor (SQAPI) Multigene Family Exhibit Differential Expression in Transgenic Tobacco Phloem and Trichome Cells

Two variants of the promoter of the squash aspartic acid protease inhibitor multigene family were isolated from Cucurbita maxima cv. ‘Supermarket’ Hybrid genomic DNA. The isolated promoters, possibly not full length, comprised a 5′-untranslated region (UTR) of 202–208 bp, contained a 63-bp upstream open reading frame (uORF) and the immediate upstream sequences of 441–445 bp. The two promoters contained several small deletions relative to each other and 22 single base differences but exhibit overall 92.5% homology over 654 bp. When the promoters were fused to a β-glucuronidase reporter gene and expressed in tobacco, one variant was highly expressed in the companion cells of the inner and outer phloem of leaves and at lower levels in other organs. The other variant was expressed at high levels in the long glandular trichomes of the leaf. Deletion analysis identified a region of ~280 bp immediately upstream of the 5′-UTR containing the TATA box that was responsible for phloem specific expression and a further region of ~180 bp that enhanced expression in one promoter and conferred trichome expression in the other. Removal of the 5′-UTR, including the uORF, inactivated the phloem promoter.     

The First Complete cDNA Sequence of the Hemocyanin from a Bivalve,the Protobranch <Emphasis Type="Italic">Nucula nucleus</Emphasis>

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M _r = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M _r = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million years ago, in conformity with fossil records from the late Cambrian. [Reviewing Editor: Dr. Rüdiger Cerff] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1 and AJ786640 for NnH2.     

Miiuy croaker (Miichthys miiuy) Peroxiredoxin2: Molecular characterization,genomic structure and immune response against bacterial infection

Peroxiredoxin2 (Prx2) protein is an important member in cellular antioxidant protein superfamily. Prx2 exists widely in prokaryotes and eukaryotes, it not only plays a part in eliminate reactive oxygen, but also takes effect in many other metabolic activities, such as stimulate epithelial cell proliferation, participate in the signal transduction in cells and so on. After molecular cloning we got that the complete cDNA sequence of Prx2 consists 882 bp, including a 5′-UTR of 46 bp, an open reading frame (ORF) of 591 bp, and a 3′-UTR of 245 bp. The complete gene of miiuy croaker Prx2 has 5 exons and 4 introns. The deduced 197 amino acid residues of miiuy croaker Prx2 consists a Val-Cys-Pro (VCP) motifs. In order to better elucidate the immune mechanisms of the Prx2 in the lower vertebrates, we conducted a research about the Prx2 gene of miiuy croaker and its expression pattern after bacterial infection. Real-time PCR (RT-PCR) results showed that expression of Prx2 was up-regulated in kidney, liver and spleen under infection with Vibrio anguillarum, and expressed level differently in ten different uninjected tissues. Our results suggested that Prx2 might be involved in immune defence in miiuy croaker.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Close Evolutionary Relatedness of α-Amylases from Archaea and Plants

The amino acid sequences of 22 α-amylases from family 13 of glycosyl hydrolases were analyzed with the aim of revealing the evolutionary relationships between the archaeal α-amylases and their eubacterial and eukaryotic counterparts. Two evolutionary distance trees were constructed: (i) the first one based on the alignment of extracted best-conserved sequence regions (58 residues) comprising β2, β3, β4, β5, β7, and β8 strand segments of the catalytic (α/β)₈-barrel and a short conserved stretch in domain B protruding out of the barrel in the β3 →α3 loop, and (ii) the second one based on the alignment of the substantial continuous part of the (α/β)₈-barrel involving the entire domain B (consensus length: 386 residues). With regard to archaeal α-amylases, both trees compared brought, in fact, the same results; i.e., all family 13 α-amylases from domain Archaea were clustered with barley pI isozymes, which represent all plant α-amylases. The enzymes from Bacillus licheniformis and Escherichia coli, representing liquefying and cytoplasmic α-amylases, respectively, seem to be the further closest relatives to archaeal α-amylases. This evolutionary relatedness clearly reflects the discussed similarities in the amino acid sequences of these α-amylases, especially in the best-conserved sequence regions. Since the results for α-amylases belonging to all three domains (Eucarya, Eubacteria, Archaea) offered by both evolutionary trees are very similar, it is proposed that the investigated conserved sequence regions may indeed constitute the ``sequence fingerprints' of a given α-amylase. Received: 3 June 1998 / Accepted: 20 August 1998     

Incorporation of a β-turn mimic in the N-terminal fragment of the B1 domain of streptococcal protein G

Summary A dibenzofuran-based β-turn mimic has been incorporated in the B1_2–29 fragment of the B1 domain of streptococcal protein G. This amino acid sequence adopts a β-hairpin structure in the complete B1 domain (B1_2–56). The modified peptide was studied by CD and NMR spectroscopy and its solution behavior was compared with the conformation adopted by the same sequence in the modified B1 domain.     

Molecular cloning,expression analysis and chromosome localization of the <Emphasis Type="Italic">Tpt1</Emphasis> gene coding for the pig translationally controlled tumor protein (TCTP)

This work describes the cloning and structural analysis of a Tpt1 cDNA coding for the porcine translationally controlled tumor protein (TCTP) molecule and its expression in porcine cells and tissues. Pig Tpt1 cDNA is 842-pb long that displays typical features of translationally controlled mRNAs, including a 5′-UTR containing a 5′-terminal oligopyrimidine tract (5′-TOP), and a 3′-UTR with a high CG-content and one AU rich element (ARE). Both 5′-UTR and 3′-UTR are highly conserved when they are compared with those of other mammals. The pig Tpt1 cDNA contains a 516-b open reading frame that encodes a predicted TCTP protein composed of 172 amino acids that exhibits extensive conservation compared with TCTP sequences from other species and a common structural feature with all the other TCTP proteins analyzed in mammals. Expression analysis demonstrated that Tpt1 mRNA is ubiquitously expressed in normal porcine tissues and cells, showing a higher expression in spleen, lymph nodes and lung, and a lower one in skin and heart. The pig Tpt1 gene localizes on the porcine chromosome 11, region p11.     

Ontogeny of respiratory area of a marine teleost, porgy,pagrus major

Relationships of respiratory areas (gill, body surface and fin areas) (A) to body mass (W) were determined with a marine teleost, the porgyPagrus major of 0.0002–1230 g (just after hatch to 3+ years old), based on the allometric formula A=αW^β. (1) Early larvae (0.0002–0.0003 g) did not have the secondary lamellae that were responsible for gas exchange at the gills. After this stage, a tetraphasic relationship was observed between lamellar area (total area of secondary lamellae, often called gill area) (GA_L) and boby mass. During the late larval and early juvenile stages, the GA_L-W relationship showed a triphasic positive allometry with β-values of 3.773, 1.561 and 1.111 corresponding to the first half of the late larval stage (0.00034–0.001g), the second half of the stage (0.001–0.01 g) and the early juvenile stage (0.02–0.1 g), respectively, During the squamated juvenile and later stages (0.1–1080g), there was a negative allometry with a β-value of 0.813. (2) A triphasic relationship was observed between the total cutaneous surface area (body surface area and fin area) (CA_b+f) and body mass. During the early larval stage, in which an increase of body mass was very small. from 0.0002 to 0.00025 g, CA_b+p/W increased with growth with a β-value of 3.986. After this stage, the CA_b+t W relationship showed a diphasic negative allometry with β-values of 0.562, during the late larval stage (0.00028–0.0045 g) and 0.652 during the early juvenile and later stages (0.0045–1230 g). (3) Based on these results, factors controlling the metabolism-size relationship are discussed.     

Cloning of a novel levoglucosan kinase gene from Lipomyces starkeyi and its expression in Escherichia coli

Levoglucosan, cellulosic pyrolysate, is converted to glucose-6-phosphate by a specific levoglucosan kinase in fungi. A novel cDNA of levoglucosan kinase gene (lgk) from yeast Lipomyces starkeyi YZ-215 was isolated by RACE method. The 1,445 bp cDNA fragment (lgk) harbouring the kinase gene exhibited one open reading frame (ORF) composed of 1,317 bp flanked by a 14 bp 5′-UTR and a 114 bp 3′-UTR, including a 25 bp poly(A) tail. The ORF encoded a 439 amino acid polypeptide with a 48.4 kDa predicted molecular mass. Analysis of amino sequence revealed that the kinase belonged to the bacterial anhydro-N-acetylmuramic acid kinase (AnmK) family, and kinase-like proteins existed in some fungi, especially in filamentous fungi such as Aspergillus. The kinase gene was transformed into Escherichia coli BL21 (DE3), recombinant E. coli could grow in M9 minimal medium with levoglucosan as a sole carbon source when induced by IPTG. In addition, the recombinant kinase was overexpressed, purified and characterized. The kinase was stable at pH 7–10 and showed maximum activity at 30°C and pH 9.0 as natural kinase, but presented higher thermostability. Kinetic constants (apparent K _m values) for LG and ATP were 105.3 ± 12.5 and 0.20 ± 0.02 mM, respectively. Furthermore, the kinase showed substrate specificity for LG. This novel levoglucosan kinase gene would be useful in constructing recombinant microbial strains for the efficient bioconversion of cellulosic pyrolysate to ethanol.