Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis)

The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach beta-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.     

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%–64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Influence of modern irrigation,drainage system and water management on spawning migration of mud loach, <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> C.

We investigated the influence of human activities on the spawning migration of the mud loach, Misgurnus anguillicaudatus. Mud loaches migrate from rivers to paddy fields for spawning. In this study, the number of adult loaches in paddy fields showed a peak in late May and rapidly decreased in July. In early July, water is discharged from paddy fields in order to allow the fields to dry for a few weeks, and water is again supplied later. The spawning season of the loaches probably spans from mid May to August; this was concluded by observing physiological indicators such as the gonad somatic index, oocyte maturity and serum levels of testosterone, estradiol-17β and 17α, 20β-dihydroxy-4-pregnen-3-one. When the loaches mature, they migrated to paddy fields if they could swim upward through the drainage ditches. However, the drainage ditches of modern paddy fields were reconstructed to deepen the water levels. The artificial drought of the rice culture management led to a discharge of water and loaches from the paddy fields during their spawning season. These results indicate that modern rice cultivation causes obstruction of the migration route and loss of spawning grounds; consequently, loaches use limited space and period of the paddy field for their spawning.     

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.)

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).     

Molecular cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in orange-spotted grouper, Epinephelus coioides

In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study a cDNA containing the orange-spotted grouper GILT (OsgGILT) coding sequence has been cloned and its complete sequence determined. The full-length cDNA of OsgGILT gene is 1066 bp nucleotides (nt) encoding a protein of 260 amino acids (aa), with a putative molecular weight of 28.7 kDa. The deduced OsgGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The result of real-time PCR showed that OsgGILT mRNA was expressed in heart, liver, brain, gill, kidney and muscle and more highly expressed in spleen. The OsgGILT expression is obviously up-regulated in spleen and kidney after induction with LPS, these results suggest that OsgGILT may be involved in the immune response to LPS challenge in orange-spotted grouper.     

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

cDNA cloning and expression analysis of a mannose-binding lectin from <Emphasis Type="Italic">Pinellia pedatisecta</Emphasis>

Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM₁ (polypeptides A) and PPA-DOM₂ (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Molecular cloning,sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.     

HCV基因组NS1区的分子克隆及序列测定

对广东省一名慢性丙型肝炎病人血清中的ＨＣＶ基因组ＮＳ１区进行分子克隆及序列测定。采用微粒吸附法提取ＨＣＶ ＲＮＡ,随机引物逆转录后进行聚合酶链反应。所用引物位于ＮＳ１区,扩增产物７８０ｂｐ在低熔点琼脂糖中电泳,加嘏相应条带处凝胶,与ｐＵＣ１８的连接批应直接在低熔点琼脂糖中完成。重组体转化ＪＭ１０９,挑取菌落增殖后提取的质粒采用ＰＣＲ和酶切法鉴定阳性克隆。将其中３２０ｂｐ的片段亚克隆到ｐＵＣ１８和ｐ     

三疣梭子蟹蜕皮抑制激素cDNA的克隆与序列分析

甲壳动物的蜕皮是由位于头胸部前鳃腔的一对Y-器通过分泌蜕皮激素（Molting hormone）来控制的（Lachaise et al．,1993）,而蜕皮激素的分泌又受到蜕皮抑制激素（Molt-inhibiting hormone,MIH）的调控（Watson et al．,2001）。MIH和性腺抑制激素（Gonad-inhibiting hormone,GIH）、甲壳动物高血糖激素（Crustacean hyperglycemic hormone,CHH）、     

Molecular cloning and characterisation of cDNAs complementary to mRNAs from wounded potato (Solanum tuberosum) tuber tissue

Five cDNA clones complementary to mRNAs representing different abundances and responses to wounding have been isolated from a library of Sau 3A fragments in the bacteriophage M13 mp8. These were characterised by hybrid-release translation and hybridisation to RNA blots. The levels of RNA complementary to two of the clones show a marked increase during the 24h after wounding, one shows a small increase and two show no appreciable changes except that caused by a general increase in the total amount of polyadenylated RNA per microgram of total RNA which increases 2.5-fold during the same period. The would-induced RNAs are not induced in diluted suspension-culture cells, but RNA complementary to each clone is present in varying levels in stems, leaves and roots of intact potato plants.Abbreviations cDNA complementary DNA - poly(A) polyadenosine - poly(A)⁺ RNA polyadenylated RNA - poly(U) polyuridine     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924-bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-PB) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis thaliana (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4), and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA) and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes, such as inducing the expression of some defense-related genes and increasing plants’ disease resistance ability. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 816–824. The text was submitted by the authors in English.