cDNA cloning and mRNA expression of the translationally controlled tumor protein(TCTP)gene from Japanese sea perch(Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Two Divergent Members of 4-Coumarate： Coenzyme A Ligase from Salvia miltiorrhiza Bunge： cDNA Cloning and Functional Study

4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.

cDNA cloning of a novel lectin that induce cell apoptosis from Artocarpus hypargyreus

We isolated a novel lectin(AHL)from Artocarpus hypargyreus Hance and showed its immunomodulatory activities.In this study,the amino acid sequence of AHL was determined by cDNA sequencing.AHL cDNA(875bp)contains a 456-bp open reading frame(ORF),which encodes a protein with 151 amino acids.AHL is a new member of jacalin-related lectin family(JRLs),which share high sequence similarities to KM+and Morniga M,and contain the conserved carbohydrate binding domains.The antitumor activity of AHL was also explored using Jurkat T cell lines.AHL exhibits a strong binding affinity to cell membrane,which can be effectively inhibited by methyl-α-D-galactose.AHL inhibits cell proliferation in a time-and dose-dependent manner through apoptosis,evidenced by morphological changes,phosphatidylserine externalization,poly ADP-ribose polymerase(PARP)cleavage,Bad and Bax up-regulation,and caspase-3 activation.We further showed that the activation of ERK and p38 signaling pathways is involved for the pro-apoptotic effect of AHL.

Purification and Characterization of a Novel Thermostable Chitinase from Thermomyces lanuginosus SY2 and Cloning of Its Encoding Gene

A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.

Cloning and Sequence Analysis of Expansin Genes from Litchi chinensis Fruit

Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments , named as Lc-Exp1 and Lc-Exp2 , were cloned. Lc-Exp1 and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryp-tophan residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Exp1 and c-Exp2. In addition, the homology between the two expansins is 71. 6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Exp1 with Fa-Exp2 or Pp-Exp1 was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Exp1 was only 77. 4% or 76.3% at amino acid sequences.

Cloning and Characterization of Porcine TSARG7 Gene and Analysis of Its Tissue-Specific Expression

TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase （PlsC） domain existing in all acyltransferase-like proteins. The porcine TSARG7 had been identified by cloning in silico but had not been confirmed experimentally. The full-length mRNA of porcine TSARG7 gene was sequenced and two splice variants were discovered. The full-length cDNA of TSARG7 variant 1 was 2 513 bp and variant 2 was 2 634 bp. The putative porcine TSARG7 proteins, which were located in the cytoplasm, encoded 458 and 456 amino acids, respectively. Real-time PCR analysis showed that TSARG7 gene was expressed in various tissues, but at different levels. The expression levels of this gene were higher in the skeletal muscle, heart, and testis than that in other tissues, suggesting that the TSARG7 gene played a role in procine skeletal muscle, heart, and testis functions.

Molecular Cloning and Tissue-specific Expression of Cu/Zn and Mn-superoxide Dismutase in the Three-keeled Pond Turtle, Chinemys reevesii

Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.

Rodent epididymal cDNAs identified by sequence homology to human and canine counterparts

<abstract>Aim: Identification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat. Methods: 'Electronic screening' of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis. Results: Rodent ESTs and genomic sequences homologous to HE3, HE4 and Ce8/Ly6G5C were identified in the public databases and the 'full-length' rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively. mRNA expression patterns were analysed in rats, including rat HEl and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis, Re3 and Re4 mRNAs were detected in all regions; Re8, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis. Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens. Conclusion: The novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.

EFFECTS OF A SYNTHETIC POLYPEPTIDE ENCODED BY THE p14-6,A cDNA CLONE WITH ANTIONCOGENE ACTIVITY, ON MALIGNANT TRANSFORMED DT CELLS

A synthetic polypeptide, pt27, which is encoded by a cDNA clone with antloncogene activity, p14-6, is found to be able to reduce remarkably the soft agar colony formation ability of part of DT cells and to raise their resistance to the ouabaln toxtcity. This shows that the pt27 peptide can affect the DT cells In a manner similar to the p14- 6 done and provides evidence that the reverting action of the p14-6 to DT cells may be exerted by the expression of its cDNA.

Isolating cDNA clones from rice induced by Magnaporthe grisea using PCR based differential screening method

The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that

Characterization and Expression Analysis of Starch Branching Enzymes in Sweet Potato

Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme （SBE） Ⅰ and Ⅱ, respectively, in sweet potato （Ipomoea batatas L.） were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid （ABA） was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

STUDY ON METASTASIS ASSOCIATED GENE SCREENED BY MONOCLONAL ANTIBODY HIL

The cDNA expression libraries derived from a highly metastatic cell subline Anip-973 and from its parental cell line, low metastatic AGZY-83a were screened by monoclonal antibodies (MoAbs) seperately against these two cell lines. A positive clone(H4-D) from the Anip-973 cDNA library was isolated and its nucleotide sequence was determined. This clone contained 978 bp with an open reading frame of 318 bp encoding a polypeptide consisting of 106 amino acids. The H4-D cDNA sequence showed 85% homology with a human propionyl-CoA carboxylase α-chain. In Western bloting analysis, the MoAb H4 recognized 2 bands(15 KDa and 27 KDa) of Anip-973 cell membrane Protein. The mRNA expression of H4-D was higher in AniP-973 cells than that in AGZY-83a cells. The metastatic Potential of Anip-973 cells was markedly decreased after being pretreated with MoAb H4. The above findings indicated that H4-D has a certain relationship with the metastatic phenotype of AniP-973 cells.

A novel ubiquitin carboxyl terminal hydroase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13suc1-agaroseaffinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accessionnumber: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functionaldomains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit theprogesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant proteinp28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminai hydrolases (UCHs). The results in this paperreveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in theprocess of progesterone-induced oocyte maturation possibly through an involvement in protein turnover anddegradation.

Identification of differentially expressed genes associated with bud dormancy release in tree peony(Paeonia suffruticosa) by suppression subtractive hybridization

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different mo- lecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

Construction of Full-length Infectious Clone for Encephalomyocarditis Virus BJC3 and Identification of the Rescued Virus

The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.

Construction of chimeric viruses based on pepper mild mottle virus using a modiffed Cre/loxP system

Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.However,stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.Here,we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus(PMMoV)and a chimeric virus with coat protein(CP)substitution assembled from separate pro-vector modules.Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

MOLECULAR CLONING OF A cDNA ENCODING FILARIAL COMMON ANTIGEN

It is estimated that more than 100 million peo-ple are infected with filaria and that 10 times as many are at risk of infection.Epidemiological

Molecular Cloning and Functional Characterization of Porcine MyD88 Essential for TLR Signaling

We isolated cDNA encoding porcine MyD88 （poMyD88） from Peyer＇s patches （Pps） of GALT. The complete open reading frame （ORF） of poMyD88 contains 879 bp encoding a deduced 293 aa residues. The amino acid sequence of poMyD88 was characterized by N-terminal death, intermediate and C-terminal Toll/IL-1 receptor （TIR） domains. The putative poMyD88 protein shares a higher level of homology with its human （87.2% amino acid identity） than with its mouse （77.4% amino acid identity） counterpart. Overexpression of poMyD88 participated in the further enhanced activation of NF-w.B in human embryonic kidney （HEK） 293 cells expressing porcine TLR2 and porcine TLR4/MD-2, but not porcine RP105/MD-1 after stimulation with the corresponding ligands. The expression levels of MyD88 were highest in the spleen and mesenteric lymph nodes （MLNs）, and lower in digestive tissues of newborn swine. In adult swine, the expression levels in the digestive tissues were lower than those in MLNs and the spleen. These results suggest that an MyD88-dependent signaling pathway is present in newborn as well as in adult swine and that it is involved in the innate immune system of these animals.