Gene Sequence, Soluble Expression and Homologous Comparison of a D-Hydantoinase fromPseudomonas putida YZ-26

A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

Cloning, Sequence Analysis, and Prokaryotic Expression of the Porcine DECR1 Gene

2,4-dienoyl-CoA reductase 1 （ DECR1 ） is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to be fully elucidated. However, it is a candidate determinant/indicator of meat quality, growth traits, and carcass quality. Here, we employed RT-PCR and rapid amplification of cDNA ends （RACE） analysis to amplify the full-length cDNA of DECR1 from Mashen pig liver, and cloned it into the expression vector pET-32a＋. After confirmation by sequencing and restriction analysis, the recombinant plasmid was transformed into E. coli BL21 cells. The cDNA of pig DECR1 contained 2,352 nucleotides, including a 987 bp open reading frame flanked by a 53 bp 5＇-untranslated region （UTR） and a 1,312 bp 3＇-UTR. The pig DECR1 coding sequence encoded 328 amino acid residues, which shared 99%, 88%, 87%, 87%, 87%, 87%, and 83% identity with those of Sus scrofa （predicted）, Bos taurus, Homo sapiens, Macaca mulatta, Pan troglodytes, Equus caballus, Canis, and Mus musculus, respectively. SDS-PAGE analysis revealed that the recombinant protein was expressed and that the expression level reached its highest level after 4 h induction. Western blot analysis indicated that the molecular weight of the expressed protein was the same as that predicted, ap- proximately 35 kDa. Collectively these data provide the basis for further studies into the physiological functions and molecular mechanisms of the pig DE- CR1 gene.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) human osteosarcoma cell. The amplified products were cloned into pGEM T Easy vector and sequenced. Then the expressed vector pBV220 hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE Sephadex A 50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz. et Migusch)

A novel HMW glutenin subunit gene 1Dy10. 1 was isolated and characterized from Xinjiang wheat （Triticum petropavlovskyi. Udacz. et Migusch） accession Daomai 2. The complete open reading frame （ORF） of 1Dy10. 1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy 10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy 10.1, an amino acid was changed from L （leucine） to P （proline） at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dyl 0.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy 12, a deletion of dipeptide GQ, which occurred in subunit IDy10, was also observed in subunit 1Dy10.1. The cloned IDylO. 1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit IDy10.1 from seed.

Isolation of a Plasmalemma Aquaporin Encoding Gene StPIP1 from Solanum tuberosum L. and Its Expression in Transgenic Tobacco

Aquaporin （AQP） belongs to a highly conserved group of membrane proteins considered as major intrinsic proteins, which facilitate water transport across biological membranes. The discovery of AQPs in plants has resulted in a paradigm shift in the understanding of plant-water relations, however, the potential relationship between the role of aquaporins in regulating plant water balance and drought tolerance still remains elusive. In this study, the gene encoding potato AQP cDNA, StPIP1 （GenBank accession no. DQ999080）, was cloned from the leaf of potato cultivar Gannongshu 2 by reverse transcription-PCR （RT-PCR）. Sequence alignment was made by BLASTn in GenBank, the phylogenetic analysis was conducted using PHYLIPWY, the 3D structure was predicted in Swiss-Model server. Subcellular localization of StPIP1 was performed by constructing CaMV35S-StPIP1-GFP and rd29A-StPIP1-GFP fusion proteins and transient expression in onion epidermis. To understand StPIP1 physiological functions in potato under various stress conditions, the StPIP1 gene in a reverse orientation was transformed into tobacco driven by the Cauliflower mosaic virus （CMV） 35S promoter. The expression levels of transgenic and wild-type plants were assessed under various abiotic stress conditions using semi-quantitative RT-PCR, and the morphological and physiological responses of transgenic plants to different stress conditions were investigated. The expression of StPIP1 mRNA decreased in transgenic plants under non-stress and stress conditions, however, the reduction was more severer under drought stress. In both non-stress and stress conditions, StPIP1 was expressed predominantly in root. The morphological and physiological investigation showed no significant differences in growth rate, germination rate, and root fresh weight （FW） between transgenic and wild-type plants when grown under favorable conditions. In contrast, under drought stress, the reduction in StPIPI expression leads to a delay in seed germination and seedling growth, accelerated seedling wilt, and leaf morphological abnormity. Under ＂enough＂ water conditions （i.e., water culture）, the aerial parts of anti-sense plants showed no differences. However, for the aerial parts to accumulate the same amount of biomass, transgenic plants needed about 3 times more abundant root system to transport water for plant growth than wild-type plants. Morphological investigation showed that the reduction in StPIP1 expression increased the root system in transgenic plants under drought stress. As a result, the increase of root mass might compensate the reduced cellular water permeability in order to ensure a sufficient water supply for the plant. Results demonstrated that StPIP1 plays an important role for water transportation in potato, especially under drought stress conditions. The reduced expression of StPIP1 decreases the cellular water transport and influences the expression of endogenous AQPs genes and thereby, has impacts on seed germination, seedling growth, and stress responses of potato to drought conditions.

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.

Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

ESTs fragments which represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fet al kidney cDNA library. Microarray was prepared by using these novel EST fragmen ts by automatic spotting. Expression patters of 79 ESTs of novel genes from huma n fetal kidney were analyzed in fetal brain and fetal heart tissues of 20\|week\ | and 26\|week\|age fetus by performing of cDNA chip hybridization. This provide s clues for studying exact functions of the novel genes. 8 genes were obtained w hich were expressed differentially in the fetal brain and heart of 20\|week\| an d 26\|week\|age respectively. Then differentially expressed genes were identifie d by Northern analysis. The more exact function of the novel genes is under stud y.

Cloning and Sequencing of the Pokeweed Antiviral Protein Gene and Its Expression in E. coli

The total RNA was isolated from pokeweed (Phytolacca americana) leaves using the method of guanidine isothiocyanite and used as a template to amplify the deleted mutant pokeweed antiviral protein (PAP) gene by RT-PCR and then the gene was cloned into the pGEMR-T vector. The sequencing results showed that the PAP gene consisted of 711nt, which was 99.6% identical to the PAP gene reported by Lin et al (1991). The IPTG-inducible expression vector containing the PAP gene was constructed and transferred into the E. coli strain BL21 (DE3)-plysS. A specific protein was produced after induction with 0.4m mol/L IPTG and its molecular weight was 26ku. The results of the double diffusion on the agar plate and the western blotting test showed that the protein produced in E. coli was highly identical with the PAP extracted by a Frenchman from French pokeweed leaves. These revealed that PAP gene was actually achieved and exactly expressed in E. coli.

Amplification，cloning，sequencing and expression of the gene encoding the major surface antigen of Toxoplasma gondii isolated

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Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in E.coli

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.

Construction of retroviral vectors to induce a strong expression of human interferon-β gene in human hepatoma cells

Establishing the hepatoma cell-specific expression of human interferon gene mediated by retroviral vectors. Methods: Human interferon-β complementary DNA (IFN-β cDNA) was inserted into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter, and MNAIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter regulated by α-fetoprotein enhancer. The retroviral constructs were respectively introduced into PA317 amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection efficiency was among (4-25)ｘ103 colonies/μg DNA/106 PA317 cells. The retrovirus infection efficiency was among (4. 5-500)ｘ104 Colony Forming Units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal cell carcinoma cells and melanoma cell lines in the presence of 4 μg/ml polybrene. Results: Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α-fetoprotein enhancer induced efficient and specific transcription and expression of IFN-β gene driven by the promoter of different origin in human hepatoma cells by which α-fetoprotein was highly produced. Conclusion: Cis-active element of α-fetoprotein gene can drive IFN-β gene specifically expressed in human hepatoma cells, which presents some valuable materials for the hepatoma-specific immune gene therapy.

Cloning and Expression Analysis of Mlo Gene from Pericallis hybrida B. Nord.

The full-length Mlo gene was obtained by reverse transcription polymerase chain reaction （RT-PCR） and RACE. The result of sequence analysis indicated that M/o gene from Pericallis hybrida B. Nord. contained about 1296bp open reading frame and encoded 431 amino acids. According to the comparison of the exogenous gene sequences by BLAST analysis and phylogenetic analysis, Mlo gene shared over 85% nucleotide homology and 98% amino acid homology. Finally, through semi-quantitative-PCR and fluorescence quantitative analysis, we found that Mlo gene showed the highest expression levels in leaves and the lowest in roots after inoculated with powdery mildew pathogen for different days.

Gene Expression Versus Sequence for Predicting Function: Glia Maturation Factor Gamma Is Not A Glia Maturation Factor

It is standard practice, whenever a researcher finds a new gene, to search databases for genes that have a similar sequence. It is not standard practice, whenever a researcher finds a new gene, to search for genes that have similar expression (co-expression). Failure to perform co-expression searches has lead to incorrect conclusions about the likely function of new genes, and has lead to wasted laboratory attempts to confirm functions incorrectly predicted. We present here the example of Glia Maturation Factor gamma (GMF-gamma). Despite its name, it has not been shown to participate in glia maturation. It is a gene of unknown function that is similar in sequence to GMF-beta. The sequence homology and chromosomal location led to an unsuccessful search for GMF-gamma mutations in glioma. We examined GMF-gamma expression in 1432 human cDNA libraries. Highest expression occurs in phagocytic, antigen-presenting and other hematopoietic cells. We found GMF-gamma mRNA in almost every tissue examined, with expression in nervous tissue no higher than in any other tissue. Our evidence indicates that GMF-gamma participates in phagocytosis in antigen presenting cells. Searches for genes with similar sequences should be supplemented with searches for genes with similar expression to avoid incorrect predictions.

Methotrexate combined with methylprednisolone for the recovery of motor function and differential gene expression in rats with spinal cord injury

Methylprednisolone is a commonly used drug for the treatment of spinal cord injury, but high doses of methylprednisolone can increase the incidence of infectious diseases. Methotrexate has anti-inflammatory activity and immunosuppressive effects, and can reduce in- flammation after spinal cord injury. To analyze gene expression changes and the molecular mechanism of methotrexate combined with methylprednisolone in the treatment of spinal cord injury, a rat model of spinal cord contusion was prepared using the PinPointTM preci- sion cortical impactor technique. Rats were injected with methylprednisolone 30 mg/kg 30 minutes after injury, and then subcutaneously injected with 0.3 mg/kg methotrexate 1 day after injury, once a day, for 2 weeks. TreadScan gait analysis found that at 4 and 8 weeks after injury, methotrexate combined with methylprednisolone significantly improved hind limb swing time, stride time, minimum longitudinal deviation, instant speed, footprint area and regularity index. Solexa high-throughput sequencing was used to analyze differential gene ex- pression. Compared with methylprednisolone alone, differential expression of 316 genes was detected in injured spinal cord treated with methotrexate and methylprednisolone. The 275 up-regulated genes were mainly related to nerve recovery, anti-oxidative, anti-inflammatory and anti-apoptotic functions, while 41 down-regulated genes were mainly related to proinflammatory and pro-apoptotic functions. These results indicate that methotrexate combined with methylprednisolone exhibited better effects on inhibiting the activity of inflammatory cytokines and enhancing antioxidant and anti-apoptotic effects and thereby produced stronger neuroprotective effects than methotrexate alone. The 316 differentially expressed genes play an important role in the above processes.

Generation and analysis of expressed sequence tags from the salt-tolerant eelgrass species, Zostera marina

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.

Expression of liver cancer associated gene HCCA3

AIM: To study and clone a novel liver cancer reisted gene,and to explore the molecular basis of liver cancer genesis.METHODS: Using mRNA differential display polymerasechain reaction (DDPCR), we investigated the difference of mRNA in human hepatocellular carcinoma (HCC) and paired surrounding liver tissues, and got a gene probe. By screening a human placenta cDNA library and genomic homologous extend, we obtained a full-length cDNA named HCCA3. We analyzed the expression of this novel gene in 42pairs of HCC and the surrounding liver tissues, and distribution in human normal tissues by means of Northern blot assay.RESULTS: A full-length cDNA of liver cancer associated gene HCCA3 has been submitted to the GeneBank nucleotide sequence databases ( Accession No. AF276707 ). The positive expression rate of this gene was 78.6% (33/42) in HCC tissues, and the clinical pathological data showed that the HCCA3 was closely associated with the invasion of tumor capsule ( P = 0.023) and adjacant small metastasis satellite nodules lesions ( P= 0.041). The HCCA3 was widely distributed in the human normal tissues, which was intensively expressed in lungs, brain and colon tissues,while lowly expressed in the liver tissues.CONCLUSION: A novel full-length cDNA was cloned and differentiated, which was highly expressed in liver cancer tissues. The high expression was closely related to the tumor invasiveness and metastasis, that may be the late heredited change in HCC genesis.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM To investigate SBA2 expression in CRC cell lines snd surgical specimens of CRC and sutologous healthy mucosa.METHODS Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples.Normalization of the results was achieved by simultaneous amplification of β-actin as an internal control.RESULTS In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and β-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts ( P ＜ 0.01 ). SBA2 expression was significantly (0.01 ＜ P ＜0.05) correlated with the grade of differentiation in CRC,with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in nonmetastasis samples (0.01 ＜ P ＜ 0.05 ).CONCLUSION SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.