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Sample records for cloning transcription expression

  1. Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo).

    Science.gov (United States)

    Sunde, Roger A; Sunde, Gavin R; Sunde, Colin M; Sunde, Milton L; Evenson, Jacqueline K

    2015-01-01

    The minimum Se requirement for male turkey poults is 0.3 μg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4) and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA) codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

  2. Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo.

    Directory of Open Access Journals (Sweden)

    Roger A Sunde

    Full Text Available The minimum Se requirement for male turkey poults is 0.3 μg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4 and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

  3. [Cloning and prokaryotic expression of transcriptional co-activator gene of Clonorchis sinensis and functional analysis of the expressed protein].

    Science.gov (United States)

    Zhang, Yong-li; Yu, Xin-bing; Wu, De; Wu, Zhong-dao; Bi, Hui-xiang

    2005-02-28

    To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function. A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamH I and Sal I, the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E. coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni2+) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC. The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel. The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.

  4. Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells

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    Fabjani Gerhild

    2008-01-01

    Full Text Available Abstract Background Many cancer cells produce interleukin-6 (IL-6, a cytokine that plays a role in growth stimulation, metastasis, and angiogenesis of secondary tumours in a variety of malignancies, including colorectal cancer. Effectiveness of IL-6 in this respect may depend on the quantity of basal and inducible IL-6 expressed as the tumour progresses through stages of malignancy. We therefore have evaluated the effect of IL-6 modulators, i.e. IL-1β, prostaglandin E2, 17β-estradiol, and 1,25-dihydroxyvitamin D3, on expression and synthesis of the cytokine at different stages of tumour progression. Methods We utilized cultures of the human colon carcinoma cell clones Caco-2/AQ, COGA-1A and COGA-13, all of which expressed differentiation and proliferation markers typical of distinct stages of tumour progression. IL-6 mRNA and protein levels were assayed by RT-PCR and ELISA, respectively. DNA sequencing was utilized to detect polymorphisms in the IL-6 gene promoter. Results IL-6 mRNA and protein concentrations were low in well and moderately differentiated Caco-2/AQ and COGA-1A cells, but were high in poorly differentiated COGA-13 cells. Addition of IL-1β (5 ng/ml to a COGA-13 culture raised IL-6 production approximately thousandfold via a prostaglandin-independent mechanism. Addition of 17β-estradiol (10-7 M reduced basal IL-6 production by one-third, but IL-1β-inducible IL-6 was unaffected. Search for polymorphisms in the IL-6 promoter revealed the presence of a single haplotype, i.e., -597A/-572G/-174C, in COGA-13 cells, which is associated with a high degree of transcriptional activity of the IL-6 gene. IL-6 blocked differentiation only in Caco-2/AQ cells and stimulated mitosis through up-regulation of c-myc proto-oncogene expression. These effects were inhibited by 10-8 M 1,25-dihydroxyvitamin D3. Conclusion In human colon carcinoma cells derived from well and moderately differentiated tumours, IL-6 expression is low and only marginally

  5. Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

    Institute of Scientific and Technical Information of China (English)

    Hualing Wu; Zhongfu Ni; Yingyin Yao; Ganggang Guo; Qixin Sun

    2008-01-01

    WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identification, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids.

  6. Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

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    Afrin, Sadia; Zhu, Jie; Cao, Hongzhe; Huang, Jingjia; Xiu, Hao; Luo, Tiao; Luo, Zhiyong

    2015-04-01

    The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  7. [Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

    Science.gov (United States)

    Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

    2003-02-01

    Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

  8. Differential expression pattern of rubber elongation factor (REF) mRNA transcripts from high and low yielding clones of rubber tree (Hevea brasiliensis Muell. Arg.).

    Science.gov (United States)

    Priya, P; Venkatachalam, P; Thulaseedharan, A

    2007-10-01

    In Hevea tree, rubber elongation factor (REF) is a key gene involved in rubber biosynthesis. Since the immaturity period for Hevea is 6 years, identification of molecular marker for latex yield potential will be beneficial for early selection of high yielding clones. The main objective of this research is to study the expression pattern of the REF gene in contrasting latex yield rubber clones (high and low yielding) by Northern blot as well as RT-PCR analysis. Accumulation of REF mRNA transcripts was significantly higher in latex cells compared to other cells of seedlings and mature Hevea trees. Northern results revealed that the level of REF gene expression in latex cells of high yielding rubber clones was significantly higher than in low yielders. According to RT-PCR results, the abundance of REF mRNA transcripts in latex cells was fivefold higher in the RRII105 clone, one of the most high yielding rubber clones. It is evident from the results that both tapping and ethephon treatment had a direct effect on induction of REF gene expression. Results demonstrate a positive correlation between REF gene expression pattern and latex yield.

  9. Molecular cloning, characterization and expression of WAG-2 alternative splicing transcripts in developing spikes of Aegilops tauschii

    Indian Academy of Sciences (India)

    SHUHONG WEI

    2016-09-01

    WAG-2 is a C-class MADS-box gene, which is orthologous to AGAMOUS (AG )inArabidopsis. The AG group C-classMADS-box genes are involved in stamen and pistil identity. In this study, two WAG-2 transcripts, namely, WAG-2f and WAG-2g, were isolated and characterized from Aegilops tauschii . The open reading frames of WAG-2f and WAG-2g were 825 and 822 bp, respectively, encoding 275 and 274 amino acid residues. BLAST searches of partial WAG-2 genomic sequence againstthe draft sequence of Ae. tauschii genome database revealed the complex structure of WAG-2 gene, which consisted of seven exons and six introns. TheWAG-2f and WAG-2g cDNAs were two alternative splicing transcripts. The alternative splicing events were produced by an alternative 5 ' splice site. The expression level of WAG-2f transcript, which was extremely weak inyoung spikes of floret primordium formation stage, increased as the spikes developed. The highest expression was observed in the spikes at the anther separation stage. Low expression levels of WAG-2f were also detected at the tetrad stage. The WAG-2g transcript was expressed at all four stages of spike development but at a relatively low level. The expression pattern of thetwo transcripts was distinctly different during floral development, thereby suggesting a functional divergence.

  10. Cloning and analysis of expression patterns and transcriptional regulation of RghBNG in response to plant growth regulators and abiotic stresses in Rehmannia glutinosa.

    Science.gov (United States)

    Zhou, Yanqing; Zhang, Yonghua; Wei, Jun; Zhang, Yu; Li, Jingyun; Wang, Wanshen; Duan, Hongying; Chen, Juanjuan

    2015-01-01

    RghBNG, a gene of unknown function, was cloned from Rehmannia glutinosa by reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA of RghBNG was 548 bp with a282-bp open reading frame. It encoded a polypeptide of 93 amino acids with a predicted molecular weight of 10.5 kDa and a theoretical isoelectric point of 9.25. Bioinformatics analysis indicated that RghBNG had no homology to any known plant genes, whereas the RghBNG polypeptide was highly similar to other plant proteins and possessed one conserved B12D protein family functional domain. Phylogenetic analysis revealed that RghBNG encoded for a dicot protein. RghBNG spatial and temporal expression patterns and responses to abiotic stresses and plant growth regulators were investigated by qRT-PCR. RghBNG transcripts were detected in roots, stems, leaves, petals, receptacles, stamens and pistils with the highest and lowest levels respectively observed in petals and leaves of mature plants. Additionally, RghBNG transcripts were detected at three developmental stages of roots, stems and leaves; the highest levels were observed in roots at seedling stage; Transcript levels changed to varying degrees in different tissues and stages; We also studied the effects of abiotic stress and plant growth regulators in roots and leaves. RghBNG expression was significantly increased (p plant growth regulators and abiotic stresses.

  11. Cloning and expression analysis of transcription factor RrTTG1 related to prickle development in rose (Rosa Rugosa

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    Feng Li-Guo

    2015-01-01

    Full Text Available A prickle is an acuminate protuberance formed by the deformation of plant trichomes together with a few cortical cells. It is a type of multicellular eglandular trichome with special morphology, which originates from the phloem but is not connected to the xylem. Rosa rugosa is an important ornamental/commercial plant and an important raw material in the food and perfume industries. However, the firm prickles on its stems are inconvenient to field management, the harvesting of flowers and garden management. The TTG1 transcription factor related to the development of prickle was isolated from R. rugosa in the present study. Its expression patterns in different tissues and varieties were analyzed. Results showed the expression level of the RrTTG1 gene was highest in the leaves, followed by the stems, but was lower in the pericarps and petals. Moreover, the higher expression level of the RrTTG1 gene in all tissues of the ‘Ciguo rose’, as compared with that of the ‘Weihai wild rose’, follows the results of field morphological observation. Therefore, the RrTTG1 transcription factor is likely to regulate the development of rose prickles. This study allows for further discussion on the molecular mechanisms of prickle formation and development in R. rugosa and provides a molecular basis for the cultivation of roses with fewer or no prickles via genetic engineering.

  12. Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection.

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    Kwan, Yee-Min; Meon, Sariah; Ho, Chai-Ling; Wong, Mui-Yun

    2015-02-01

    Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection. Copyright © 2014 Elsevier GmbH. All rights reserved.

  13. The cloning of the cdk2 transcript and the localization of its expression during gametogenesis in the freshwater giant prawn, Macrobrachium rosenbergii.

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    Chen, Jie; Liu, Ping; Li, Zhen; Chen, Ying; Qiu, Gao-Feng

    2013-08-01

    Cyclin-dependent kinases (cdks) are key regulators of the cell cycle. In mammals, cdk2 plays an essential role in the meiosis of spermatocytes and oocytes. To investigate the role of cdk2 kinase during gametogenesis in crustaceans, we cloned a complete cDNA sequence of cdk2 from the freshwater giant prawn, Macrobrachium rosenbergii, and examined its localization and expression in the developing gonads. The prawn cdk2 cDNA is 1,745 bp in length and encodes a putative protein of 305 amino acids. The deduced protein contains a conserved cyclin binding motif PSTAIRE and shares high homology with reported cdk2 kinases of other species. RT-PCR analysis showed a wide distribution of the cdk2 mRNA in all tested organs including the testis, ovary, heart, muscles, hepatopancreas and gills, and the highest level of expression in the ovary and testis. Localization by in situ hybridization of cdk2 mRNA in the ovary showed high expression in the ooplasm of previtellogenic and the nuclei of late vitellogenic oocytes. In testicular sections, cdk2 transcript is low in spermatogonia, high in spermatocytes, but reduced in spermatids and sperm. The high expression of the cdk2 transcripts in meiotic spermatocytes and oocytes indicated that the cdk2 gene has the conservative function in the germ cells meiosis during gametogenesis.

  14. Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats

    NARCIS (Netherlands)

    Meng, L.; Jia, R.X.; Sun, Y.; Wang, Z.Y.; Wan, Y.J.; Zhang, Y.L.; Zhong, B.S.; Wang, F.

    2014-01-01

    Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung devel

  15. Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals.

    Science.gov (United States)

    D'Onofrio, C; Colantuoni, V; Cortese, R

    1985-08-01

    Human plasma retinol binding protein (RBP) is coded by a single gene and is specifically synthesized in the liver. We have characterized a lambda clone, from a human DNA library, carrying the gene coding for plasma RBP. Southern blot analysis and DNA sequencing show that the gene is composed of six exons and five introns. Primer elongation and S1 mapping experiments allowed the definition of the initiation of transcription and the identification of the putative promoter. The 5'-flanking region of the RBP gene was fused upstream to the coding sequence of the bacterial enzyme chloramphenicol acetyl transferase (CAT): the chimeric gene was introduced, by calcium phosphate precipitation, into the human hepatoma cell line Hep G2 and into HeLa cells. Efficient expression of CAT was obtained only in Hep G2. Primer elongation analysis of the RNA extracted from transfected Hep G2 showed that initiation of transcription of the transfected chimeric gene occurs at a position identical to that of the natural gene. Transcriptional analysis of Bal31 deletions from the 3' end of the RBP 5'-flanking DNA allowed the identification of the RBP gene promoter.

  16. cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hong; Thalmann, I.; Thalmann, R. [Washington Univ., St. Louis, MO (United States)] [and others

    1995-06-10

    We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a sub-unit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear. 40 refs., 5 figs.

  17. Comparative transcriptional activity of five promoters in BAC-cloned MDV for the expression of the hemagglutinin gene of H9N2 avian influenza virus.

    Science.gov (United States)

    Ma, Chengtai; Zhang, Zhenjie; Zhao, Peng; Duan, Luntao; Zhang, Yaoyao; Zhang, Fushou; Chen, Wenqing; Cui, Zhizhong

    2014-09-01

    On the basis of recent studies, much attention has been given to recombinant MDV (rMDV)-based vaccines. During the construction of rMDV, the activity of promoters to transcribe foreign genes is one of the major factors that can affect protective efficacy. To investigate the transcription activity and efficacy of five different promoters, the advantage of an existing rMDV BAC infectious clone that had been previously constructed was used to construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain was inserted into the US2 region under five selected promoters. These five promoters included three MDV endogenous promoters (the promoter for the gB gene and a bi-directional promoter in both directions for pp38 (ppp38) and 1.8 kb RNA transcripts (p1.8 kb)), and two exogenous promoters (CMV and SV40). Among these five promoters, the CMV promoter demonstrated the highest activity, followed by p1.8 kb and SV40, which had a similar transcriptional activity level. Two of the MDV endogenous promoters showed much lower transcriptional activities, particularly the promoter ppp38, which had the lowest activity. The results of the in vivo experiment proved that none of the three recombinant viruses of rGX-CMV-HA, rGX-SV40-HA and rGX-p1.8kb-HA provided protection in SPF chickens. Chickens vaccinated with rGX-pPP38-HA induced 50% and rGX-gB-HA induced 25% protection against the challenge with H9N2, respectively.

  18. Cloning, expression, purification and crystallization of a transcriptional regulatory protein (Rv3291c) from Mycobacterium tuberculosis H37Rv.

    Science.gov (United States)

    Shrivastava, Tripti; Kumar, Sandeep; Ramachandran, Ravishankar

    2004-10-01

    Rv3291c, the translational product of the Mycobacterium tuberculosis Rv3291c gene, is an 18 kDa protein. It is a putative transcriptional regulatory protein belonging to the leucine-responsive regulatory protein/asparagine synthase C (Lrp/AsnC) family, which are proteins that have been identified in archaea and bacteria. Rv3291c probably plays a significant role during the persistent/latent phase of M. tuberculosis, as supported by its up-regulation several-fold during this stage. Orthorhombic crystals of recombinant Rv3291c have been grown from trisodium citrate dihydrate-buffered solutions containing monoammonium dihydrogen phosphate. Diffraction data extending to 2.7 A have been collected from a single crystal with unit-cell parameters a = 99.6, b = 100.7, c = 100.6 A. Assuming an octamer in the asymmetric unit results in a Matthews coefficient (VM) of 1.75 A3 Da(-1), corresponding to a solvent content of about 30%.

  19. Molecular cloning, tissue expression and regulation of liver X receptor (LXR) transcription factors of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Cruz-Garcia, L; Minghetti, M; Navarro, I; Tocher, D R

    2009-05-01

    Fish are important sources of high quality protein, essential minerals such as iodine and selenium, vitamins including A, D and E, and omega-3 fatty acids in the human diet. With declining fisheries worldwide, farmed fish constitute an ever-increasing proportion of fish in the food basket. Sustainable development of aquaculture dictates that diets will have to contain increasing levels of plant products that are devoid of cholesterol, but contain phytosterols that are known to have physiological effects in mammals. Liver X receptors (LXR) are transcription factors whose activity is modulated by sterols, with activation inducing cholesterol catabolism and de novo fatty acid biosynthesis in liver. Transcriptomic analysis has shown that substitution of fish meal and oil with plant products induces genes of cholesterol and fatty acid metabolism in salmonids. Here we report the cloning of LXR cDNAs from two species of salmonid fish that are important in aquaculture. The full-length cDNA (mRNA) of LXR obtained from salmon was shown to be 3766 bp, which included a 5'-untranslated region (UTR) of 412 bp and a 3'-UTR of 1960 bp and an open reading frame (ORF) of 1394 bp, which specified a protein of 462 amino acids. The trout LXR full-length cDNA was 2056 bp, including 5'- and 3'-UTRs of 219 and 547 bp, respectively, and an ORF of 1290 bp, which specified a protein of 427 amino acids. The protein sequences included characteristic features of mammalian LXRs, including the DNA binding (DBD), containing P-box, ligand binding (LBD) and activation function-2 (AF-2) domains, D-box, D (hinge) region, and eight cysteines that belong to the two zinc fingers. Phylogenetic analysis clustered the salmonid LXRs together, more closely with zebrafish and more distantly from medaka and stickleback. A pair-wise comparison among vertebrate LXR sequences showed the amino acid sequence predicted by the salmon LXR ORF showed greatest identity to that of trout 97%, and 97%, 87% and 81% identity

  20. Anoxia-induced transcriptional upregulation of sarp-19: cloning and characterization of a novel EF-hand containing gene expressed in hepatopancreas of Littorina littorea.

    Science.gov (United States)

    Larade, Kevin; Storey, Kenneth B

    2004-04-01

    Many marine molluscs have well-developed biochemical adaptations that allow them to live without oxygen for long periods of time, but very little is currently known about the molecular biology underlying these processes. Differential screening of a cDNA library derived from the hepatopancreas of the marine snail Littorina littorea revealed a novel anoxia-induced gene, sarp-19 (snail anoxia-responsive protein, 19 kDa). Examination of the sarp-19 transcript revealed an open reading frame that encoded a protein of 168 amino acids containing an N-terminal signal sequence and two putative EF-hand domains. Expression analysis of transcript levels established that sarp-19 accumulated over a time course of anoxia exposure, reaching a maximum 5.6-fold increase after 96 h compared with aerobic controls. However, transcript levels were reduced by 50% within 1 h when aerobic conditions were reestablished. Nuclear runoff assays confirmed transcriptional upregulation of sarp-19 during anoxia exposure, and organ explant experiments showed that the gene was also responsive to anoxia exposure in vitro. sarp-19 transcripts were also elevated in response to freezing, suggesting that the protein may have a role in the physiological responses of this intertidal snail to both aerial exposure and winter freezing. Hepatopancreas explants treated with a calcium ionophore showed increased levels of the sarp-19 transcript, suggesting a possible feedback mechanism regulated by levels of intracellular calcium. Expression was also responsive to tissue incubation with cyclic GMP and phorbol 12-myristate 13-acetate but was not affected by cyclic AMP, implicating involvement of protein kinases G and C but not protein kinase A in the expression of sarp-19. The SARP-19 protein may play a role in calcium-activated signaling during anoxia exposure in L. littorea.

  1. Cloning-free regulated monitoring of reporter and gene expression

    Directory of Open Access Journals (Sweden)

    Demirkaya Omer

    2009-03-01

    Full Text Available Abstract Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

  2. Steroidogenic acute regulatory protein in white sturgeon (Acipenser transmontanus): cDNA cloning, sites of expression and transcript abundance in corticosteroidogenic tissue after an acute stressor.

    Science.gov (United States)

    Kusakabe, Makoto; Zuccarelli, Micah D; Nakamura, Ikumi; Young, Graham

    2009-06-01

    The white sturgeon, Acipenser transmontanus, is a primitive bony fish that is recognized as an important emerging species for aquaculture. However, many aspects of its stress and reproductive physiology remain unclear. These processes are controlled by various steroid hormones. In order to investigate the regulation of steroidogenesis associated with acute stress in sturgeon, a cDNA-encoding steroidogenic acute regulatory protein (StAR) was isolated from white sturgeon. The putative amino acid sequence of sturgeon StAR shares high homology (over 60%) with other vertebrates. Phylogenetic analysis grouped sturgeon StAR within Actinopterygii, but it was clearly segregated from teleost StARs. RT-PCR analysis revealed that transcripts were most abundant in yellow corpuscles found throughout the kidney and weaker signals were detected in gonad and kidney. Very weak signals were also detected in brain and spleen by quantitative real-time PCR. In situ hybridization revealed that StAR is expressed in the cells of yellow corpuscles. No significant changes in StAR gene expression were detected in response to an acute handling stress. These results suggest that StAR is highly conserved throughout vertebrates, but the expression of the functional protein during the stress response may be partially regulated post-transcriptionally.

  3. Neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting.

    Science.gov (United States)

    MacDonald, Erin; Volkoff, Hélène

    2009-04-01

    cDNAs encoding for neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) were cloned in an elasmobranch fish, the winter skate. mRNA tissue distribution was examined for the three peptides as well as the effects of two weeks of fasting on their expression. Skate NPY, CART and CCK sequences display similarities with sequences for teleost fish but in general the degree of identity is relatively low (50%). All three peptides are present in brain and in several peripheral tissues, including gut and gonads. Within the brain, the three peptides are expressed in the hypothalamus, telencephalon, optic tectum and cerebellum. Two weeks of fasting induced an increase in telencephalon NPY and an increase in CCK in the gut but had no effects on hypothalamic NPY, CART and CCK, or on telencephalon CART. Our results provide basis for further investigation into the regulation of feeding in winter skate.

  4. Cloning of human RTEF-1, a transcriptional enhancer factor-1-related gene preferentially expressed in skeletal muscle: evidence for an ancient multigene family.

    Science.gov (United States)

    Stewart, A F; Richard, C W; Suzow, J; Stephan, D; Weremowicz, S; Morton, C C; Adra, C N

    1996-10-01

    Transcriptional Enhancer Factor-1 (TEF-1) is a transcription factor required for cardiac muscle gene activation. Since ablation of TEF-1 does not abolish cardiac gene expression, we sought to identify a human gene related to TEF-1 (RTEF-1) that might also participate in cardiac gene regulation. A human heart cDNA library was screened to obtain a full-length RTEF-1 cDNA. Fluorescence in situ hybridization assigned the RTEF-1 gene to chromosome 12p13.2-p13.3. In contrast, PCR screening of human/rodent cell hybrid panels identified TEF-1 on chromosome 11p15.2, between D11S1315 and D11S1334, extending a region of known synteny between human chromosomes 11 and 12 and arguing for an ancient divergence between these two closely related genes. Northern blot analysis revealed a striking similarity in the tissue distribution of RTEF-1 and TEF-1 mRNAs; skeletal muscle showed the highest abundance of both mRNAs, with lower levels detected in pancreas, placenta, and heart. Phylogenetic analysis of all known TEF-1-related proteins identified human RTEF-1 as one of four vertebrate members of this multigene family and further suggests that these genes diverged in the earliest metazoan ancestors.

  5. Cloning and prokaryotic expression of HGLP cDNA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A novel cDNA, HGLP, which encodes a G- protein coupled receptor (GPCR) like protein, has been isolated and cloned. The coding region of the human HGLP predicts a 7-transmembrane region protein with motifs of rhodopsin-like GPCR superfamily. Northern blot analysis reveals a 3-kb transcript in various human tissues examined. The N- and C-terminal coding regions of HGLP, which are deduced as non-transmembrane regions, have been amplified by PCR and cloned into pET30a+ vector. Then the recombinant proteins are highly expressed in E. coli.

  6. 梅花 CBF 转录因子的克隆及表达%Cloning and Expression of CBF Transcription Factor from Prunus mume

    Institute of Scientific and Technical Information of China (English)

    张杰; 张启翔; 杨炜茹

    2012-01-01

    根据GenBank中与梅花同属的桃、甜樱桃等已发表CBFs转录因子序列设计简并引物,采用PCR和RTPCR方法,从梅花基因组DNA和cDNA中克隆CBF转录因子片段.结果表明,两种途径获得的CBF基因序列一致,基因全长821 bp,编码238个氨基酸,其氨基酸序列具有典型的CBF蛋白特征,包含保守的AP2/EREB DNA 结合结构域及CBF家族蛋白特征短多肽序列(PKK/RPAGRxKFxETRHP和DSAWR).氨基酸相似性分析结果表明,该基因与欧洲甜樱桃、矮扁桃等CBF转录因子相似性较高.相对荧光定量PCR结果显示,4℃低温胁迫下,其表达量符合CBF转录因子表达特点,随着胁迫时间的增长表达量呈上升趋势,8h时达峰值,说明该基因在低温胁迫下上调表达.%Primers were designed according to the CBF transcription factors of Peach (Prunus persica), Sweet cherry (Prunus avium) et al from GenBank. Fragments of CBF gene were isolated from Prunus mume by PCR and RT-PCR, The CBF gene was 821 bp long, encoding a putative protein of 238 amino acids;The amino acids sequence owns the characteristics of the CBF protein,which contains an AP2/EREB DNA-binding domain and two special short amino acids sequences;Similarity analysis showed that the nu-cleotide were highly similarity to that of P. avium, P. tenella et al. Relative real-time PCR experiment showed the expression of PmCBFl was coincidence with the expression characteristics of the CBF gene after exposed to 4℃. The expression of PmCBFl increased at beginning and achieved the highest after 8 hour,indicating PmCBFl was induced under low temperature stress.

  7. Cloning and expression analysis of an ERF transcription factor in soybean%一个大豆ERF转录因子的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    张大勇; 许玲; 易金鑫; 徐照龙; 何晓兰; 刘晓庆; 黄益洪; ZULFIQAR A; 马鸿翔

    2011-01-01

    Total RNA was isolated from soybean root tissues treated with 20% PEG-6000 solution and reversely transcribed into cDNA using RT-PCR method. An ethylene responsive factor (ERF) gene (designated as GmERF7 because of being located on the seventh chromosome, locus name; Glyma07g02930) was cloned. The sequence analysis indicates that GmERF7 contains an intron of 237 bp and an ORF (open reading frame) of 585 bp. It encodes a protein (molecular weight: 21.99×103) of 194 amino acids with theoretical pI of 7.06. Sequence analysis of amino acids demonstrated its high similarity with those from other plant species. The phylogenetic analysis depicted it's nearer genetic distance with Medicago truncatula and Ricinus communis and farther with Vitis vinifera and Arabidopsis thaliana. Semi-quantitative RT-PCR showed that this gene mainly expressed in leaf and pod tissues of soybean. The expression of GmERF7 in root and leaf could be induced after 6 h of 20% PEG treatment indicating its probable function during the drought stress. The subcellular localization using onion epidermal cells showed that GmERF7 protein was mainly localized at the nucleus and cell membrane after N-terminus signal peptide, and thus indicated that GmERF7 was a regulator for downstream genes. Real-time RT-PCR analysis indicated the down-regulation of gene at 10 min, then up-regulation at 30 min followed by down-regulation up to 2 h of exogenous application of ABA, implying that the gene maybe participate in transduction pathway of ABA-dependent signal for plant tolerance to adverse environment.%利用RT-PCR方法从聚乙二醇(PEG)处理的大豆根部组织中克隆了1个大豆乙烯响应因子(ERF)基因,由于其位于大豆基因组第7染色体上,故命名为GmERF7,基因座为Glyma07g02930.序列分析结果表明:该基因含有1个237 bp长的内含子,开放阅读框(ORF)长585 bp,编码194个氨基酸,蛋白质分子量为2.199× 104,理论等电点为7.06;通过氨基酸序列比对发现,

  8. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  9. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  10. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  11. SSH adequacy to preimplantation mammalian development: Scarce specific transcripts cloning despite irregular normalisation

    Directory of Open Access Journals (Sweden)

    Renard JP

    2005-11-01

    Full Text Available Abstract Background SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. Results Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs tester-specific transcripts. Conclusion The close agreement between our hybridization and sequencing results shows that the

  12. Cloning, high-level expression, purification and characterization of a ...

    African Journals Online (AJOL)

    Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakøC, ... African Journal of Biotechnology ... Hence in this study, we reported the cloning, high-level expression, purification and characterization of ...

  13. Cloning and expression of human colon mast cell carboxypeptidase

    Institute of Scientific and Technical Information of China (English)

    Zhang-Quan Chen; Shao-Heng He

    2004-01-01

    AIM: To clone and express the human colon mast cell METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to conrtruct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P.pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli was purified with amylose affinity chromatography and heparin agarose chromatogphy.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.carboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the fonctional study on hMC-CP.

  14. X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

    Directory of Open Access Journals (Sweden)

    Chi-Hun Park

    Full Text Available To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF, and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process

  15. 八棱海棠中转录因子基因MrDREBA6的克隆及表达分析%Cloning and expression of transcription factor gene MrDREBA6 from Malus micromalus

    Institute of Scientific and Technical Information of China (English)

    付晓燕; 熊爱生; 姚泉洪; 彭日荷; 章镇; 乔玉山; 周军; 朱波; 高峰; 田永生; 赵伟

    2009-01-01

    DREB晒类基因是植物中重要的一类转录因子基因,其编码的产物广泛参与生长发育和各类生理过程.根据拟南芥和水稻DREB类基因序列,设计2个兼并引物,利用RACE法从八棱海棠cDNA文库中扩增出一编码DREB类转录因子的cDNA序列.命名为MrDREBA6.从cDNA序列、推测的氨基酸序列、进化树、功能域和分子结构模型进行预测和较为全面的分析.表明MrDREBA6属于DREB类转录因子的A6亚族.蛋白质三级结构与AtRAP2.4和AtERFl相似.荧光定量PCR分析MrDREBA6在各种处理下表达水平显示,MrDREBA6受到PEG、ABA、高盐和低温的诱导表达.另外.组织表达特异性检测表明,正常生长条件下MrDREBA6基因在叶中表达较高,在根和茎中的表达量略低.%MrDREB gene is an important family of transcription factors in plant, encoding transcriptional regulators with a va-riety of functions involved in the developmental and physiological processes. Here, using two degenerate primers according to the sequences of DREB genes from Arabidopsis and rice,a cDNA encoding putative DREB protein was cloned from cDNA of Malus micromalus Makino by RACE,named as MrDREBA6. Then,cDNA and deduced amino acid sequence,phylogenetic tree,function domain and three-dimension structure were predicted and analyzed. MrDREBA 6 from Malus micromalus Maki-no was classified into A6 subgroup of DREB family transcription factor. MrDREBA6,AtRAP2.4 and AtERFI have similar three--dimension structure. The results of Real-Time-Quantitative PCR analysis for Malus micromalus Makino exposed to various treatments demonstrated that MrDREBA 6 gene was induced by PEG, ABA, high-salinity and low-temperature stress-es. Expression profiling analyses of MrDREBA 6 in different organs indicated that MrDREBA 6 gene was mainly expressed in leaf, and faintly expressed in root and stem under normal growth condition.

  16. Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts

    NARCIS (Netherlands)

    R. Kraaij (Robert); M. Verhoef-Post (Miriam); J.A. Grootegoed (Anton); A.P.N. Themmen (Axel)

    1998-01-01

    textabstractGlycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormon

  17. 番茄低温响应转录因子SlNAC41克隆及表达分析%Cloning and Expression Analysis of a Cold-Responsive Transcription Factor SlNAC41 in Tomato

    Institute of Scientific and Technical Information of China (English)

    刘辉; 王涛涛; 张俊红; 欧阳波; 李汉霞

    2014-01-01

    NAC transcription factors play vital roles in regulating plant growth and developmental processes as well as in response to biotic and abiotic stresses. In our previous study, Unigene SGN-U212711 was found to be strongly induced by low temperature through analyzing the gene expression proifles of tomato seedlings under cold stress. In this study, we cloned this gene (designated as SlNAC41) from tomato. SlNAC41 had an open reading frame (ORF) of 1 173 bp encoding 390 amino acids with a typical NAM domain in its N-terminal, sug-gesting that it belonged to NAC transcription factor family. The molecular weight and isoelectric point of SlNAC41 protein was 43.5 kDa and 5.2, respectively. Quantitative real-time PCR analysis showed that SlNAC41 was expressed in all tested tomato tissues, with the highest expression in lfowers and the lowest expression in mature red fruits. Expression of SlNAC41 was induced by cold, drought, salt, methyl viologen (MV), abscisic acid (ABA) and ethephon (ETH) treatments, especially was strongly induced by cold and drought stresses. Promoter sequence analyzed by PLACE and PlantCARE showed that there were many cis-acting regulatory elements involved in response to light, pathogen infection, hormones, cold, dehydration and salt stresses. These results suggest that SlNAC41 may play important roles in regulating biotic and abiotic stress responses in tomato.%NAC转录因子在调控植物生长发育、生物及非生物逆境应答中发挥着重要作用。前期,我们通过对番茄幼苗在低温胁迫下的基因表达谱进行分析,发现Unigene SGN-U212711受低温诱导表达强烈。本研究从番茄中克隆了该基因,命名为SlNAC41,其开放阅读框(ORF)1173 bp,编码390个氨基酸,蛋白N端具有典型的NAM结构域,属于NAC转录因子家族成员。预测SlNAC41蛋白分子量为43.5 kDa,等电点为5.2。实时荧光定量PCR分析表明, SlNAC41在番茄各组织均有表达,在花中的表达量最高,在红熟果中

  18. Cloning and expression of Kluyveromyces fragilis LAC4 gene

    Institute of Scientific and Technical Information of China (English)

    霍克克; 李育阳

    1995-01-01

    The genomic library of Kluyveromyces fragilis was constructed in E.coli TG1,and 5β-galactosidase gene(LAC4)clones have been obtained from the library by complementation of theKluyveromyces lactis lac4-8 mutation.The studies on the structure and the function of the LAC4 gene revealedthat(i)the gene can also complement E.coli lacZ mutation;(ii)the physical map of the K.fragilis LAC4gene was very similar to that of K.lactis;(iii)the β-galactosidase levels expressed by the clone strains weremuch higher than that expressed by the original strain;(iv)the variation of the β-galactosidase level of differ-ent clone strains induced by lactose or galactose was related to the retained degree of the 5’ flanking region ofLAC4 gene,suggesting that there migth he a lactose specific transcription activating element in the region.

  19. 茄子花青素合成相关基因SmMYB的克隆与表达分析%Cloning and Expression Analysis of an Anthocyanin-related Transcription Factor Gene SmMYB in Eggplant

    Institute of Scientific and Technical Information of China (English)

    邵文婷; 刘杨; 韩洪强; 陈火英

    2013-01-01

    MYB gene's cDNA and gDNA sequences in full length were cloned from eggplant {Solarium melongenaL.) cultivars'YZ14'and'YZ3'using RT-PCR and RACE. Sequence analysis shows that the full length of cDNA is 1 035 bp long, and the open reading frame (ORF) is 837 bp long, encoding 278 amino acids. The protein ispelectric point is at pH8.47. The SmMYB protein contains two typical DNA-binding domains and has 71% homologies with anthocyanin biosynthesis-related R2R3-MYB transcription factor in capsicum. Furthermore, the protein is located mostly in the nucleus, in agreement with the regulatory function of MYB transcription factors. The corresponding gDNA is 3 834 bp in length, consisting of three exons and two introns. Fluorescence semi-quantitative PCR analysis indicates that SmMYB is expressed in all organs of the plant, including roots, stems, leaves, petals, and peels. However, the expression level of SmMYB is tissue-specific, and highly correlated with the concentration of anthocyanin in peel after shading treatment. Therefore, the SmMYB is speculated to be a MYB transcription factor gene, which positively regulates the biosynthesis of anthocyanin in eggplant.%以茄子(Solanum melongenaL.) ‘YZ14’ (紫茄)和‘YZ3’ (白茄)为试验材料,采用同源克隆与RACE (rapid amplification of cDNA ends)相结合的方法克隆了茄子MYB基因cDNA及gDNA全长序列,命名为SmMYB.序列分析结果表明:该基因cDNA全长1 035 bp,开放阅读框837 bp,编码278个氨基酸,与辣椒(Capsicum annuum L.)MYB转录因子氨基酸序列相似性达71%;成熟蛋白的等电点为8.47,具有2个典型的DNA-binding结构域且亚细胞定位于细胞核; gDNA全长3 834 bp,包含3个外显子及2个内含子.荧光半定量检测结果表明:SmMYB在茄子根、茎、叶、花瓣、果皮中均有表达,但表达水平具有组织特异性;遮光处理后紫色茄子果皮中该基因表达量变化与花青素合成量变化趋势相似.推测SmMYB为一个MYB转

  20. Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

    Science.gov (United States)

    Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

    2014-09-01

    An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones. © 2014

  1. Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer.

    Science.gov (United States)

    Seim, Inge; Lubik, Amy A; Lehman, Melanie L; Tomlinson, Nadine; Whiteside, Eliza J; Herington, Adrian C; Nelson, Colleen C; Chopin, Lisa K

    2013-04-01

    Ghrelin is a multifunctional hormone, with roles in stimulating appetite and regulating energy balance, insulin secretion and glucose homoeostasis. The ghrelin gene locus (GHRL) is highly complex and gives rise to a range of novel transcripts derived from alternative first exons and internally spliced exons. The wild-type transcript encodes a 117 amino acid preprohormone that is processed to yield the 28 amino acid peptide ghrelin. Here, we identified insulin-responsive transcription corresponding to cryptic exons in intron 2 of the human ghrelin gene. A transcript, termed in2c-ghrelin (intron 2-cryptic), was cloned from the testis and the LNCaP prostate cancer cell line. This transcript may encode an 83 amino acid preproghrelin isoform that codes for ghrelin, but not obestatin. It is expressed in a limited number of normal tissues and in tumours of the prostate, testis, breast and ovary. Finally, we confirmed that in2c-ghrelin transcript expression, as well as the recently described in1-ghrelin transcript, is significantly upregulated by insulin in cultured prostate cancer cells. Metabolic syndrome and hyperinsulinaemia have been associated with prostate cancer risk and progression. This may be particularly significant after androgen deprivation therapy for prostate cancer, which induces hyperinsulinaemia, and this could contribute to castrate-resistant prostate cancer growth. We have previously demonstrated that ghrelin stimulates prostate cancer cell line proliferation in vitro. This study is the first description of insulin regulation of a ghrelin transcript in cancer and should provide further impetus for studies into the expression, regulation and function of ghrelin gene products.

  2. Cloning and expression of mamba toxins.

    Science.gov (United States)

    Smith, L A; Olson, M A; Lafaye, P J; Dolly, J O

    1995-04-01

    Mamba venoms contain pharmacologically active proteins that interfere with neuromuscular transmission by binding to and altering the normal functioning of neuronal proteins involved, directly or indirectly, with regulating nerve transmission. Of the mamba toxins studied to date, many act on voltage-sensitive K+ channels, nicotinic or muscarinic acetylcholine receptors, or acetylcholinesterase. In an attempt to clone, characterize, and express the genes encoding these toxins, as well as other genes specifying activities not completely elucidated as yet, a cDNA library was constructed from mRNA isolated from the glands of the black mamba. Clones from the library harboring sequences encoding 14 different mamba toxins were isolated and characterized by nucleotide sequence analysis. Genes coding for three proteins, dendrotoxins (DTX) K, I, and E, were expressed as maltose-binding (MBP) fusion proteins in the periplasmic space of Escherichia coli. The DTXK-MBP fusion protein was affinity purified, cleaved from its chaperon, and the recombinant DTXK purified from MBP. Recombinant DTXK was shown to be identical to native DTXK in its N-terminal sequence, chromatographic behavior, convulsion-inducing activity, and binding to voltage-activated K+ channels in bovine synaptic membranes. Computer modeling was employed to create three-dimensional structures of DTXK and DTX1 from the X-ray crystal structure of alpha-DTX utilizing both structural and sequence homologies. Comparisons were made between the three toxins, providing a framework for site-directed mutagenesis.

  3. Porcine gamma-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression

    DEFF Research Database (Denmark)

    Frandsen, Pernille Munk; Madsen, Lone Bruhn; Bendixen, Christian

    2009-01-01

    which shows a high similarity to bovine (90%), human (87%) and mouse (83%) γ-synuclein. A genomic clone containing the entire porcine SNCG gene was isolated and its genomic organization determined. The gene is composed of five exons, the general structure being observed to be very similar...... reports the cloning and characterization of the porcine (Sus scrofa) γ-synuclein cDNA (SNCG). The SNCG cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine SNCG cDNA codes for a protein of 126 amino acids...... to that of the human SNCG gene. Expression analysis by quantitative real-time RT-PCR revealed the presence of SNCG transcripts in all examined organs and tissues. Differential expression was observed, with very high levels of SNCG mRNA in fat tissue and high expression levels in spleen, cerebellum, frontal cortex...

  4. Cloning and functional characterisation of avian transcription factor E2A

    Directory of Open Access Journals (Sweden)

    Meyer Kerstin B

    2004-06-01

    Full Text Available Abstract Background During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. Results Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. Conclusions The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.

  5. Cloning and functional characterisation of avian transcription factor E2A.

    Science.gov (United States)

    Conlon, Thomas M; Meyer, Kerstin B

    2004-06-14

    During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.

  6. [Cloning and functional analysis of Phyllostachys edulis MYB transcription factor PeMYB2].

    Science.gov (United States)

    Xiao, Dong-Chang; Zhang, Zhi-Jun; Xu, Ying-Wu; Yang, Li; Zhang, Feng-Xue; Wang, Chao-Li

    2013-10-01

    MYB-type transcription factor is one of the largest families in plants, which plays important roles in accepting stress signals from environment and regulating the expression of stress-tolerant genes. In this paper, using homologous cloning and RACE technology, a MYB-type transcription factor, designated PeMYB2, was cloned from Phyllostachys edulis. The results of bioinformatics showed that PeMYB2 is a typical R2R3-MYB. It contained two tandem repeats in its N-terminus, and a membrane protein DUF3651 in its C-terminus. In addition, phylogenetic analysis indicated that PeMYB2 shared the highest homology with 85.98% to OsMYB18 protein from Oryza sativa spp. Japonica. In addition, a yeast one-hybrid assay showed that PeMYB2 could activate the expression of downstream genes. After PeMYB2 was transformed into Arabidopsis thaliana, seven PeMYB2 transgenic Arabidopsis lines were obtained. Phenotypic analysis of the transgenic and wild-type Arabidopsis showed that over-expression of PeMYB2 caused delayed flower or dwarfism in transgenic Arabidopsis. Under the abiotic stress conditions, such as salt and cold stresses, the over-expression of PeMYB2 in Arabidopsis had higher survival rate than the wild-type Arabidopsis. Expression analysis of saline stress response marker genes in the transgenic and wild-type plants under the salt stress condition showed that PeMYB2 regulated the expression of NXH1, SOS1, RD29A, and COR15A. As the result, PeMYB2 might play an important role in various responses to abiotic stresses in P. edulis.

  7. Expression of chromatin modification genes in organs of cloned cattle that died within hours after birth

    Institute of Scientific and Technical Information of China (English)

    LI Shijie; LIAN Zhengxing; LI Dongjie; YU Shuyang; ZHANG Lei; DAI Yunping; LI Rong; FEI Jing; LI Ning

    2006-01-01

    Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned animals died shortly after birth and displayed organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we have examined expression patterns of four genes that modified chromatin (DNMT1, PCAF,MeCP2 and EED) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves produced by artificial insemination (AI) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of DNMT1 and PCAF were found in some studied tissues, but the expression of MeCP2 and EED had similar levels to those of the normal controls. The expression of DNMT1 showed a higher level in heart, liver and brain of both cloned bovines. A higher expression level of PCAF was seen in heart and liver of both cloned bovines, but a lower level was seen only in spleen of adult fibroblast (AF) cell-derived clones. Our results suggest that aberrant expression in gene that modified chromatins were found in cloned bovine tissues of neonatal death. Because DNMT1 and PCAF play an important role in DNA methylation and histone acetylation on nuclear chromatin respectively, and normal expression of DNMT1 and PCAF is needed for precious reprogramming of donor nuclear, the aberrant transcription patterns of DNMT1 and PCAF in these clones 5 contribute to the defects of organs reported in neonatal death of clones.

  8. Cloning and Expression Characteristics of the Pig Stra8 Gene

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang

    2014-07-01

    Full Text Available Stra8 (Stimulated by Retinoic Acid 8 is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE, the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper’s gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p < 0.05 increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar.

  9. Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium-dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha.

    Science.gov (United States)

    Doyen, Périne; Bigot, Aurélie; Vasseur, Paule; Rodius, François

    2008-01-01

    Glutathione S-transferases (GST) and glutathione peroxidases (GPx) are essential components of cellular detoxification systems. We identified GST and GPx transcripts in the freshwater bivalve Dreissena polymorpha, their full-length coding sequences were obtained by reverse-transcription PCR using degenerated primers followed by 5' and 3' RACE-PCR (rapid amplification of cDNA ends-PCR). The cDNA identified encoded proteins of 205 and 243 amino acids corresponding respectively to a pi-class GST and a selenium-dependent GPx. The comparison of the deduced amino acid sequences with GST and GPx from other species showed that the residues essential to the enzymatic function of these two proteins are highly conserved. We studied their expression pattern in the digestive gland, the gills and the excretory system of D. polymorpha. The results showed that pi-GST mRNA expression is higher in the digestive gland than in the gills or the excretory system. Se-GPx transcripts are expressed at high, medium and very low levels in the digestive gland, the excretory system and the gills, respectively.

  10. Cloning and Expression of Osteonectin Gene from Rats

    Institute of Scientific and Technical Information of China (English)

    ZHOU Lingde; YUAN Lin; YAN Yuhua; LI Shipu

    2006-01-01

    Total cellular RNA was extracted from the osteoblast cells of newborn rats' calvarial bones, and the cDNA containing open-reading frame of osteonectin was amplified by reverse transcription polymerase chain reaction (RT-PCR). The obtained product was named On. The On fragment was inserted into pBT-T vector. Then, the On was subcloned, in-frame fused to 3'-end of the GST gene of the prokaryotic expression vector pGEX-KG, and the resulting recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS competent cells. A 60 kD fusion protein was expressed after IPTG induction. The On fragment was sequenced, and the sequencing result shows that it shares 99.8% homology with the sequence published in GenBank. The On SDS-PAGE analysis exhibits that the On was expressed with the GST gene. There is 10% fused protein in the total E.coli proteins, and the fusion protein is a soluble protein. These experimental results imply that On from Wistar rats was cloned successfully and expressed efficiently.

  11. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    Science.gov (United States)

    2011-09-01

    Army Research Laboratory: 2010; p 19. 18. Sambrook, J.; Fritsch, E. F.; Maniatis , T. Molecular Cloning : A Laboratory Manual. 2nd ed.; Cold Spring...on a rotating platform until not viscous (adapted from Molecular Cloning : A Laboratory Manual) (18). Each solution was centrifuged at 45,000 x g for... Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

  12. 青花菜转录因子基因 BoWRKY2的克隆与表达分析%Cloning and expression analysis of a transcription factor gene BoWRKY2 from broccoli

    Institute of Scientific and Technical Information of China (English)

    蒋明; 陈贝贝; 管铭; 李金枝; 黄笑梅; 顾云吉

    2015-01-01

    Summary Broccoli ( Brassica oleracea var . italica) , which belongs to Cruciferae family , is a cash crop widely cultivated in China , and it is regarded as one of the most consumed vegetables in the world . The flower head of broccoli is rich in minerals , vitamins , fibers as well as anti‐oxidants , so it is recognized as a healthy vegetable with anti‐cancer properties . As a major broccoli production center in China , the average plantation areas reach to 8 000 hm2 in Taizhou of Zhejiang Province . However , broccoli cultivation suffered from plant diseases of downy mildew and stalk break which were caused by Hyaloperonospora parasitica and Sclerotinia sclerotiorum , respectively , resulting in yield and quality loss . WRKY transcription factors played important roles in plant stress responses , and WRKY domains were defined as an approximately 60‐amino acid motif named WRKYGQK as well as a zinc finger structure of C‐X4‐5‐C‐X2223‐H‐X1‐HattheirC‐terminus.TheWRKYdomainbindsspecificallytoDNAsequenceof(T)(T)TGAC(C/T) known as the W‐box which exists in defense‐related promoters . Enhanced disease resistance by overexpression of WRKY genes in different crop plants has been reported in recent years . Inthisstudy,aWRKYgenedesignatedasBoWRKY2,wasisolatedfromB.oleraceavar.italica.Basedon sequence analysis , expression patterns of BoWRKY2 were detected using reverse transcription‐polymerase chain reaction(RT‐PCR) methodwhilechallengedby H.parasiticaand S.sclerotiorum,respectively. The results showed that the genome DNA sequence was 1 507 bp in length with two introns and a complete coding sequence of 987 bp , and the length of two introns were 425 and 95 bp , respectively ; BoWRKY2 encoded 328 amino acids with a WRKYGQK residue and a zinc finger structure of C‐X5‐C‐X23‐H‐X1‐H . The WRKY domain located between 247 and 313 residues , and several DNA binding sites were found at sites of 66 , 67 , 69 , 71 , 74 , 281 , 282 , 283 and 317

  13. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    Science.gov (United States)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  14. Characterization of the molecularly cloned murine alpha-globin transcription factor CP2.

    Science.gov (United States)

    Lim, L C; Fang, L; Swendeman, S L; Sheffery, M

    1993-08-25

    We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.

  15. An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression

    OpenAIRE

    Magis, Wendy; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    1996-01-01

    The mouse metallothionein-I (mMT-I) promoter is activated by the metal response element-binding transcription factor (MTF), which binds metal response elements (MREs) when stimulated with heavy metals. We analyzed eight K562 erythroleukemia cell clones, each carrying a single integrated copy of an mMT-I/β-geo construct, using a system that can independently assess the level of β-geo expression and the rate at which it is silenced. In these clones, basal expression ...

  16. Cloning and Expression Profiling of the Transcription Factor CBF Gene from Rosa hybrida%月季CBF转录因子基因的克隆及表达分析

    Institute of Scientific and Technical Information of China (English)

    翟俊峰; 王法微; 王南; 宗俊梅; 李海燕

    2012-01-01

    We firstly cloned the partial fragment of CBF from Rosa hybrida 'Hanjin 4' using homology cloning methods according to AP2/EREBP conserved sequences. Then, two pairs of specific primers for inverse PCR were designed to clone the 5' and 3' end sequences of CBF gene, respectively. The resulting 5' and 3' end sequences and the partial fragment were combined to a full length sequence, which was named RhCBF and submitted to GenBank (Accession number: EF582843 ). The full-length of cDNA is 758 bp and the open reading frame (ORF) is 603 bp, encoding 200 amino acids; According to Real-time PCR analysis using a pair of specific primers, we found that salt, low temperature stresses could induce the expression of RhcCBF dramatically, but the rnRNA level of RhCBF could hardly induced by drought treatment. These results show that RhCBF would play a critical role in salt and low temperature stresses.%根据CBF(c.repeat binding factor)AP2/EREBP保守区设计1对简并引物,采用PCR方法对月季‘寒锦4号’(Rosahybrida‘Hanjin4’)CBF转录因子基因中间片段进行克隆;再根据中间片段区域设计了两对特异引物,采用反向PCR方法对该基因的5r和3端的序列进行克隆,将中间片段与反向PCR产物拼接得到CBF基因全长序列,命名为RhCBF,GenBank注册编号为EF582843;该基因序列长758bp,ORF为603bp,编码200个氨基酸;同时,根据基因序列设计1对特异引物,利用荧光定量PCR分析月季CBF在不同逆境胁迫下的表达情况。结果显示低温和盐均可以诱导RhCBF的表达,而干旱处理不能诱导其表达。

  17. Cloning and expression of porcine SRPK1 gene

    African Journals Online (AJOL)

    Academic Journals

    2012-01-10

    Jan 10, 2012 ... cloned by real time polymerase chain reaction (RT-PCR), yet coding sequence ... phosphorylation of the RNA splicing factors with RS ... basic molecular information that is useful for the further .... of transcription, such as: HSF1, HSF2, Ik-1, IK-2, SRY, ... protein and human SRPK1 in the structure data base of.

  18. Cloning and expression of swine myostatin gene and its application in animal immunization trial

    Institute of Scientific and Technical Information of China (English)

    MA; Xianyong; CAO; Yongchang; SHU; Dingming; BI; Yingzuo

    2005-01-01

    We have amplified swine myostatin (MSTN) gene by reverse transcription polymerase chain reaction (RT-PCR) and cloned it into pGEM-T Easy vector. The cloned swine MSTN gene consists of 1128 nucleotides, which has been submitted to GenBank (acquired registered code- AY448008). The cloned swine MSTN gene was successfully expressed in E. coli without the first 25 amino acids. Crude extraction of the expressed recombinant MSTN protein was used to immunize mice to investigate the effects on their bodyweights. We show here that the body weights of the immunized mice were higher than that of the controls, even though the difference was not significant. Surprisingly, the progenies of the immunized mice also were heavier than the controls. Especially at day 3, the average body weight of the immunized mice was 10.5% higher than that of the controls , which is significant (p < 0.05).

  19. Cloning, expression and location of RNase9 in human epididymis

    Directory of Open Access Journals (Sweden)

    Lin YQ

    2008-11-01

    Full Text Available Abstract Background Mammalian spermatozoa become fully motile and fertile during transit through the luminal fluid of the epididymis. At least 200 proteins are present in the epididymal lumen, but the potential roles of these luminal proteins in male fertility are unknown. Investigation of the function of these proteins will elucidate the mechanism of sperm maturation, and also provide new drug targets for male contraception. We cloned RNase9 from a human epididymis cDNA library for characterization and analysis of its functions. Findings It was predicted that human RNase9 gene was located on chromosome 14q11.2 and encoded a 205 amino acids protein with a signal peptide of 26 amino acids at the N-terminus. The protein had eight conserved cysteine residues characteristic of the RNase A family members and several potential post-translational modification sites. At the transcriptional level, RNase9 was expressed in a wide variety of tissues, and the expression was higher in men than in boys. RNase9 was localized to the post-equatorial region of the sperms' head. Immunofluorescence staining showed that RNase9 protein was present mostly in the epithelium of the epididymal tubule. Recombinant RNase9 had no ribonuclease activity. In addition, RNase9 had no detectable effect on sperm motility and fertilization as demonstrated by blocking spermatozoa with anti-RNase9 polyclonal serum. Conclusion RNase9 is expressed in a wide variety of tissues. It is located on the post-equatorial region of the sperm head and the epithelium of epididymal tubule. Although RNase9 belongs to the RNase A family, it has no ribonuclease activity.

  20. Cloning and characterization of a putative transcription factor ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... bZIP transcription factors have been reported to play important roles in ... ding to protein structural features, Arabidopsis bZIP ... reactions were set up using Ex Taq DNA polymerase (TaKaRa) and ... RNA was isolated from maize samples using the TRIzol reagent ... 2– t method (Livak and Schmittgrn, 2001).

  1. Expression Analysis and Cloning of GRAS Transcription Factor Gene from Halostachys caspica%新疆盐穗木GRAS转录因子基因克隆及表达分析

    Institute of Scientific and Technical Information of China (English)

    周莲洁; 杨中敏; 张富春; 王艳

    2013-01-01

    An cDNA fragment was isolated from Halostachys caspica by suppression subtractive hybridization and its full-length cDNA with 2 090 bp was cloned by SMARTTM RACE,which consisted of a 1 635 bp open reading frame encoding 544 amimo acids with molecular weight of 61.503 kD and an isoelectric point of 6.1,a 294 bp 5'-UTR and 161 bp 3'-UTR.The deduced amino acid sequence got together with that of GRAS family protein VvSCL13 from Vitis vinifera and had a C-terminal conserved domain,so it was named HcSCL13 gene (GenBank accession number KC68640).Real-time PCR method was performed to investigate the expression profile of HcSCL13 gene under salt stress.HcSCL13 showed up-regulated expression patterns under salt treatment.Based on our results,we concluded that HcSCL13 gene might be involved in salt response and one of important components for the salt tolerant pathway in H.caspica.%利用抑制消减杂交法从藜科盐穗木属盐生植物盐穗木中分离得到了一个盐胁迫响应的表达序列标签(EST)片段,结合SMARTTM RACE技术获得了盐穗木GRAS转录因子基因的cDNA.序列分析表明,该基因全长2 090 bp,含有1 635 bp的阅读框,294 bp的5′-UTR和161 bp的3'-UTR,编码544个氨基酸,分子质量为61.503kD,理论等电点为6.1.系统进化树和Blast同源序列比对分析结果显示,该基因编码的蛋白具有GRAS家族特有的C端保守结构域,并与葡萄GRAS家族蛋白VvSCL13聚集在一起,故将该基因命名为HcSCL13(GenBank登录号KC68640).实时荧光定量qRT-PCR分析表明,HcSCL13基因在盐胁迫后表达呈明显上调,初步推测Hc-SCL13基因可能与盐穗木的耐盐性相关.

  2. Cloning and functional analysis of FLJ20420: a novel transcription factor for the BAG-1 promoter.

    Directory of Open Access Journals (Sweden)

    Hongyu Liu

    Full Text Available BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5' cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ∼26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix, except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski. Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA -transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p = 0.0006. The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p = 0.0001. Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal

  3. Cloning and expression of special F protein from human liver

    Institute of Scientific and Technical Information of China (English)

    Shu-Ye Liu; Xin-Da Yu; Chun-Juan Song; Wei Lu; Jian-Dong Zhang; Xin-Rong Shi; Ying Duan; Ju Zhang

    2007-01-01

    AIM:To clone human liver special F protein and to express it in a prokaryotic system.METHODS:Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this,cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E coli BL21 (DEB) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein.RESULTS:The cDNA clone of human liver special F protein (1134bp) was successfully produced,with the cDNA sequence being published in Gene-bank:DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted.CONCLUSION:F protein expresses cDNA clone in a proKaryotic system,which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.

  4. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  5. Cloning and expression of aequorin photoprotein using intein tag

    Directory of Open Access Journals (Sweden)

    Elah sadat Seyed Hosseini

    2015-01-01

    Full Text Available Background: Intein (INT, is the internal parts of the protein which can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. This protein sequence and their characteristic of self-cleavage by thiol induction, temperature and pH changes is used for protein purification. The advantage of this method compared to the other protein purification methods is that it doesn’t require any protease enzyme and protease removal steps that make this method important economically. In this study, aequorin photoprotein was hybridized with INT in molecular form and its expression was evaluated. Materials and Methods: In this study, aequorin coding gene that was cloned in pET21-a in the previous studies, was cloned in pTYB21 vector containing INT tag by specific primers and restriction enzymes. Then the resulting pTY-aequarin was transformed to the ER2566 expression strain and cloning accuracy was confirmed by electrophoresis, western blotting and sequencing. Results: The photoprotein aequorin was cloned into SapI/PstI restriction site of pTYB21 plasmid accurately and successfully. Aequorin- INT hybrid protein expression confirmed using traditional methods. Conclusion: The photoprotein aequorin constract in fused with INT confirmed by molecular methods. Also rate of Aequorin- INT expression determined about %25 of cell total protein.

  6. Cloning and expression of Neisseria meningitides luxS gene

    Institute of Scientific and Technical Information of China (English)

    Bahram Kazemi; Nazila Baghalzadeh-Mohammadi; Reza Hadavi; Mojgan Bandehpour; Elham Ghayour; Majid Moghbeli; Kazem Parivar

    2008-01-01

    Neisseria meningitides is a Gram-negative bacterium which is an important causative agent of septicemia and meningitis.Numerous pathogenic bacteria contain luxS,which is required for autoinducer-2 production.Neis-seria meningitides contains a functional copy of luxS that is necessary for full meningococcal virulence.Neisse-ria meningitides DNA was extracted and its luxS gene was amplified by nested PCR.PCR product was purified and cloned in to pQE-30 expression vector.Recombinant plasmid was transformed and mass cultured.luxS was expressed in E.coli and confirmed by western blot analysis.In this study,N.meningitides luxS was amplified, cloned and expressed successfully.The sequencing of PCR product confirmed that amplified gene was luxS. Gene was expressed and observed in SDS-PAGE.Protein was reacted by his tag monoclonal antibody through western blot analysis.

  7. Cloning and expression of prion protein encoding gene of flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; SUN Xiuqin; ZHANG Jinxing; ZAN Jindong

    2008-01-01

    The prion protein (PrP) encoding gene of flounder (Paralichthys olivaceus) was cloned.It was not interrupted by an intron.This gene has two promoters in its 5' upstream,indicating that its transcription may be intensive,and should have an important function.It was expressed in all 14 tissues tested,demonstrating that it is a house-keeping gene.Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  8. Cloning and Expression of the Lactococcus lactis purDEK Genes, Required for Growth in Milk

    DEFF Research Database (Denmark)

    Nilsson, Dan; Kilstrup, Mogens

    1998-01-01

    An operon containing the genes purD and purE and part of the purK gene was cloned from the facultative anaerobic gram positive bacterium Lactococcus lactis by complementation of the purD mutation in Escherichia coli SO609. The genes encode enzymes in the de novo pathway of purine nucleotides....... The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of purD indicated that a region of 145 bp is enough to give regulated expression of the reporter lacLM genes, which...

  9. Cloning and Expression Profiling Analysis of a Transcription Factor ChNAC1 in Hazelnut(Corylus heterophylla Fisch.)%平榛NAC转录因子的分离及表达特性分析

    Institute of Scientific and Technical Information of China (English)

    赵天田; 陈新; 刘庆忠; 王贵禧; 梁丽松; 马庆华

    2012-01-01

    NAC transcription factors are a family of functionally diverse proteins. These plant-specific NAC domain genes play an important role in response to various stresses. A cDNA encoding the NAC-like gene homologue was isolated from hazelnut( Corylus heterophylla Fisch. ) by RACE-PCR and designated ChNACl ( GenBank Accession No. HQ639415). Sequence analysis showed that cDNA of ChNACl was 1154 bp long and contained a single open reading frame. The predicted ChNACl protein has 291 amino acids with an estimated molecular mass of 33. 31 kD and an isoelectric point of 6. 66 kD,qRT-PCR analysis showed that the expression of ChNACl was induced by low temperature and peaked at 4 h after exposed to low temperatures of 4℃. The transcripts of ChNACl appeared in many hazelnut tissues including male inflorescence, bark, flower bud and seeds, but mostly accumulated in male inflorescence. These results suggest that ChNACl may function in cold stress signal transduction pathway.%NAC转录因子是近十年来新发现的具有多种生物功能的植物特异转录因子,在植物生长发育、激素调节和抵抗逆境等方面发挥着重要的作用.本研究基于Solexa技术对平榛花芽转录组文库进行分析,结合RACE-PCR扩增,从平榛中克隆了一个与NAC类基因同源的cDNA序列ChNAC1,该序列长度为1154bp,具有长度为876bp的完整开放阅读框架,推测编码蛋白含有291个氨基酸,具有N-末端同源性较高且十分保守的NAC结构域和一个位于C-末端的高度可变区域.qRT-PCR分析表明,ChNAC1可以在4℃低温胁迫条件下上调表达,在4h时出现表达峰值.组织表达分析结果表明,ChNAC1在雄花序中表达最高,其次是花芽、树皮和种子.推测ChNAC1可能参与植物响应低温反应过程.ChNAC1基因的克隆及表达分析为进一步阐明和探讨平榛NAC转录因子的功能奠定了基础.

  10. Cloning and expression of the rabbit prostaglandin EP2 receptor

    OpenAIRE

    Guan, Youfei; Stillman, Brett A.; Zhang, Yahua; Schneider, André; Saito, Osamu; Davis, Linda S.; Redha, Reyadh; Breyer, Richard M.; Breyer, Matthew D.

    2002-01-01

    Background Prostaglandin E2 (PGE2) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP2 receptor in regulating fertility, vascular tone and renal function. Results The full-length rabbit EP2 receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP2 exhibited specific [3H]PGE2 binding ...

  11. Cloning and expression of murine immune interferon cDNA.

    OpenAIRE

    1983-01-01

    The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line. A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma. Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins. The mature murine IFN-gamma encoded is 136...

  12. A weakened transcriptional enhancer yields variegated gene expression.

    Directory of Open Access Journals (Sweden)

    Cathy Collins

    Full Text Available Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, approximately 1.4 kb 3' of the promoter, but inactive approximately 6 kb 3', indicating that the activity of the core enhancer was distance-dependent. A longer segment--the core enhancer plus approximately 1 kb of 3' flanking material, including the 3' matrix attachment region--was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3' flank includes binding sites for at least two activators.

  13. Molecular cloning and expression profiling of multiple Dof genes of Sorghum bicolor (L) Moench.

    Science.gov (United States)

    Gupta, Shubhra; Arya, Gulab C; Malviya, Neha; Bisht, Naveen C; Yadav, Dinesh

    2016-08-01

    DNA binding with one finger (Dof) proteins represent a family of plant specific transcription factors associated with diverse biological processes, such as seed maturation and germination, phytohormone and light mediated regulation, and plant responses to biotic and abiotic stresses. In present study, a total of 21 Dof genes from Sorghum bicolor were cloned, sequenced and in silico characterized for homology search, revealing their identity to Dof like proteins. The expression profiling of SbDof genes using quantitative RT-PCR in different tissue types and also under drought and salt stresses was attempted. The SbDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth condition. Two of the SbDof genes namely SbDof8 and SbDof12 showed comparatively high level of transcript abundance in all the tissue types tested; whereas some of the SbDof genes showed a distinct tissue specific expression pattern. Further a total of 13 SbDof genes showed differential expression when subjected to either of the abiotic stress i.e. drought or salinity. Three of the SbDof genes namely SbDof12, SbDof19 and SbDof24 were found to be up-regulated in response to drought and salt stress. Comparative analysis of SbDof genes expression revealed existence of a complex transcriptional and functional diversity across plant growth and developmental stages.

  14. 枳NLP转录因子克隆及其在不同水分条件下的表达%Cloning and Expression ofPoncirus Trifoliata (L.) Raf. NIN- Like Transcription Factors under Different Water Conditions

    Institute of Scientific and Technical Information of China (English)

    曹雄军; 卢晓鹏; 熊江; 李静; 吴倩; 周芳芳; 谢深喜

    2016-01-01

    Objective]The objective of this study was to analyze the expression ofPoncirus trifoliata (L.) Raf. NIN-like transcription factors to discuss the regulation mechanism of nitrogen assimilation under different water conditions in citrus.[Method] Primers were designed to amplify the sequences of theNLPgenes CDS ofPoncirus trifoliate(L.) Raf.based on orange genome databases and molecular biology. Alignment of the sequences was performed using ClustalX and phylogenetic analysis of this alignment was conducted using MEGA. Analysis of the RelativeNLP genes expression data under different water conditions using Real-Time Quantitative PCR.[Result]FourPoncirus trifoliata (L.) Raf.NLP genes:PtNLP2,PtNLP4,PtNLP7andPtNLP8were obtained. The sequence alignment analysis showed that the identity of all NIN-like proteins was 45.13%, and possessed both the RWP-RK and PB1 domain, and all NIN-like proteins were high identity to sweet orange, respectively 97.57%, 96.47%, 99% and 97.33%. Phylogenic analysis has indicated that the 4Poncirus trifoliata (L.) Raf.NLP genes can be classified into four groups with Arabidopsis (PtNLP2andAtNLP1/2,PtNLP4andAtNLP4/5,PtNLP7andAtNLP6/7,PtNLP8andAtNLP8/9). There were differences in the expression pattern ofNLP genes inPoncirus trifoliata (L.) Raf. leaves and roots. The results indicated that with the decrease of soil water, the expression level ofNLP genes inPoncirus trifoliata (L.) Raf. leaves were up-regulated. ThePtNLP2, PtNLP4,PtNLP7 andPtNLP8 expression level were the highest when the relative water capacity was 15.4%, they were 2.9, 3.5, 5.9 and 2.8 folds higher respectively compared to the control (the relative water holding capacity was 61.0%). After that, the gene expression level began to down-regulate and had no statistically significant difference between the control and the relative water holding capacity of 9.4%. While the expression level ofNLP genes inPoncirus trifoliata (L.) Raf. roots of control were the highest, and the gene

  15. Cloning and prokaryotic expression of rat homolog of Serpina3n and its expression change during liver regeneration.

    Science.gov (United States)

    Wang, G P; Zhang, X S; Li, Y H; Zheng, J L; Tang, C Z; Zhang, W X

    2012-09-03

    A strikingly upregulated expressed sequence tag was screened from regenerating rat liver at 8 h in a 0-4-8-12 h short-interval successive partial hepatectomy model from a previous study. In the present study, a full-length open reading frame (ORF) corresponding to this expressed sequence tag was predicted through electronic cloning and was subsequently cloned from an 8-h rat regenerating liver and deposited in GenBank (accession No. HM448398). Sequence analysis of HM448398 and the predicted ORF revealed that the two ORFs may be different transcripts of a gene. The sequence of HM448398 was highly homologous to that of rat Serpina3n, suggesting that it may be a homolog of Serpina3n. The pGEX-2TK prokaryotic expression vector for this ORF was constructed, and the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis manifested that the recombinant expression vector could express the glutathione-S-transferase-fused rat homolog of Serpina3n in an insoluble form in BL21. The target fusion protein was purified with affinity chromatography and was used as antigen to immunize rabbits for the production of polyclonal antibodies. Immunohistochemistry and real-time reverse transcription polymerase chain reaction analysis revealed that the gene was highly expressed in the priming and termination phases of liver regeneration. These findings lay a solid foundation for further study of roles of HM448398 using knock-in and RNA interference methods during liver regeneration.

  16. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D;

    1990-01-01

    antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  17. Production of cloned pigs with targeted attenuation of gene expression.

    Directory of Open Access Journals (Sweden)

    Vilceu Bordignon

    Full Text Available The objective of this study was to demonstrate that RNA interference (RNAi and somatic cell nuclear transfer (SCNT technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE, a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA expression vector under the control of RNA polymerase III (U6 promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

  18. Transcriptional regulation of human thromboxane synthase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K.D.; Baek, S.J.; Fleischer, T [Univ. of Maryland Medical School, Baltimore, MD (United States)] [and others

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  19. RNA-Seq for enrichment and analysis of IRF5 transcript expression in SLE.

    Directory of Open Access Journals (Sweden)

    Rivka C Stone

    Full Text Available Polymorphisms in the interferon regulatory factor 5 (IRF5 gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE. IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s, it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1 SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2 an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3 an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.

  20. Cloning and expression of mouse peroxiredoxin I in IEC-6 Cells.

    Science.gov (United States)

    Zhang, Bo; Su, Yong-Ping; Wang, Tao; Wang, Feng-Chao; Ai, Guo-Ping; Xu, Hui; Wang, Jun-Ping; Huang, Yue-Sheng; Jiang, Jian-Xin

    2004-07-15

    To clone and express mouse peroxiredoxin I in IEC-6 cells. Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced, pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCR and Western blot. A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin I. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified, which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin I in IEC-6 cells. Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.

  1. Cloning and expression of mouse peroxiredoxin I in IEC-6 Cells

    Institute of Scientific and Technical Information of China (English)

    Bo Zhang; Yong-Ping Su; Tao Wang; Feng-Chao Wang; Guo-Ping Ai; Hui Xu; Jun-Ping Wang; Yue-Sheng Huang; Jian-Xin Jiang

    2004-01-01

    AIM: To clone and express mouse peroxiredoxin I in IEC-6cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced,pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCR and Western blot.RESULTS: A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin I. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRⅠ-BamHⅠ fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified,which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin I in IEC-6 cells.CONCLUSION: Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.

  2. Cloning and expression of transaldolase from potato.

    Science.gov (United States)

    Moehs, C P; Allen, P V; Friedman, M; Belknap, W R

    1996-11-01

    We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.

  3. Teratogenic factors affect transcription factor expression.

    Science.gov (United States)

    Kojima, Takuya; Asano, Shinya; Takahashi, Naoki

    2013-01-01

    Chemical compounds are produced every day, many with adverse effects on human health, and hence it is vital to predict the risks to humans simply, rapidly, and accurately. Teratogens have a serious impact on fetal development. This has been studied mainly by phenotypic analysis of experimental animals. However, since phenotypes can vary within different species, we established a new evaluation system based on our recent finding that teratogens influence Hox gene expression in mice. Similarly to the Hox gene expression changes, the expression patterns of several transcription factors involved in development, including the Dlx, Irx, Sall, and T-box families, were altered after 6 h of exposure to retinoic acid (RA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The expression changes in Dlx4, Dlx6, Irx5, Sall2, Sall3, Sall4, Tbx10, and Tbx22 were linked to teratogen-induced phenotypes, and our results indicate that expression changes in developmental transcription factors can help to predict teratogenic risk.

  4. Molecular cloning and expression analysis on LPL of Coilia nasus.

    Science.gov (United States)

    Wang, Meiyao; Xu, Dongpo; Liu, Kai; Yang, Jian; Xu, Pao

    2016-06-01

    Coilia nasus is one important commercial anadromous species which mainly distributed in the Yangtze River in China. At present, it has been on the "National Key Protective Species List" because of its severe resource damage. Lipid metabolism is very important during its long-distance migration. To make further research on lipid metabolism of C. nasus, we cloned lipoprotein lipase gene with homologous cloning method. A full-length cDNA of LPL of C. nasus was cloned from liver which covered 3537 bp with a 1519 bp open reading frame encoding 505 deduced amino acids whose molecular mass was 57.5 kDa and theoretical isoelectric point was 7.58. The deduced amino acids had high similarity with the reported LPL sequence of other species. It had typical conserved domain of LPL protein containing catalytic triad, N-linked glycosylation sites and conserved heparin-binding site, etc. We adopted quantitative real-time RT-PCR method to detect the mRNA expression of LPL of C. nasus in ten tissues including mesenteric adipose, liver, muscle, stomach, spleen, heart, head kidney, trunk kidney, gill and brain with β-actin as internal reference. LPL expressed in all the detected tissues. The highest expression was in mesenteric adipose, and followed by liver, muscle, stomach. Lipid expressed lowly in spleen, heart, head kidney, trunk kidney, gill and brain. The research on the cloning and differential expression of LPL of C. nasus will lay foundation for further research on lipid metabolism of C. nasus.

  5. Expression of Two N1 Clones with Single Amino Acid Dissimilarity of Avian Influenza H5N1 Virus

    Directory of Open Access Journals (Sweden)

    RISZA HARTAWAN

    2012-12-01

    Full Text Available Two clones of N1 gene derived from isolate A/Dk/Tangerang/Bbalitvet-ACIAR-TE11/2007 (H5N1 exhibit single mismatch of amino acid sequence at position 242 that is threonine and methionine for the clone #3 and #5, respectively. In order to evaluate the effect of the amino acid substitution, these clones were inserted into two different expression vectors that are pEGFP-C1 and pcDNA-3.3 TOPO® TA cloning. Subsequently, the respective recombinant clones were transfected into eukaryotic cells, including CEF, RK13 and VERO using Lipofectamine ‘plus’ reagent. As a result, the clone #3 retaining atypical sequence showed lower expression level rather than the clone #15 in both vectors and all type of cells. The 3D conformational modelling revealed that the mutation occurs in the inner part of glycoprotein embedded within envelope or matrix. Therefore, the missense mutation seems has no effect on the antigenic properties of neuraminidase but this substitution by any means causes lethal mutagenesis in the individual gene expression by reducing level of protein transcript.

  6. Cloning and characterization of zebra fish SPATA4 gene and analysis of its gonad specific expression.

    Science.gov (United States)

    Liu, Shangfeng; Liu, Bowen; He, Shan; Zhao, Ying; Wang, Zhao

    2005-06-01

    The spermatogenesis associated 4 gene (SPATA4, previously named TSARG2) was first cloned in human tissues and was reported to be a candidate spermatocyte apoptosis-related gene that is expressed specifically in testis. Analysis of SPATA4 expression and regulation in zebra fish may provide insight into the understanding of the complicated process of gonadogenesis. In this study, we cloned and characterized the SPATA4 gene from zebra fish (Danio rerio), which is homologous to human and mouse SPATA4. Zebra fish SPATA4 consists of six exons separated by five introns, as all SPATA4 genes in vertebrates. A promoter region was predicted using homologous blast and cloned for further study, and possible transcription factors were analyzed in this region. The putative protein encoded by this gene was analyzed using bioinformatics methods. Multi-tissue RT-PCR results demonstrated that the zebra fish SPATA4 gene is expressed specifically in testis and slightly in ovary. Analysis of the SPATA4 sequence and its spatial expression pattern indicate that this gene is highly conserved and may play an important role in the process of zebra fish gonadogenesis.

  7. Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

    Science.gov (United States)

    Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

    2009-05-01

    Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation.

  8. Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio.

    Science.gov (United States)

    Fiengo, Marcella; Donizetti, Aldo; del Gaudio, Rosanna; Minucci, Sergio; Aniello, Francesco

    2012-06-01

    We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

  9. Molecular cloning and in vitro expression of a silent phenoxazinone synthase gene from Streptomyces lividans.

    Science.gov (United States)

    Madu, A C; Jones, G H

    1989-12-14

    Phenoxazinone synthase (PHS) catalyzes a step in actinomycin D biosynthesis in Streptomyces antibioticus. Two sequences from Streptomyces lividans that hybridize to the phs gene of S. antibioticus have been cloned in Escherichia coli K-12 using the plasmid pBR322. Although there was some similarity in the restriction maps of the two cloned fragments, neither insert appeared to be a direct subset of the other nor of the S. antibioticus phs gene. In vitro expression studies, in a streptomycete coupled transcription-translation system, showed that a 3.98-kb SphI fragment encoded a PHS-related protein. These observations provide additional support for the existence of silent genes for antibiotic production in streptomycetes.

  10. Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

    Science.gov (United States)

    Bautsch, W; Emde, M; Kretzschmar, T; Köhl, J; Suckau, D; Bitter-Suermann, D

    1992-06-01

    A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

  11. Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

    Institute of Scientific and Technical Information of China (English)

    Juan Wang; Weibin Sun; Chun Lu; Guixia Tang

    2005-01-01

    Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2 (hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

  12. Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display.

    Directory of Open Access Journals (Sweden)

    Haichun Gao

    Full Text Available A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway cloning system. A total of 3584 individual ORFs (85% have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST-tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray and other proteomic tools (e.g., mass spectrometry-based proteomic analysis, and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro.

  13. Cloning, expression, and polymorphism of the porcine calpain10 gene

    Institute of Scientific and Technical Information of China (English)

    Xiuqin Yang; Di Liu; Hao Yu; Lijuan Guo; Hui Liu

    2008-01-01

    Calpains are calcium-regulated protcases involved in cellular functions that include muscle proteolysis both ante- and postmortem. This study was designed to clone the complete coding sequence of the porcine calpain10 gene, CAPN10, to analyze its expression characteristics and to investigate its polymorphism. Two isoforms of the CAPN10 gene, CAPN10A and CAPN10B, were obtained by reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods combined with in silico cloning. RT-PCR results indicated that CAPN10 mRNA was ubiquitously expressed in all tissues examined and, with increasing age,the expression level increased in muscles at six different growth points. In the same tissues, the expression level of CAPN10A was higher than that of CAPN10B. In addition,three single nucleotide polymorphisms were detected by the PCR-single-stranded conformational polymorphism method and by comparing the sequences of Chinese Min pigs with those of Yorkshire pigs. C527T mutation was a missense mutation and led to transforming Pro into Leu at the 176th amino acid. The results of the current study provided basic molecular information for further study of the function of the porcine CAPN10 gene.

  14. Cloning and activity analysis of in vitro expression of plant NAD-IDH genes

    Institute of Scientific and Technical Information of China (English)

    CHEN Defu; CHEN Xiwen

    2004-01-01

    The plant NAD+-dependent isocitrate dehydrogenase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidopsis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added together to the in vitro system with 36 μg/mL of protein disulfide isomerase, the expressed products had NAD-IDH enzymatic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.

  15. Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation.

    Science.gov (United States)

    Clos, J; Westwood, J T; Becker, P B; Wilson, S; Lambert, K; Wu, C

    1990-11-30

    We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila. The predicted sequence of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization. A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats. Drosophila HSF produced in E. coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro. In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction. These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo.

  16. Streptokinase: cloning, expression, and excretion by Escherichia coli.

    Science.gov (United States)

    Malke, H; Ferretti, J J

    1984-06-01

    Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage lambda replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene ( skc ). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.

  17. Cloning and characterization of neuropeptide Y (NPY) and cocaine and amphetamine regulated transcript (CART) in Atlantic cod (Gadus morhua).

    Science.gov (United States)

    Kehoe, Amy S; Volkoff, Hélène

    2007-03-01

    Neuropeptide Y (NPY) and cocaine and amphetamine regulated transcript (CART) are two neuropeptides involved in the regulation of feeding in both mammals and fish. NPY stimulates feeding whereas CART inhibits feeding. In this study, we have cloned the full-length cDNA and complete genomic DNA sequences for NPY and CART in Atlantic cod. Atlantic cod preproNPY share a 45-85% identity with preproNPY from other fish whereas preproCART shows a 70% identity to CART peptides from zebrafish and goldfish. RT-PCR revealed that NPY mRNA is expressed in brain, in particular the forebrain, and in peripheral tissues, including intestine and kidney. CART mRNA is expressed throughout the brain and in ovaries. In order to assess the role of these peptides in the regulation of feeding, we examined changes in mRNA expression in the forebrain before, during and after a meal. NPY and CART mRNA both undergo peri-prandial changes in expression, with NPY levels being elevated around meal time and CART showing a decline 2 h after a meal. Food deprivation for 7 days induced a decrease in CART mRNA expression in the brain but did not affect NPY mRNA expression. Overall, our results suggest that NPY and CART are conserved peptides that might be involved in the regulation of feeding and other physiological functions in Atlantic cod.

  18. Comparative transcriptional analysis reveals differential gene expression between asymmetric and symmetric zygotic divisions in tobacco.

    Directory of Open Access Journals (Sweden)

    Tian-Xiang Hu

    Full Text Available Asymmetric cell divisions occur widely during many developmental processes in plants. In most angiosperms, the first zygotic cell division is asymmetric resulting in two daughter cells of unequal size and with distinct fates. However, the critical molecular mechanisms regulating this division remain unknown. Previously we showed that treatment of tobacco zygotes with beta-glucosyl Yariv (βGlcY could dramatically alter the first zygotic asymmetric division to produce symmetric two-celled proembryos. In the present study, we isolated zygotes and two-celled asymmetric proembryos in vivo by micromanipulation, and obtained symmetric, two-celled proembryos by in vitro cell cultures. Using suppression-subtractive hybridization (SSH and macroarray analysis differential gene expression between the zygote and the asymmetric and symmetric two-celled proembryos was investigated. After sequencing of the differentially expressed clones, a total of 1610 EST clones representing 685 non-redundant transcripts were obtained. Gene ontology (GO term analysis revealed that these transcripts include those involved in physiological processes such as response to stimulus, regulation of gene expression, and localization and formation of anatomical structures. A homology search against known genes from Arabidopsis indicated that some of the above transcripts are involved in asymmetric cell division and embryogenesis. Quantitative real-time PCR confirmed the up- or down-regulation of the selected candidate transcripts during zygotic division. A few of these transcripts were expressed exclusively in the zygote, or in either type of the two-celled proembryos. Expression analyses of select genes in different tissues and organs also revealed potential roles of these transcripts in fertilization, seed maturation and organ development. The putative roles of few of the identified transcripts in the regulation of zygotic division are discussed. Further functional work on these

  19. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  20. Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate.

    Science.gov (United States)

    Zhao, Xueqing; Yuan, Zhaohe; Feng, Lijuan; Fang, Yanming

    2015-07-01

    Exterior fruit color is an important trait for the evaluation of pomegranate fruit quality, but the molecular mechanism underlying the variation in color between red- and white-fruited pomegranate is poorly understood. In this study, full-length cDNA clones encoding enzymes involved in anthocyanin biosynthesis-such as chalcone synthase, chalcone isomerase, flavanone 3-hydoxylase, dihydroflavonol 4-reductase, anthocyanidin synthase (ANS), UDP-glucose-flavonoid 3-O-glucosyltransferase, and the R2R3 MYB transcription factor PgMYB-were isolated from fruit peels. In addition, transcript levels of anthocyanin biosynthetic genes were quantitatively measured by real-time PCR in red and white fruits. In both cultivars, two expression peaks for structural genes were detected during fruit development, whereas only one peak was observed-during early development-for PgMYB. While PgMYB is important for flavonoid biosynthesis, other transcription factors appear to also be necessary for the regulation of anthocyanin biosynthesis. No anthocyanins were detected in the white cultivar. Peels of white fruits contained transcripts of all identified genes except for PgANS, suggesting that the lack of PgANS expression may be the main factor responsible for the absence of anthocyanins in white pomegranate. PgANS may be the key gene involved in anthocyanin biosynthesis in pomegranate fruit.

  1. Cloning and tissue expression of CAST transcript II in goat%山羊CAST基因Ⅱ型转录本的克隆及在山羊不同组织中的表达

    Institute of Scientific and Technical Information of China (English)

    赵伯阳; 汪代华; 徐刚毅; 赵文伯; 郑程莉

    2011-01-01

    钙蛋白酶抑制蛋白((Calpastatin,CAST)基因是与畜禽肉质性状密切相关的重要候选基因.文章根据牛和绵羊CAST基因mRNA,应用RACE技术首次成功克隆了山羊CAST基因11型转录本(以下简称CAST II基因)全长cDNA,对序列及编码的氨墓酸进行了生物信息学分析.结果显示,该基因cDNA全长2 474 by,完整的开放阅读框(ORF)为558-2 252 by,编码564个氨基酸.氮基酸序列中存在4个保守结构域和1个保守七肤序列;蛋白质二级结构以无规卷曲和a一螺旋为主,富含疏水区,存在多个磷酸化位点以及蛋白激酶C(Protein kinase C,PKC)的磷酸化位点.通过实时荧光定量RT-PCR技术分析了CAST 11基因在天府肉羊部分组织中的表达情况.结果表明:CAST 11基因在所选择的天府肉羊7种组织中均有表达,半岁各组织中,眼肌的表达量最高,与腿肌差异显著(P<0.05),极显著高于内脏各组织(P<0.01);在眼肌组织中,CAST 11 A因的表达量随着年龄的增长而增加,3岁时的表达量最高.%Calpastatin (CAST) gene is closely related with meat quality in livestock and poultry.Based on the bovine and ovine mRNA sequences, the cDNA of CAST Ⅱgene in goat was amSplified successfully for the first time by using RACE-PCR.Results showed that CASTⅡ of goat was 2474 bp in length with an open reading frame (ORF) 1695 bp long and encoded 564 amino acids, and there were four conserved domains and one conserved seven-peptide domain m amino acids sequences.Bioinformation analysis indicated that its secondary structures mainly were random coil and helical regions, and contained rich hydrophobic regions, certain phosphorylation sites, and protein kinase C (PKC) sites.Meanwhile,analysis of tissue expression of the gene in Tianfu meat goat demonstrated it was expressed in seven selected tissues.When the goat was of 6-month age, the highest expression was observed in longissimusdorsi, which was significantly higher than that of crureus (P<0

  2. Cloning and expression of the rat homologue of the Huntington disease gene

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, I.; Epplen, J.T.; Riess, O. [Ruhr-Univ. Bochum (Germany)] [and others

    1994-09-01

    Huntington`s disease (HD) is an autosomal dominant neurodegenerative disorder which is manifested usually in adult life. The age of onset is variable and leads to progressive symptoms including involuntary choreatic movements and various cognitive and psychiatric disturbances. Recently, a gene (IT15) was cloned containing a (CAG){sub n} repeat which is elongated and unstable in HD patients. IT15 is widely expressed in human tissues but unrelated to any known deduced protein sequence. To further investigate the HD gene, 15 rat cDNA libraries were screened. 24 clones have been identified covering the Huntingtin gene. Comparison of the Huntingtin gene between human and rat revealed homologies between 80% and 87% at the DNA level and about 90% at the protein level. These analyses will help to define biologically important sequence regions, e.g., via evolutionary conservation. One clone contains the (CAG){sub n} repeat which consists of eight triplets compared to seven triplets in the mouse and a median of 17 in human. As in humans there are two transcripts arising from differential 3{prime}-polyadenylation. In the 3{prime}UTR a stretch of about 280 bp is exchanged for a 250 bp fragment with no homology in rodents and man. The cDNA clones are currently used to study Huntingtin gene expression during development in rodent tissues. RNA in situ hybridization of embryonic sections shows predominant signals in all neuronal tissues. In contrast to previously published data Huntingtin mRNA expression in testis is increased in spermatocytes vs. spermatogonia.

  3. Cloning and GST-fused expression in E. coli of mouse β-1,4-galactosyltransferase

    Institute of Scientific and Technical Information of China (English)

    龚兴国; 钟文涛; 吴文英

    2004-01-01

    β-1,4-galactosyltransferase (β4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of β4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse β4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopro-pyl-β-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse β4Gal-T. The transcriptional product of β4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.

  4. Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. tritici, the take-all fungus.

    Science.gov (United States)

    Litvintseva, Anastasia P; Henson, Joan M

    2002-03-01

    Gaeumannomyces graminis var. tritici, a filamentous ascomycete, is an important root pathogen of cereals that causes take-all disease and results in severe crop losses worldwide. Previously we identified a polyphenol oxidase (laccase) secreted by the fungus when induced with copper. Here we report cloning and partial characterization of three laccase genes (LAC1, LAC2, and LAC3) from G. graminis var. tritici. Predicted polypeptides encoded by these genes had 38 to 42% amino acid sequence identity and had conserved copper-binding sites characteristic of laccases. The sequence of the LAC2 predicted polypeptide matched the N-terminal sequence of the secreted laccase that we purified in earlier studies. We also characterized expression patterns of these genes by reverse transcription-PCR. LAC1 was transcribed constitutively, and transcription of LAC2 was Cu inducible. All three genes were transcribed in planta; however, transcription of LAC3 was observed only in planta or in the presence of host (wheat) plant homogenate.

  5. An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression

    Science.gov (United States)

    Magis, Wendy; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    1996-01-01

    The mouse metallothionein-I (mMT-I) promoter is activated by the metal response element-binding transcription factor (MTF), which binds metal response elements (MREs) when stimulated with heavy metals. We analyzed eight K562 erythroleukemia cell clones, each carrying a single integrated copy of an mMT-I/β-geo construct, using a system that can independently assess the level of β-geo expression and the rate at which it is silenced. In these clones, basal expression and rate of silencing vary widely and independently with integration site. This implies that the rates of transcription and of silencing are separate properties determined by interaction of the regulatory elements of the transgene with the site of integration. Induction of the mMT-I promoter with zinc both increases expression level and strongly retards silencing of β-geo expression. At a given integration site, expression level and silencing are affected coordinately by induction. Taken together with earlier studies of distant metal-responsive elements, these results suggest that distance from the promoter may determine whether a factor can increase transcription rate. Stimulation of an MRE can both increase transcription and overcome repressive effects of chromatin; we suggest that these functions are linked. PMID:8943035

  6. Cloning and expression analysis of MBLL cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.

  7. Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening.

    Science.gov (United States)

    Rastogi, R; Dulson, J; Rothstein, S J

    1993-11-01

    Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from oat and Escherichia coli. Degenerate oligonucleotides corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction amplification of tomato (Lycopersicon esculentum Mill.) genomic DNA, and a 1.05-kb fragment was obtained. This genomic DNA fragment encodes an open reading frame of 350 amino acids showing about 50% identity with the oat ADC protein. Using this fragment as a probe, we isolated several partial ADC cDNA clones from a tomato pericarp cDNA library. The 5' end of the coding region was subsequently obtained from a genomic clone containing the entire ADC gene. The tomato ADC gene contains an open reading frame encoding a polypeptide of 502 amino acids and a predicted molecular mass of about 55 kD. The predicted amino acid sequence exhibits 47 and 38% identify with oat and E. coli ADCs, respectively. Gel blot hybridization experiments show that, in tomato, ADC is encoded by a single gene and is expressed as a transcript of approximately 2.2 kb in the fruit pericarp and leaf tissues. During fruit ripening the amount of ADC transcript appeared to peak at the breaker stage. No significant differences were seen when steady-state ADC mRNA levels were compared between normal versus long-keeping Alcobaca (alc) fruit, although alc fruit contain elevated putrescine levels and ADC activity at the ripe stage. The lack of correlation between ADC activity and steady-state mRNA levels in alc fruit suggests a translational and/or posttranslational regulation of ADC gene expression during tomato fruit ripening.

  8. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  9. Cloning and expression of the enzymatic region of Streptococcal hyaluronidase

    Directory of Open Access Journals (Sweden)

    Nafiseh Al-Sadat Mirjamali

    2014-09-01

    Full Text Available Objective(s: Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity. Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied. Results: Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair was inserted correctly into the recombinant vector. The expressed protein (65 KDa was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient’s sera. Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

  10. Molecular cloning, characterization, and expression of wheat cystatins.

    Science.gov (United States)

    Kuroda, M; Kiyosaki, T; Matsumoto, I; Misaka, T; Arai, S; Abe, K

    2001-01-01

    We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47-68% amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63-67% similalities to one another while 93% of amino acids were identical between WC1 and WC3. This suggested that WCI, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.

  11. Cloning and tissue expression characterization of the chicken APOB gene.

    Science.gov (United States)

    Zhang, Sen; Shi, Hui; Li, Hui

    2007-01-01

    Apolipoprotein B (APOB) serves an essential role in the assembly and secretion of triglyceride-rich lipoproteins and lipids transport. This study was designed to clone the full-length cDNA of the chicken APOB gene, to characterize the expression profile, and investigate the differential expression between layer and broiler of the chicken APOB gene. The full-length cDNA sequence (14,150-bp) that contained a 13,896-bp ORF encoding 4,631 amino acids was obtained by RT-PCR, RACE, and bioinformatics analysis. qReal-Time PCR analysis showed that the chicken APOB gene was highly expressed in kidney, liver, and intestine. The results of differential expression showed that the APOB gene was more highly expressed in intestine and kidney in Bai'er layer than in broiler, but there was no significant difference in liver between the two breeds. The results of this study provided basic molecular information for studying the role of APOB in the energy transportation in avian species.

  12. Implementation of semi-automated cloning and and prokaryotic expression screening: the impact of SPINE

    NARCIS (Netherlands)

    Alzari, P.M.; Folkers, G.E.|info:eu-repo/dai/nl/162277202

    2006-01-01

    The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques

  13. Implementation of semi-automated cloning and and prokaryotic expression screening: the impact of SPINE

    NARCIS (Netherlands)

    Alzari, P.M.; Folkers, G.E.

    2006-01-01

    The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques

  14. Cloning, Physical Mapping and Expression Analysis of a Wheat Mlo-like Gene

    Institute of Scientific and Technical Information of China (English)

    Ling YU; Ji-Shan NIU; Pei-Du CHEN; Zheng-Qiang MA; Da-Jun LIU

    2005-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) was performed to isolate a cDNA clone using specific primers designed based on the barley (Hordeum vulgare L.) Mlo gene cDNA sequence.A full-length cDNA encoding an Mlo-like protein was isolated and characterized in a Triticum aestivum L.-Haynaldia villosa L. 6VS/6AL translocation line. The putative protein consists of 534 amino acid residues,which contain a nuclear localization motif (NLS), nine casein kinase Ⅱ motifs (S/T-X-X-D/E) and seven protein kinase C motifs (S/T-X-R/K). It is highly homologous to other plant Mlo proteins. Thus, this clone was designated as Ta-Mlo (GenBank accession No. AF384144). Northern blotting analysis showed that the transcription of Ta-Mlo was enhanced slightly by Blumeria graminis (DC) EO Speer f. sp. tritici. Western blotting analysis showed that the protein expression product of the Ta-Mlo gene in wheat seedling leaves is a membrane-bound protein. The protein could be induced by B. graminis. Southern blotting analysis indicated that there is one copy of the Ta-Mlo gene in each wheat genome. Ta-Mlo was localized on specific chromosomal regions of 2AL, 2BL, and 2DL in wheat.

  15. Cloning and Expression of Ontak Immunotoxin Using Intein Tag

    Directory of Open Access Journals (Sweden)

    SA Moosavizadeh

    2016-06-01

    Full Text Available Introduction: Inteins (INT are internal parts of a number of proteins in yeast and some other unicellular eukaryotes, which can be separated from the immature protein during protein splicing process. After identifying the mechanism of intein action, applications of these sequences are be considered in the single- step purification of recombinant proteins and different intein tags were developed. The most important advantage of using intein tags in purification of recombinant proteins than other affinity tags is no requirement of expensive protease enzymes and following additional steps to remove protease that make intein tags economically are considered more important. In the present study, denileukin diftitox immunotoxin (brand name Ontak, be fused with an intein tag and it was inserted in pTXB1 plasmid. Methods: In this study, with respect to multiple cloning sites (MCS of pTXB1, specific primers were designed. Polymerase Chain Reaction (PCR was performed and encoding sequence of ONTAK was cloned using restriction sites of NdeI and SapI. Recombinant vector (PTX-IDZ was transformed into E. coli strain ER2566 and expression of gene was studied. Results: The accuracy of recombinant construct was confirmed by PCR and enzymatic digestion. The produced recombinant proteins were confirmed by SDS-PAGE and Western blotting. Conclusion: Restriction site of SapI guarantees no additional residues incorporate in primary protein sequence. Also, the expression of this construct was analyzed in compare with fused protein to poly-His tag. According to the appropriate expression of fused protein in both constructs it was expected that one step- purification of considered drug protein will be success in the following steps.

  16. Cloning and expression study of the lobster (Homarus americanus) vitellogenin: Conservation in gene structure among decapods.

    Science.gov (United States)

    Tiu, Shirley Hiu Kwan; Hui, Ho-Lam; Tsukimura, Brian; Tobe, Stephen S; He, Jian-Guo; Chan, Siu-Ming

    2009-01-01

    This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.

  17. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    Directory of Open Access Journals (Sweden)

    Hiroshi Kondo

    2015-01-01

    Full Text Available Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12 were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC, an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5, an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1 expression levels were enhanced. After treatment with dexamethasone (DEX, 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX, and keratinocyte growth factor (KGF, surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

  18. Tumoral Environment Triggers Transcript Anomalies in Established Tumors: Induction of Altered Gene Expression and of Aberrant, Truncated and B2 Repeat-Containing Gene Transcripts

    Directory of Open Access Journals (Sweden)

    Pieter Rottiers

    1999-12-01

    Full Text Available In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR-based subtraction suppression hybridization (SSH to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies.

  19. Molecular cloning and ontogenesis expression of fatty acid transport protein-1 in yellow-feathered broilers.

    Science.gov (United States)

    Song, Yuzhen; Feng, Jiaying; Zhou, Lihua; Shu, Gang; Zhu, Xiaotong; Gao, Ping; Zhang, Yongliang; Jiang, Qingyan

    2008-06-01

    Fatty acid transport protein-1 (FATP-1) is one of the important transporter proteins involved in fatty acid transmembrane transport and fat deposition. To study the relationship between FATP-1 mRNA expression and fat deposition, chicken (Gallus gallus) FATP-1 sequence was first cloned by rapid amplification of cDNA ends (RACE). Tissue samples of chest muscle, leg muscle, subcutaneous fat, and abdominal fat were collected from six male and six female broilers each, at 22 days, 29 days, and 42 days, respectively. The tissue specificity and ontogenesis expression pattern of the FATP-1 mRNA of yellow-feathered broilers was studied by real-time reverse transcription polymerase chain reaction (RT-PCR), and the fat deposition laws in different tissues were also compared. A 2,488 bp cDNA sequence of chicken FATP-1 was cloned by RACE (GenBank accession no. DQ352834), including 547 bp 3' end untranslated region (URT) and 1,941 bp open reading frame (ORF). Chicken FATP-1 encoded 646 amino acid residues, which shared 83.9% and 83.0% identity with those of human and rat, respectively. The results of quantitative PCR demonstrated a constant FATP-1 mRNA expression level in the chest muscle and subcutaneous fat of both male and female broilers at three stages, whereas the expression level of the FATP-1 mRNA in the leg muscle at 42 days was significantly higher than that at 22 days or 29 days. In the abdominal fat of male broilers, the gene expression significantly increased with age, whereas the female broilers showed a dramatic downregulation of FATP-1 expression in abdominal fat at 42 days. This suggested a typical tissue- and gender-specific expression pattern of chicken FATP-1, mediating the specific process of fatty acid transport or utilization in muscle and adipose tissues.

  20. Molecular cloning and ontogenesis expression of fatty acid transport protein-1 in yellow-feathered broilers

    Institute of Scientific and Technical Information of China (English)

    Yuzhen Song; Jiaying Feng; Lihua Zhou; Gang Shu; Xiaotong Zhu; Ping Gao; Yongliang Zhang; Qingyan Jiang

    2008-01-01

    Fatty acid transport protein-1 (FATP-1) is one of the important transporter proteins involved in fatty acid transmembrane transport and fat deposition. To study the relationship between FATP-1 mRNA expression and fat deposition, chicken (Gallus gallus) FATP-1 sequence was first cloned by rapid amplification of cDNA ends (RACE). Tissue samples of chest muscle, leg muscle, subcutaneous fat, and abdominal fat were collected from six male and six female broilers each, at 22 days, 29 days, and 42 days, respectively. The tissue specificity and ontogenesis expression pattern of the FATP-1 mRNA of yellow-feathered broilers was studied by real-time reverse transcription polymerase chain reaction (RT-PCR), and the fat deposition laws in different tissues were also compared. A 2,488 bp cDNA sequence of chicken FATP-1 was cloned by RACE (GenBank accession no. DQ352834), including 547 bp 3' end untranslated region (URT) and 1,941 bp open reading frame (ORF). Chicken FATP-1 encoded 646 amino acid residues, which shared 83.9% and 83.0% identity with those of human and rat, respectively. The results of quantitative PCR demonstrated a constant FATP-1 mRNA expression level in the chest muscle and subcutaneous fat of both male and female broilers at three stages, whereas the expression level of the FATP-1 mRNA in the leg muscle at 42 days was significantly higher than that at 22 days or 29 days. In the abdominal fat of male broilers, the gene expression significantly increased with age, whereas the female broilers showed a dramatic downregulation of FATP-1 expression in abdominal fat at 42 days. This suggested a typical tissue-and gender-specific expression pattern of chicken FATP-1, mediating the specific process of fatty acid transport or utilization in muscle and adipose tissues.

  1. Rapid Characterization of S. mansoni Expression Library Clones of Potential Interest

    Directory of Open Access Journals (Sweden)

    KANAMURA Herminia Yohko

    1997-01-01

    Full Text Available A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

  2. Expression liver-directed genes by employing synthetic transcriptional control units

    Institute of Scientific and Technical Information of China (English)

    Marie-Luise Lemken; Wolfgang A. Wybranietz; Ulrike Schmidt; Florian Graepler; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer

    2005-01-01

    AIM: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver,thereby compensating for the lack of specificity of currently available gene therapeutic vector systems.METHODS: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neor fusion gene cassette was generated,and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis.CONCLUSION: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver

  3. Cloning and embryonic expression of zebrafish PLAG genes.

    Science.gov (United States)

    Pendeville, Hélène; Peers, Bernard; Kas, Koen; Voz, Marianne L

    2006-03-01

    PLAG transcription factors play important roles in oncogenesis. To date three members of this subfamily of zinc finger proteins have been identified in humans and mice: PLAG1, PLAGL1 and PLAGL2. In this study, we identified zebrafish orthologs of PLAG1 and PLAGL2 and a novel member of this family, PLAGX. We examined the temporal expression of these three genes by quantitative real time RT-PCR and found that all three genes are maternally provided, expressed at low level during early somitogenesis and, during late somitogenesis and beyond, PLAG expression increases to reach a plateau level around 5 dpf. Whole mount in situ experiments revealed that PLAG1, PLAGL2 and PLAGX display a similar pattern of expression characterized by a low ubiquitous expression overcame by high expression in some restricted compartments such as the ventricular zone of the brain, the pectoral fin buds, the developing pharyngeal arches and the axial vasculature. We show that this pattern resembles the one observed for the proliferative marker PCNA, suggesting that the PLAG genes are expressed more strongly in zones of active proliferation. This hypothesis was proven for the ventricular zone shown to be a highly proliferative zone using the anti-phosphohistone H3 antibody that detects cells in mitosis.

  4. Cloning and quantitative expression analysis of drought-induced genes in soybean.

    Science.gov (United States)

    Stolf-Moreira, R; Medri, M E; Neumaier, N; Lemos, N G; Brogin, R L; Marcelino, F C; de Oliveira, M C N; Farias, J R B; Abdelnoor, R V; Nepomuceno, A L

    2010-05-11

    We determined the expression levels of DREB transcription factor (Gmdreb1) and of the genes Gmgols, Gmpip1b, Gmereb, and Gmdefensin in drought-tolerant (MG/BR46-Conquista) and drought-sensitive (BR16) genotypes of soybean, during drought. The trial was carried out in a controlled-environment chamber, set up to provide drought conditions. Sequences of Arabidopsis thaliana DREB-family proteins were used to build a phylogenetic tree through the alignment of the conserved regions near the AP2 domain. We found that Gmdreb1 is similar to Atrap2.1, which is located near the AtDREB1 and AtDREB2 families. The amplified fragment was cloned and sequenced; alignment with the sequence available at Genbank showed total similarity. Expression analysis showed that under drought: a) Gmdreb1 expression increased in leaves and roots of both genotypes and expression level changes occurred that were correlated with the length of the water-deficit period; b) there were increased expression levels of Gmdefensin in roots of MG/BR46; c) expression of Gmgols increased in leaves and roots of the two genotypes; d) Gmpip1b expression generally increased, except in roots of BR16, and e) the same was found for Gmereb, except in roots of MG/BR46.

  5. Molecular Cloning, Expression and Characterization of Ribokinase of Leishmania major

    Institute of Scientific and Technical Information of China (English)

    Patrick. O.J. OGBUNUDE; Nadia LAMOUR; Michael P. BARRETT

    2007-01-01

    Ribokinase (EC 2.1.7.15) from Leishmania major was cloned, sequenced and overexpressed in Escherichia coli. The gene expressed an active enzyme that had comparable activity to the same enzyme studied in E. coli. It specifically phosphorylated D-ribose. Under defined conditions, the Km for the substrates D-ribose and ATP were 0.3±0.04 mM and 0.2±0.02 mM, respectively. The turnover numbers of the enzyme for the substrates were 10.8 s-1 and 10.2 s-1, respectively. The enzyme product ribose 5-phosphate inhibited the phosphorylation of D-ribose with an apparent Ki of 0.4 mM, which is close to the Km (0.3 mM) of D-ribose, suggesting that it might play a role in regulating flux through the enzyme.

  6. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    Science.gov (United States)

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  7. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter.

    Science.gov (United States)

    Liu, Dan; Liu, Xin; Wu, Ye; Wang, Wen; Ma, Xinliang; Liu, Huirong

    2016-01-16

    HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at -1205~-838 bp and -146~+93 bp, with the -838~-649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at -709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.

  8. Molecular cloning and expression analysis of cDNA ends of chicken neuropathy target esterase.

    Science.gov (United States)

    Chang, Ping-An; Sun, Quan; Ni, Xiao-Min; Qv, Feng-Qiong; Wu, Yi-Jun; Song, Fang-Zhou

    2008-03-10

    Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in human and some sensitive animals. Adult hens are usually the animal model for experimental studies of OPIDN. However, little is known about the sequence and characteristics of chicken NTE. We report here the cloning of the 5' and 3' cDNA ends of chicken NTE through rapid amplification of cDNA ends (RACE) and their expression profiles in different tissues with northern blotting. The cloned 3' cDNA end of chicken NTE is 801 base pair (bp) in length with an open reading frame (ORF) of 379 bp. It contains a termination codon (TAG) and a 422-nucleotide noncoding sequence with the polyA sequence (GenBank accession no. DQ126678). The chicken NTE 5' cDNA end is 665 bp in length with an ORF of 552 bp. It contains an initiation codon (ATG) and a 113-bp untranslated region (GenBank accession no. DQ126677). The deduced proteins from 5' and 3' cDNA ends have a high degree of homology to humans and mouse NTE at the amino acid level. Chicken NTE is suggested to be a transmembrane protein by the transmembrane helix prediction of the deduced N-terminal sequence. The chicken NTE gene is expressed as a 4.5k b transcript in different tissues, including brain, kidney, liver and testis. Moreover, the mRNA expression of chicken NTE is highest in brain, and the mRNA levels of chicken NTE in testis, kidney and liver are about 75%, 47% and 24% of that in brain, respectively. These results should be helpful in cloning chicken full-length NTE gene.

  9. Molecular cloning of partial cDNAs for rat DNA topoisomerase II isoforms and their differential expression in brain development.

    Science.gov (United States)

    Tsutsui, K; Tsutsui, K; Okada, S; Watanabe, M; Shohmori, T; Seki, S; Inoue, Y

    1993-09-05

    cDNA segments for DNA topoisomerase II were amplified from rat brain RNA after reverse transcription by the polymerase chain reaction, using degenerate oligonucleotide primers deduced from the conserved regions of topoisomerase II of higher eukaryotes. The cDNA product from a successful amplification was homogeneous in length but heterogeneous in sequence. Restriction mapping of the cloned cDNA fragments revealed that they consisted of two distinct sequence groups. DNA sequencing of representative clones from each group, designated A and B, showed that they are highly homologous to cDNAs of human topoisomerase II isoforms, alpha and beta, respectively. Northern blot analysis indicated that the transcript level for rat topoisomerase II alpha was high in embryonic brain and in the cerebellum of 2-day newborns, followed by rapid decrease to a undetectable level at 4 weeks after birth. In contrast, rat topoisomerase II beta transcript was present throughout the embryonic and postnatal stages. In the developing cerebellum, cells expressing topoisomerase II alpha were confirmed exclusively to the outer mitotic zone of the external granular layer, whereas the transcript of topoisomerase II beta was detected over the entire cortical region. These results clearly indicate that the isoform alpha is expressed only in proliferating cells. The differential expression of topoisomerase II isozymes was also observed among developed tissues. Therefore, the isozymes are most likely to be involved in the following different physiological processes: topoisomerase II alpha in cell proliferation, and topoisomerase II beta in some processes unrelated to cell proliferation.

  10. Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Shun-Ying Jin; Yan-Li Liu; Li-Na Xu; Yong Jiang; Ying Wang; Bao-Xue Yang; Hong Yang; Tong-Hui Ma

    2006-01-01

    AIM: To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system.METHODS: A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells.RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot.RESULTS: An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut.Northern blot showed a single 3.0 kb transcript in selected digestive organs, pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Tmmunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins.CONCLUSION: pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.

  11. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  12. Cloning and characterization of GRIPE, a novel interacting partner of the transcription factor E12 in developing mouse forebrain.

    Science.gov (United States)

    Heng, Julian Ik Tsen; Tan, Seong-Seng

    2002-11-08

    The helix-loop-helix (HLH) family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and hematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. Although studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD are intimately involved in neuronal fate determination, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. To address this, a yeast two-hybrid interaction screen was employed to identify dimerization partners to E12. Screening of an embryonic day 11.5 forebrain library resulted in the cloning of GRIPE, a novel GAP-related interacting protein to E12. GRIPE binds to the HLH region of E12 and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidentally detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were recapitulated through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein dimerization of which with E12 has important consequences for cells undergoing neuronal differentiation.

  13. Cloning, expression and characterization of phycoerythrin gene from Ceramium boydenn.

    Science.gov (United States)

    Zhang, Xiaowen; Zhao, Fangqing; Qin, Song; Yan, Binlun

    2006-04-01

    Phycobiliproteins function as a major light harvesting protein-pigment complex in the cyanobacteria and the eukaryotic algae. Phycoerythrin (PE) is a kind of phycobiliproteins, widely located in all rhodophytes, some species of cyanobacteria and cryptophytes, and different ecotypes of Prochlorococcus populations. PeBA encoding beta and alpha subunits of PE from Ceramium boydenn was cloned and sequenced in this research. A peBA specific PCR primer was synthesized, based on the peBA gene conserved sequences. The beta subunit encoding gene (peB) contained an open reading frame of 534 bp, while the alpha subunit (peA) was 495 bp. Recombinant expression plasmid pET-peAB was constructed and expressed in Escherichia coli BL21. The molecular weight of expressive product of peB and peA was about 23.3 and 18.2 KD, respectively. Results of codon usage analysis show that G + C content is heterogeneous among different groups of PE and spacers have dramatically lower G + C contents than coding regions. Also there is a high variance in G + C content among sequences at the third position sites. It is also found in this paper that several sequence regions, which might reflect functional or structural requirements of the PE organization, and several residues known for their functional importance are conserved in almost all the sequences.

  14. Cloning and Expression Profiles of Myf5 Gene of Yak

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    Yaqiu Lin

    2015-01-01

    Full Text Available To reveal the sequence characteristic and expression pattern of Myf5 gene in Jiulong yaks (Bos grunniens, a full-length cDNA of Myf5 was cloned from yak muscle tisssue by RT-PCR. The cDNA obtained was 821bp nucleotide (nt long with an ORF of 768 bp which encoding 255 amino acids. Compared with cattle, sheep, pig, horse, human, pygmy chimpanzee, mouse, rat and dog, the homology of amino acid sequences were higher (89-9%, but lower in Zebrafish (60%. SQ RT-PCR analysis showed that Myf5 gene expression was observed only in longissimus muscle, but not be detected in heart, liver, kidney, spleen and adipose tissues. The expression level of Myf5 gene in longissium muscle of 0.5 and over 9 years old yaks was significantly higher than those of 3.5-5.5 years old yaks (p<0.05. These results suggest that Myf5 may play an important role in the regulation of muscle growth and development of yak.

  15. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts.

    Science.gov (United States)

    Wrenzycki, C; Wells, D; Herrmann, D; Miller, A; Oliver, J; Tervit, R; Niemann, H

    2001-07-01

    experiment 4, the RA of DNMT transcripts was decreased in in vivo-derived blastocysts compared to those produced in vitro. Mash2 expression was increased in in vivo embryos and those IVP embryos produced in medium containing Sigma BSA. The RA of Hsp was higher in IVP embryos produced in serum containing medium than in those produced in Sigma BSA or in vivo. In vivo embryos and those produced in Life Technologies BSA had the lowest expression of IF transcripts. Expression of all other genes was not affected by variation in NT methodology or IVP culture systems throughout experiments 1-4. In conclusion, depending on steps of the cloning procedure NT-derived embryos display marked differences from their IVP- and in vivo-derived counterparts. An aberrant expression pattern in NT embryos was found with respect to genes thought to be involved in stress adaptation, trophoblastic function, and DNA methylation during preimplantation development.

  16. Cloning and Identification of Porcine SMPX Differentially Expressed in F1 Crossbreds and Their Parents

    Institute of Scientific and Technical Information of China (English)

    Zhu-Qing REN; Yuan-Zhu XIONG; Chang-Yan DENG; Ming-Gang LEI

    2006-01-01

    In order to investigate porcine heterosis on the molecular basis, Large White (L), a European purebred, and Meishan (M), a Chinese indigenous purebred, were hybridized directly and reciprocally to produce F1 hybrids, Large White×Meishan (LM) and Meishan×Large White (ML) pigs. Using mRNA differential display, we found an expression sequence tag (EST) differentially expressed in F1 hybrids and their parents, designated as EST55, which was homologous to human and murine skeletal muscle protein (SMPX), and the full-length cDNA of porcine SMPX was cloned by the rapid amplification of cDNA end (RACE) method. Translation of the mRNA transcript revealed an open reading frame (ORF) of 86 amino acid residues encoding a nuclear location signal peptide, two overlapping casein kinase Ⅱ phosphorylation sites and one N-glycosylation site with theoretical molecular weight of 9.3 kDa. Alignment analysis revealed that the deduced protein sequence shared 94%, 83% and 78% homology with that of its human, mouse and rat counterparts, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that it was expressed predominantly in skeletal and heart muscles, whereas at a moderate level in backfat,spleen, stomach and uterus tissues. Two single nucleotide polymorphism (SNPs), located in 5'- and 3'-untranslated region (UTR), respectively, were identified by PCR and sequencing. Phylogenetic tree and the secondary structure prediction were also performed. The possible relationship between porcine SMPX and heterosis was discussed.

  17. Rapid and efficient assembly of transcription activator-like effector genes by USER cloning.

    Science.gov (United States)

    Wang, Song; Li, Wei; Wang, Shuo; Hu, Baoyang

    2014-06-20

    Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.

  18. Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hai-Yan Hu; Xiao-Ming Wang; Wei Wang; Xian-Xi Liu; Chun-Ying Jiang; Yan Zhang; Ji-Feng Bian; Yi Lu; Zhao Geng; Shi-Lian Liu; Chuan-Hua Liu

    2003-01-01

    AIM: To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma.METHODS: Total RNA was extracted from colon cancer tissues and amplified by reverse-transcription PCR with two primers, which span the whole coding region of ODC. The synthesized ODC cDNA was cloned into vector pQE-30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30-ODC. The sequence of inserted fragment was confirmed by DNA sequencing,the fusion protein including 6His-tag was facilitated for purification by Ni-NTA chromatographic column.RESULTS: ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing. The DNA sequence matching on NCBI Blast showed 99 % affinity. The vector was transformed into E.coli M15 and expressed. The expressed ODC protein was verified with Western blotting.CONCLUSION: The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.

  19. Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

    Science.gov (United States)

    Liu, Chun-Hung; Chang, Chin-Chyuan; Chiu, Yun-Chih; Cheng, Winton; Yeh, Maw-Sheng

    2011-12-01

    Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.

  20. Cloning and Expression of an Ogura Cytoplasmic Male Sterile (OguCMS)-related MYB Transcription Factor in Brassica oleracea var.capitata%甘蓝胞质雄性不育(OguCMS)相关的MYB转录因子BoMYB1的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    张磊; 康宗利; 刘海霞; 康俊根

    2012-01-01

    Ogura cytoplasmic male sterile (OguCMS) is the most widely used male sterile type in cabbage breeding. MYB transcription factors play a key role in regulation of plant defense response and multiple development processes. In present experiment, a R2R3-MYB transcription factor which down regulated 10.3 times in cabbage (Brasska okracea var. capitata) OguCMS lines was cloned by SMART RACE strategy.The full-length cDNA of B0MYB1 was 1 141 bp, which contained a 196 bp long 5' untranslated region, a 246 bp long 3' untranslated region and a 699 bp long open reading frame (GenBank accession number: JN703995). It was localized in the nucleus by subcellular localization prediction. It was an anther preferentially expressed gene in cabbage, which reached its expression peak in the late development. It was induced by the regulation of plant hormones salicylic acid(SA) and jasmonate methyl (JA-ME), and consequently regulated the expression of anther development genes. The experimental results suggests that B0MYB1 may be one of the important genes which involved in OguCMS anther development.%萝卜胞质雄性不育(OguCMS)是目前甘蓝中应用较广的雄性不育类型,MYB转录因子具有调控植物防御应答反应和多个发育过程的作用.本实验以在甘蓝(Brassica oleracea var.capitata)OguCMS花药中下调10.3倍的EST序列为信息探针,结合电子克隆及RACE技术,得到一个与甘蓝OguCMS雄性不育相关的MYB转录因子全长cDNA,命名为BoMYB1(GenBank登录号:JN703995).经亚细胞定位预测,该基因定位于细胞核,全长1 141 bp,包含一个长度为196 bp的5’非翻译区、246 bp的3’非翻译区和一个699 bp的开放阅读框.该基因在花药中具表达优势,并在花药发育晚期出现表达高峰,受植物激素水杨酸(SA)和茉莉酸甲酯(JA-ME)的调控,诱导花药发育基因的表达.实验结果提示,BoMY B1可能是参与OguCMS花药发育的重要基因之一.

  1. Identification and transcriptional profiling of differentially expressed genes associated with resistance to Pseudoperonospora cubensis in cucumber.

    Science.gov (United States)

    Li, Jian-Wu; Liu, Jun; Zhang, He; Xie, Cong-Hua

    2011-03-01

    To identify genes induced during Pseudoperonospora cubensis (Berk. and Curk.) Rostov. infection in cucumber (Cucumis sativus L.), the suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from cucumber seedlings inoculated with the pathogen as a tester and cDNA from uninfected cucumber seedlings as a driver. A forward subtractive cDNA library (FSL) and a reverse subtractive cDNA library (RSL) were constructed, from which 1,416 and 1,128 recombinant clones were isolated, respectively. Differential screening of the preferentially expressed recombinant clones identified 58 unique expressed sequence tags (ESTs) from FSL and 29 from RSL. The ESTs with significant protein homology were sorted into 13 functional categories involved in nearly the whole process of plant defense such as signal transduction and cell defense, transcription, cell cycle and DNA processing, protein synthesis, protein fate, proteins with binding functions, transport, metabolism and energy. The expressions of twenty-five ESTs by real-time quantitative RT-PCR confirmed that differential gene regulation occurred during P. cubensis infection and inferred that higher and earlier expression of transcription factors and signal transduction associated genes together with ubiquitin/proteasome and polyamine biosynthesis pathways may contribute to the defense response of cucumber to P. cubensis infection. The transcription profiling of selected down-regulated genes revealed that suppression of the genes in reactive oxygen species scavenging system and photosynthesis pathway may inhibit disease development in the host tissue.

  2. Cloning, characterization and expression analysis of porcine microRNAs

    Directory of Open Access Journals (Sweden)

    Desilva Udaya

    2009-02-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig. Results By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined. Conclusion Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.

  3. 转录因子基因Ta WRKY72b-1的克隆、表达及在烟草中表达对植株磷效率的影响%Cloning and Expression of Wheat Transcription Factor Gene TaWRKY72b-1 and Its Effect on Phosphorus Use Efficiency in Transgenic Tobacco Plants

    Institute of Scientific and Technical Information of China (English)

    苗鸿鹰; 赵金峰; 李小娟; 孙昭华; 路文静; 谷俊涛; 郭程瑾; 肖凯

    2009-01-01

    Phosphorus is one of the indispensable elements in plant growth and development, which is the main component for ATP, nucleic acid, and lecithin. Since phosphorus usually exists in the hard-absorption compounds in soil to cause plant suffering from phosphorus deficiency during growing stage. So plant morphological and physiologically develops the adaptation to low phosphorus stress. Transcription regulation plays an important role in responding to deficient-P cue in plants. Several transcription factors mediating the deficient@ signal transduction have been reported in Arabidopsis and rice. But no similar studies have been conducted in wheat by now. In this study, an expressed sequence tag (EST) homologous to Arabidopsis WRKY75 was identified based on sequencing of clones from a subtraetive root cDNA library, in which the differential expressed genes responding to low-P were enriched. The EST gene with high similarity to wheat WRKY72b (GenBank accession No. EF368383), was cloned and referred to TaWRKY72b-1. TaWRKY72b-1 had two base differences with WRKY72b at the cDNA sequence, with an open reading frame (OR.F) of 621 bp and encoding a polypeptide of 206 amino acids. TaWRKY72b-1 contained one of conserved WRKY motif and one of C_2H_2 zinc finger motif. Phylogenetic tree analysis indicated that TaWRKY72b-1, wheat WRKY72a and barley WRKY12 were possibly derived from one ancestor. Compared with those in sufficient-P (2 mmol L~(-1) P) condition, the transcripts of TaWRKY72b-1 in roots and leaves under the defieient-P (20 μmol L~(-1) P) condition were all dramatically increased, suggesting that TaWRKY72b-1 gene was involved in the response to low P stress in the plants. Under deficient-P condition, the expression of TaWRKY72b-1 in transgenic tobacco plants obviously increased plant dry weights, plant accumulative phosphorus amount, and phosphorus utilization efficiency compared with that in CK (empty vector transformed plants). Therefore, the TaWRKY72b-1 gene has

  4. Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes

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    Latruffe Norbert

    2004-03-01

    Full Text Available Abstract Background In rats, two peroxisomal 3-ketoacyl-CoA thiolase genes (A and B have been cloned, whereas only one thiolase gene is found in humans. The aim of this study was thus to clone the different mouse thiolase genes in order to study both their tissue expression and their associated enzymatic activity. Results In this study, we cloned and characterized two mouse peroxisomal 3-ketoacyl-CoA thiolase genes (termed thiolase A and B. Both thiolase A and B genes contain 12 exons and 11 introns. Using RNA extracted from mouse liver, we cloned the two corresponding cDNAs. Thiolase A and B cDNAs possess an open reading frame of 1272 nucleotides encoding a protein of 424 amino acids. In the coding sequence, the two thiolase genes exhibited ≈97% nucleotide sequence identity and ≈96% identity at the amino acid level. The tissue-specific expression of the two peroxisomal 3-ketoacyl-CoA thiolase genes was studied in mice. Thiolase A mRNA was mainly expressed in liver and intestine, while thiolase B mRNA essentially exhibited hepatic expression and weaker levels in kidney, intestine and white adipose tissue. Thiolase A and B expressions in the other tissues such as brain or muscle were very low though these tissues were chiefly involved in peroxisomal disorders. At the enzymatic level, thiolase activity was detected in liver, kidney, intestine and white adipose tissue but no significant difference was observed between these four tissues. Moreover, thiolase A and B genes were differently induced in liver of mice treated with fenofibrate. Conclusion Two mouse thiolase genes and cDNAs were cloned. Their corresponding transcripts are mostly expressed in the liver of mice and are differently induced by fenofibrate.

  5. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    Science.gov (United States)

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  6. Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.

    Directory of Open Access Journals (Sweden)

    Manika Indrajit Singh

    Full Text Available We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3. Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR". We show the cloning, expression and purification of Green Fluorescent Protein (GFP as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3 with or without a hexa-histidine tag.

  7. Cloning and expression analysis of an o-methyltransferase (OMT) gene from Chinese shrimp, Fenneropenaeus chinensis.

    Science.gov (United States)

    Li, Dian-Xiang; Du, Xin-Jun; Zhao, Xiao-Fan; Wang, Jin-Xing

    2006-09-01

    O-methyltransferase (OMT) is ubiquitously present in diverse organisms and plays an important regulatory role in plant and animal growth, development, reproduction and defence and has also been implicated in human emotion and disease. A putative o-methyltransferase (OMT) gene has been cloned from the haemocytes of bacteria-infected Chinese shrimp (Fenneropenaeus chinensis) by suppression subtractive hybridisation (SSH) coupled with the SMART cDNA method. The isolated 944 bp full-length cDNA contains a single 666bp open reading frame (ORF) encoding a putative OMT protein of 221 amino acids. The predicted protein has a molecular weight of 24,572.06 Da and a pI of 5.27 as well as ten phosphorylation sites. Northern blot and in situ hybridisation analyses demonstrated that the OMT transcripts were constitutively expressed in tissue of shrimp challenged by bacterial infection and in unchallenged shrimp tissue. Constitutive OMT transcript was found in areas such as haemocytes, heart, hepatopancreas, stomach, gill, intestine and ovary. However, the OMT transcripts were upregulated in hepatopancreas and stomach in challenged shrimp.

  8. INHIBITORY ROLE OF TRANSCRIPTION FACTOR COUP-TFⅡ IN EXPRESSION OF HTERT IN HELA CELLS

    Institute of Scientific and Technical Information of China (English)

    Qiang Wang; Zeng-liang Bai; Li Xuan; Lin Hou; Bo Zhang

    2004-01-01

    Objective To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity.Methods The full cDNA of COUP-TFⅡ was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFⅡ fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFⅡ with hTERT promoter in vitro was identified by lectrophoretic mobility shift assay and Footprint. The role of COUP-TFⅡ in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA.Results COUP-TFⅡ could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter.Luciferase reporter assay indicated COUP-TFⅡ could suppress hTERT promoter activity and stable introduction of COUP TFⅡ into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity.Conclusion The human COUP-TFⅡ can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.

  9. Cloning and Expression Profiles of TaWRKY46,a WRKY Type Transcription Factor Gene in Wheat(Triticum aestivum L.)%小麦转录因子基因TaWRKY46的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    丁长欢; 孙昭华; 李小娟; 肖凯

    2012-01-01

    在富集低磷胁迫特异表达基因的小麦根系cDNA差减杂交文库中,鉴定了1个小麦WRKY型转录因子基因(TaWRKY46).依据该基因的cDNA序列,在石新828中克隆了该基因.TaWRKY46的cDNA长度为872 bp,开放阅读框为669 bp,编码222个氨基酸残基,氨基酸组成上含有保守的WRKY基序和C2H2基序.系统进化分析表明,TaWRKY46与大麦WRKY34可能来自相同的祖先.与对照供磷水平(2 mmol/L Pi)相比,低磷处理(20 μmol/L Pi)使根系中TaWRKY46的转录本数量明显增多,表明TaWRK46对低磷胁迫逆境产生了明显应答.此外,TaWRKY46对氮、钾、锌和钙养分胁迫也表现明显的上调表达特征.表明该基因编码蛋白在介导上述养分逆境信号的转导中具有重要作用.此外,TaWRKY46对干旱、盐分等非生物逆境和脱落酸(ABA)也产生明显应答.因此,TaWRKY46是小麦种属中应答多种非生物逆境的重要调控转录因子,在增强小麦抵御养分胁迫和非生物逆境的能力中可能具有重要作用.%One WRKY type transcription factor gene designated as TaWRKY46 was identified in a root subtrae-tive cDNA library in which the differential expressed genes responding to the low-Pi stress were enriched. Based on the eDNA sequence of TaWRKY46, this gene was cloned in wheat cultivar Shixin828. The cDNA of TaWRKY46 was 872 bp in length, with an open reading frame of 669 bp and an encoded polypeptide of 222 aa. TaWRKY46 contains a conserved WRKY motif and a C2H2 conserved domain. Phylogenetic analysis displays a high similarity between TaWRKY46 and HvWRKY34, a WRKY transcription family gene in barley , suggesting them to be possibly derived from a same ancestor. Compared with those in sufficient-P (2 mmol/L Pi) condition,the transcripts of TaWRKY46 in roots under the deficient-P (20 |x"mol/L Pi) condition were dramatically increased, suggesting that TaWRKY46 gene was involved in the response to low P stress in the plants. In the meantime, TaWRKY46 was

  10. Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

    Science.gov (United States)

    Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

    2008-01-01

    In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

  11. [Cloning and expression of Streptococcus salivarius urease gene in Escherichia coli].

    Science.gov (United States)

    Wang, Yan; Feng, Xi-ping; Xie, You-hua; Tao, Dan-ying; Luan, Xiao-ling

    2010-08-01

    To clone Streptococcus salivarius (Ss) 57. I urease gene, which can express ureolytic activity in Escherichia coli (Ec) without adding extra nickel ions. Urease gene was cloned by polymerase chain reaction in three separate parts. The three separate plasmids were digested by specific restriction enzymes and ligated together. The expression of the complete urease gene in Ec was detected by phenol red assay and pH analysis. Urease gene of Ss 57.I was eventually cloned and proved correct. Urease activity of the obtained clone was positive in Ec. Without adding extra NiCl(2), the recombinant Ec could hydrolyze urea to produce ammonia, resulting in the increase of pH value. The clone of Ss urease gene obtained in this study could express ureolytic activity in Ec without adding extra nickel ions. The current clone can be used to construct ureolytic effector strain used in replacement therapy in caries prevention.

  12. Genetic control of human brain transcript expression in Alzheimer disease.

    Science.gov (United States)

    Webster, Jennifer A; Gibbs, J Raphael; Clarke, Jennifer; Ray, Monika; Zhang, Weixiong; Holmans, Peter; Rohrer, Kristen; Zhao, Alice; Marlowe, Lauren; Kaleem, Mona; McCorquodale, Donald S; Cuello, Cindy; Leung, Doris; Bryden, Leslie; Nath, Priti; Zismann, Victoria L; Joshipura, Keta; Huentelman, Matthew J; Hu-Lince, Diane; Coon, Keith D; Craig, David W; Pearson, John V; Heward, Christopher B; Reiman, Eric M; Stephan, Dietrich; Hardy, John; Myers, Amanda J

    2009-04-01

    We recently surveyed the relationship between the human brain transcriptome and genome in a series of neuropathologically normal postmortem samples. We have now analyzed additional samples with a confirmed pathologic diagnosis of late-onset Alzheimer disease (LOAD; final n = 188 controls, 176 cases). Nine percent of the cortical transcripts that we analyzed had expression profiles correlated with their genotypes in the combined cohort, and approximately 5% of transcripts had SNP-transcript relationships that could distinguish LOAD samples. Two of these transcripts have been previously implicated in LOAD candidate-gene SNP-expression screens. This study shows how the relationship between common inherited genetic variants and brain transcript expression can be used in the study of human brain disorders. We suggest that studying the transcriptome as a quantitative endo-phenotype has greater power for discovering risk SNPs influencing expression than the use of discrete diagnostic categories such as presence or absence of disease.

  13. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    Science.gov (United States)

    Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

    2015-09-01

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  14. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia)

    2015-09-25

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  15. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos

    Science.gov (United States)

    Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D. Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  16. High-throughput cloning and expression in recalcitrant bacteria

    NARCIS (Netherlands)

    Geertsma, Eric R.; Poolman, Bert

    2007-01-01

    We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism

  17. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  18. Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141

    Directory of Open Access Journals (Sweden)

    Wenqian Deng

    2009-01-01

    Full Text Available ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE. The open reading frame (ORF encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.

  19. A novel cytochrome P450 gene from Catharanthus roseus cell line C20hi: cloning and characterization of expression

    Directory of Open Access Journals (Sweden)

    Lihong He

    2012-06-01

    Full Text Available An expressed sequence tag (EST obtained from a subtractive-suppression hybridization cDNA library constructed using Catharanthus roseus cell line C20hi and its parental cell line C20D was used to clone a full-length cytochrome P450 cDNA of cyp71d1. The encoded polypeptide contained 507 amino acids with 39–56% identity to other CYP71D subfamily members at the amino acid level. Expression characteristics of cyp71d1 were determined using semi-quantitative RT-PCR. The cyp71d1 transcript was expressed in all three cell lines with the highest level in the cell line C20hi. In the mature C. roseus plant, the cyp71d1 cDNA was highly expressed in petals, roots and stems, but very weakly expressed in young leaves. Its transcription level increased with the development of flowers. 2,4-D could down-regulate the transcription of cyp71d1, as did KT, but only to a minor degree. Neither light nor yeast elicitor could induce the transcription of cyp71d1.

  20. Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, S.W.; Rogers, J.C. (Washington State Univ., Pullman, WA (United States). Inst. of Biological Chemistry)

    1999-04-01

    The authors cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Ncotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of re-expression of mRNA for the aleurone RNase revealed that, like the pattern for [alpha]-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase [alpha]-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

  1. CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

    Science.gov (United States)

    Borovsky, Dov; Sterner, Andeas; Powell, Charles A

    2016-01-01

    The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh.

  2. Transcriptional control of hepatocanalicular transporter gene expression

    NARCIS (Netherlands)

    Muller, M

    2000-01-01

    Transport processes for larger organic solutes at the canalicular membrane are mainly driven by members of the superfamily of ATP-binding cassette (ABC) transporters. The funct ions of these transporters range from bile component secretion to xenobiotica and phase II-conjugate export. The transcript

  3. Dissecting specific and global transcriptional regulation of bacterial gene expression

    NARCIS (Netherlands)

    Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

    2013-01-01

    Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an experiment

  4. 珍稀泌盐植物长叶红砂两个WRKY转录因子的克隆及表达分析%Cloning and expression analysis of two WRKY transcription factors from the rare recretohalophyte Reaumuria trigyna

    Institute of Scientific and Technical Information of China (English)

    王佳; 郑琳琳; 顾天培; 王学峰; 王迎春

    2014-01-01

    Reaumuriatrigyna is an endangered small shrub thriving in vast arid areas of Inner Mongolia.This species has developed a distinct morphology and physiology to adapt to the semi-desert environment.The WRKY gene family are plant-specific transcription factors which play important roles in the regulation of plant development,metabolism,and diverse biotic and abiotic stresses.Based on R. trigyna transcriptome data, two cDNA fragments (annotated as WRKY transcription factors )which were significantly upregulated after salt treatment were selected.Using rapid amplification of cDNA ends (RACE),these two WRKY transcription factors were cloned from R. trigyna.Blasted in the NCBI database,the homology to ArabidopsisAtWRKY33 were 79% and 87%,so they were named RtWRKY33-1 and RtWRKY33-2 (GenBank accession number KF421158 and KF421159),respectively.The full-length of the RtWRKY33-1 was 2163 bp and included a 1681 bp open reading frame (ORF)which encoded 573 amino acids.The full-length of RtWRKY33-2 was 2155 bp and included a 1776 bp ORF encoding 591 amino acids.Amino acid sequence analysis indicated that they were typical of class I WRKY transcription factors.Protein structure prediction analysis found that the primary structure and secondary structure in the WRKY domain of the amino acid sequence had structures characteristic of high similarity.However,in the non-conserved domains,especially the N-terminus (1 to 80 aa range),be-fore the first WRKY domain (190 to 240 aa range)and between two WRKY domains (430 to 450 aa range), there were significant differences that may influence the function of these two genes.Semi-quantitative RT-PCR analysis showed that four abiotic stresses can induce the expression of these two genes.However,Rt-WRKY33-2 was mainly inducted by salt,cold and ABA,while RtWRKY33-2 responded faster to drought stress.These two genes in R. trigyna may play a different rolein abiotic stress responses.This research is a basis for study on the regulation mechanism

  5. Leucine zipper transcription factor-like 1 expression in gastric ...

    African Journals Online (AJOL)

    gastric cancer and its relationship to relative adhesion molecule ... Keywords: Leucine zipper transcription factor-like1 expression, Gastric cancer, Relative adhesion molecule, Protein ..... cell lung cancer (NSCLC) and recurrence [17].

  6. Molecular cloning and expression of interleukin 1beta (IL-1β) from red seabream (Pagrus major)

    Institute of Scientific and Technical Information of China (English)

    CAI Zhonghua; SONG Linsheng; GAO Chunping; WU Longtao; QIU Lihua

    2004-01-01

    The interleukin 1β (IL-1β) Cdna was cloned from the red seabream (Pagrus major) by homology cloning strategy.A Cdna fragment was amplified by PCR using two degenerated primers,which were designed according to the conserved regions of other known IL-1β sequences,and elongated by 3′ ends and 5′ ends RACE PCR to get the full length coding sequence of red seabream IL-1β (RS IL-1β).The sequence contained 1252 nucleotides that included a 5′ untranslated region (UTR) of 84 bp,a 3′ UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 Kd and putative isoelectric point Pi of 5.29.The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature,but lacked the signal peptide and the clear ICE cut site,which were common in other nonmammalian IL-1β genes.The RS IL-1β had the highest homology with piscine IL-1β according to phylogenetic tree analysis.The transcript expression was detected in blood,brain,gill,heart,head kidney,kidney,liver,muscle and spleen in the pathogen challenged and healthy red seabream by RT-PCR.Results showed that the RS IL-1β Mrna was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish,and the expression could be enhanced by pathogen challenging.

  7. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Z.; Chen, S.; Wilson, D.B.

    1999-11-01

    An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake system that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.

  8. Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos

    Directory of Open Access Journals (Sweden)

    Peng Xiu-li

    2011-08-01

    Full Text Available Abstract Background The very low density lipoprotein receptor gene (VLDLR, a member of the low density lipoprotein receptor (LDLR gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce. Methods Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE. Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR. Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM procedure of the SAS® software package. Results In duck, two VLDLR transcripts were identified, one transcript (variant-a containing an O-linked sugar domain and the other (variant-b not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13 and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P Conclusions Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance

  9. A novel approach to sequence validating protein expression clones with automated decision making

    Directory of Open Access Journals (Sweden)

    Mohr Stephanie E

    2007-06-01

    Full Text Available Abstract Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence

  10. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    Science.gov (United States)

    Wang, Qing; Ning, Xuanxuan; Pei, Dong; Zhao, Jianmin; You, Liping; Wang, Chunyan; Wu, Huifeng

    2013-05-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

  11. Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

    Institute of Scientific and Technical Information of China (English)

    WANG Qing; NING Xuanxuan; PEI Dong; ZHAO Jianmin; YOU Liping; WANG Chunyan; WU Huifeng

    2013-01-01

    Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis.In this study,two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR).The two cDNAs were named MgTrx1 and MgTrx2,respectively.The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa,respectively.Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys.In addition,MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria.Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined.The MgTrxl transcript was most abundant in hemocytes and gills,whereas the MgTrx2 transcript was most abundant in gonad,hepatopancreas,gill and hemocytes.Following Vibrio anguillarum challenge,the expression of MgTrxl was up-regulated and reached its peak,at a value 10-fold the initial value,at 24 h.Subsequently,expression returned back to the original level.In contrast,the expression level of MgTrx2 was down-regulated following bacterial stimulation,with one fifth of the control level evident at 12 h post challenge.These results suggest that MgTrxl and MgTrx2 may play important roles in the response of M.galloprovincialis to bacterial challenge.

  12. Cloning and Expression Analysis of Wheat Stress-responsive Transcription Factor Gene TaSNA C1%小麦胁迫响应转录因子基因TaSNAC1的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    单丽伟; 宋鹏; 刘夏燕; 张超; 卫晓彬; 韩兆雪; 郭蔼光; 范三红

    2012-01-01

    Stress responsive NAC transcription factors involve in plant abiotic stress tolerance. Overexpression of SNAC1 significantly enhances drought, cold and salinity resistance in transgenic rice(Oryza saliva). In this study, TaSNA Cl was obtained from common wheat (Triticum aestivum) by homology-based cloning, its sub-celluar localization was analyzed, and its expression patterns in different tissues and under PEG or salt stress were investigated by quantitative RT-PCR. The cDNA of amplified TaSNA Cl including complete CDS was 1 076 bp in size, and the gDNA was 1 222 bp including a 146 bp intron (GenBank accession No. JN621240). TaSNACl encoded a protein with 329 amino acids, which showed 97.3%, 86.3%, 81.1%, 79.1% and 79.2% identity with SNAC1 of barley (Hordeum vulgare), false brome (Brachupodium distachyon), rice, maize (Zea mays) and sorghum (Sorghum bicolor), respectively. Results of phylogenetic analysis showed that TaSNACl was different from other wheat NAC transcription factors, it was clustered into a separate clade with other grass stress-responsive NAC. Structure prediction showed that TaSNACl might form a dimer, including a untypical nuclear localization signal (NLS) and a typical no apical meristem (NAM) domain. The core motif "WKATGXDK100-107" was located in a p sheet, which formed a concave surface and confered the ability of DNA binding. Based on transient expression assay using A rabidopsis thaliana mesophyll protoplasts, we found TaSNACl localized in the nucleus specifically. The expression levels of TaSNACl in both leaf and root were increased significantly in similar pattern during the application of high salt, and the increase in root was more dramatic (upto -60 folds in root and ~10 folds in leaf). Under PEG stress, the transcripts of TaSNA Cl were elevated quickly and sharply in root, but the change in leaf was delayed and the amplitude was decreased (about 15 folds in root and 6 folds in leaf). These data suggest that TaSNA Cl plays a vital role

  13. TANDEM REPEATING OF THE EXPRESSION CARTRIDGE—— A NOVEL STRATEGY TO ENHANCE THE EXPRESSION EFFICIENCY OF CLONED GENE IN ESCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    林缨; 卢圣栋

    1996-01-01

    A novel strategy to enhance the expression eHiciency of cloned target gene in Escher&hia colt was developed. The whole expression cartridge, consisting of promoter, SD sequence, target gene and transcription terminator, was tandem repeatedly engineered into a expression plasmld. Consequently, the copynumber of specific gene was increased substantially, leading to the improvemem of expression efficiency.Using this approach, a recombinant plasmid, designed as pLYD, was constructed and transformated into the Escherichia coti strain DHSa. Upon induction, the desired protein was synthesized in a considerable level and accumulated up to 63% of the total cell proteins, The pretent study reveated that taildem repeating of expression cartridge provided a convenient means to improve expression level efficiently.

  14. Jun N-Terminal Kinase 1 Mediates Transcriptional Induction of Matrix Metal loproteinase 9 Expression

    Directory of Open Access Journals (Sweden)

    David L. Crowe

    2001-01-01

    Full Text Available Tumor cell invasion and metastasis require precise coordination of adherence to extracellular matrix (ECM and controlled degradation of its components. Invasive cells secrete proteolytic enzymes known as matrix metal lop roteinases (MMPs which degrade specific basement membrane molecules. Expression of these enzymes is regulated by multiple signaling mechanisms, including the mitogen-activated protein kinase (MAPK pathway. One of the terminal effectors of this signaling cascade is jun N-terminal kinase 1 (JNK1 which phosphorylates the transcription factor c-jun, a component of the AP-1 complex. MMP-9 expression is regulated by two well-characterized AP-1 sites in the promoter of this gene. To determine how JNK1 activity regulated MMP-9 expression in human squamous cell carcinoma lines, we overexpressed this kinase in SCC25 cells. JNK1 overexpression induced MMP-9 protein levels and activity in this cell line. Elevated MMP-9 expression correlated with increased invasion of reconstituted basement membranes by JNK1 -overexpressiog clones. Site-directed mutagenesis of the MMP-9 promoter revealed that JNK1 cooperated with its transcription factor target c-jun to increase MMP-9 expression at the transcriptional level via the proximal AP-1 site. These results suggest that elevated JNK1 expression may contribute to increased MMP-9 activity and ECM invasion by tumor cells.

  15. Molecular cloning, expression analysis and sequence prediction of ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... comparison of the amino acid sequences from C/EBPβ cloned in this study and those from different ... subcutaneous fat was the highest among all the analyzed tissues, and the relative quantity ..... Carbohydrate Metabolism.

  16. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    Science.gov (United States)

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions.

  17. Cloning and structural and expressional characterization of BcpLH gene preferentially expressed in folding leaf of Chinese cabbage

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Vegetative growth of Chinese cabbage undergoes the four successive stages which are characterized with the definite types of juvenile,rosette,folding and head leaves.From shoot tips of Chinese cabbage at early folding stage,we constructed a cDNA library and screened the differentially expressed cDNA clones using the cDNAs derived from developing folding leaves and rosette leaves as probes.One complete length of cDNA clone is designated as BcpLH.Computer alignment matched BcpLH to the domains of double-stranded RNA binding (DBRM) and the homologous regions were recognized between BcpLH and human and mouse double-stranded RNA-binding protein TRBP.PCR expression analysis shows that during vegetative growth BcpLH gene was expressed preferentially in folding leaves at folding stage.Transcripts of BcpLH gene were increased when plants were sprayed with IAA.It is deduced that BcpLH gene may be related to initiation of folding leaf and leafy head and induced by auxin in the aspect of transcriptional expression.

  18. Cloning and developmental expression of the murine neurofilament gene family.

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); D.N. Meijer (Dies); D. Flavell (David); J. Hurst; F.G. Grosveld (Frank)

    1986-01-01

    textabstractDNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the e

  19. Leptin Genes in Blunt Snout Bream: Cloning, Phylogeny and Expression Correlated to Gonads Development

    Directory of Open Access Journals (Sweden)

    Honghao Zhao

    2015-11-01

    Full Text Available To investigate the leptin related genes expression patterns and their possible function during the gonadal development in fish, the cDNA and genomic sequences of leptin, leptin receptor (leptinR, and leptin receptor overlapping transcript like-1 (leprotl1 were cloned and their expression levels were quantified in the different gonadal development stages of Megalobrama amblycephala. The results showed that the full length cDNA sequences of leptin, leptinR and leprotl1 were 953, 3432 and 1676 bp, coding 168, 1082, and 131 amino acid polypeptides, and the genomic sequences were 1836, 28,528 and 5480 bp, which respectively had 3, 15 and 4 exons, respectively. The phylogenetic analysis revealed that three genes were relatively conserved in fish species. Quantitative real-time PCR results showed that the three genes were ubiquitously expressed in all examined tissues during the different gonadal development stages. The leptin and leptinR took part in the onset of puberty, especially in female M. amblycephala, by increasing the expression levels in brain during the stage I to III of ovary. The expression levels of leptin and leptinR had significant differences between male and female in hypothalamic-pituitary-gonadal (HPG axis tissues (p < 0.05. The leptinR had the same variation tendency with leptin, but the opposite changes of expression levels were found in leprotl1, which may resist the expression of leptinR for inhibiting the function of leptin in target organ. These findings revealed details about the possible role of these genes in regulating gonadal maturation in fish species.

  20. The cloning, genomic organization and tissue expression profile of the human DLG5 gene

    Directory of Open Access Journals (Sweden)

    Gibbs Richard A

    2002-02-01

    Full Text Available Abstract Background Familial atrial fibrillation, an autosomal dominant disease, was previously mapped to chromosome 10q22. One of the genes mapped to the 10q22 region is DLG5, a member of the MAGUKs (Membrane Associated Gyanylate Kinase family which mediates intracellular signaling. Only a partial cDNA was available for DLG5. To exclude potential disease inducing mutations, it was necessary to obtain a complete cDNA and genomic sequence of the gene. Methods The Northern Blot analysis performed using 3' UTR of this gene indicated the transcript size to be about 7.2 KB. Using race technique and library screening the entire cDNA was cloned. This gene was evaluated by sequencing the coding region and splice functions in normal and affected family members with familial atrial fibrillation. Furthermore, haploid cell lines from affected patients were generated and analyzed for deletions that may have been missed by PCR. Results We identified two distinct alternately spliced transcripts of this gene. The genomic sequence of the DLG5 gene spanned 79 KB with 32 exons and was shown to have ubiquitous human tissue expression including placenta, heart, skeletal muscle, liver and pancreas. Conclusions The entire cDNA of DLG5 was identified, sequenced and its genomic organization determined.

  1. CLONING AND EXPRESSION OF A GENE MEDIATINGγ-RADIATION-INDUCED APOPTOSIS IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To identify the member of the caspase family proteases involved in γ-radiation-induced apoptosis in HL-60 cells and to study the expression of the caspase gene in normal, apoptotic cells and in immortal tu mor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were pre sent in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-60 cells. Caspase-3 mRNA in apoptotic HL-60 cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identified with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly ex pressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-60 cells than in control HL-60 cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-60 cells. The high level of expression of caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radiation, their inherent ability to survive, and apop tosis.

  2. Cloning and expression analyses of the anthocyanin biosynthetic genes in mulberry plants.

    Science.gov (United States)

    Qi, Xiwu; Shuai, Qin; Chen, Hu; Fan, Li; Zeng, Qiwei; He, Ningjia

    2014-10-01

    Anthocyanins are natural food colorants produced by plants that play important roles in their growth and development. Mulberry fruits are rich in anthocyanins, which are the most important active components of mulberry and have many potentially beneficial effects on human health. The study of anthocyanin biosynthesis will bring benefits for quality improvement and industrial exploration of mulberry fruits. In the present study, nine putative genes involved in anthocyanin biosynthesis in mulberry plants were identified and cloned. Sequence analysis revealed that the mulberry anthocyanin biosynthetic genes were conserved and had counterparts in other plants. Spatial transcriptional analysis showed detectable expression of eight of these genes in different tissues. The results of expression and UPLC analyses in two mulberry cultivars with differently colored fruit indicated that anthocyanin concentrations correlated with the expression levels of genes associated with anthocyanin biosynthesis including CHS1, CHI, F3H1, F3'H1, and ANS during the fruit ripening process. The present studies provide insight into anthocyanin biosynthesis in mulberry plants and may facilitate genetic engineering for improvement of the anthocyanin content in mulberry fruit.

  3. Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba Ⅰ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP Ⅱ for Uroplakin Ⅱ was successfully constructed.After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

  4. Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum.

    Directory of Open Access Journals (Sweden)

    Yupeng Yin

    Full Text Available BACKGROUND: One important protein family that functions in nucleotide excision repair (NER factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like, has been shown to be another developmentally related member of the SNF2 family. METHODOLOGY/PRINCIPAL FINDINGS: In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. CONCLUSIONS/SIGNIFICANCE: We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.

  5. Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2.

    Science.gov (United States)

    Cangelosi, Chris; Hannagan, Susan; Santiago, Clayton P; Wilson, James W

    2015-11-01

    Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria.

  6. Transcriptional and epigenetic regulation of PPARγ expression during adipogenesis

    OpenAIRE

    Lee, Ji-Eun; Ge, Kai

    2014-01-01

    The nuclear receptor PPARγ is a master regulator of adipogenesis. PPARγ is highly expressed in adipose tissues and its expression is markedly induced during adipogenesis. In this review, we describe the current knowledge, as well as future directions, on transcriptional and epigenetic regulation of PPARγ expression during adipogenesis. Investigating the molecular mechanisms that control PPARγ expression during adipogenesis is critical for understanding the development of white and brown adipo...

  7. Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription.

    Science.gov (United States)

    Kutky, Meherzad; Hudak, Katalin A

    2017-09-01

    HIV-1 transcription is primarily controlled by the virally encoded Tat protein and its interaction with the viral TAR RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In this study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant ( Phytolacca americana ), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1, NF-κB and SP-1, which modulate HIV-1 transcription by binding to the viral LTR, were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein. ©2017 The Author(s).

  8. RNA transcripts of full-length cDNA clones of rabbit hepatitis E virus are infectious in rabbits.

    Science.gov (United States)

    Cossaboom, Caitlin M; Huang, Yao-Wei; Yugo, Danielle M; Kenney, Scott P; Piñeyro, Pablo; Matzinger, Shannon R; Heffron, C Lynn; Pierson, F William; Meng, Xiang-Jin

    2014-11-07

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.

  9. Transcription mediated insulation and interference direct gene cluster expression switches.

    Science.gov (United States)

    Nguyen, Tania; Fischl, Harry; Howe, Françoise S; Woloszczuk, Ronja; Serra Barros, Ana; Xu, Zhenyu; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-11-19

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.

  10. Molecular cloning and expression analysis of a gene for sucrose transporter from pear (Pyrus bretschneideri Rehd.) fruit.

    Science.gov (United States)

    Zhang, Huping; Zhang, Shujun; Qin, Gaihua; Wang, Lifen; Wu, Tao; Qi, Kaijie; Zhang, Shaoling

    2013-12-01

    Here we report the cloning of a sucrose transporter cDNA from pear (Pyrus bretschneideri Rehd. cv 'Yali') fruit and an analysis of the expression of the gene. A cDNA clone, designated PbSUT1 was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. PbSUT1 potentially encoded a protein of 499 amino acid residues with a predicted molecular mass of 53.4 kDa and an isoelectric point (pI) of 9.21. Phylogenetic analysis revealed that the PbSUT1 belonged to type III SUTs and was more closely related to the MdSUT1 from apple fruit. Some major facilitator superfamily (MFS)-specific sequence motifs were found in the predicted PbSUT1 peptides, and an MFS_1 domain was located at the amino acid positions of 29-447 of the sequence. A study of gene expression along fruit development showed that PbSUT1 transcripts are present at all stages but significantly increase before fruit enlargement and during the ripening process with increasing sucrose levels. In contrast, the expression levels don't change much during the period of rapid fruit growth. This work shows that sucrose transporter may play a role in the accumulation of sugars during maturation and in maintaining the internal cellular distribution.

  11. Cloning, multicopy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in yarrowia lipolytica

    CSIR Research Space (South Africa)

    Ramduth, D

    2008-05-01

    Full Text Available cloned and functionally expressed in Y. lipolytica, under the control of a growth inducible hp4d promoter. The transformation experiments yielded only two positive multicopy transformants, which were assessed in flask cultures. The selected transformant...

  12. Expression of cloned genes of transgenic microorganisms introduced into man-made ecosystems

    Science.gov (United States)

    Maksimova, E. E.; Popova, L. Yu.

    Modeling of transgenic microorganism introduction into small man-made ecosystems can help forecast changes in expression of cloned genes under different conditions of existence. Introduction of the E. coli Z905/pPHL7 strain containing a plasmid with luminescent system genes of luminous bacteria led to changes in cell and colony morphology, reduction in metabolic activity of cells, and, as a result, a lower level of expression of cloned gene. A low concentration of nutrients has been shown to favor greatly the phenotypic change of cells of the recombinant strain. Expression of cloned genes changed due to: a lower concentration of plasmid DNA, a change in regulation of cloned genes, and a change in cells of biosynthesis of substrates needed for expression of luminescent genes. The conducted investigations can provide a basis for the use of marker transgenic microorganisms in closed ecosystems of different types.

  13. Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid.

    Science.gov (United States)

    Steger, G; Tabler, M; Brüggemann, W; Colpan, M; Klotz, G; Sänger, H L; Riesner, D

    1986-12-22

    The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (-) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromatography (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.

  14. Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines.

    Science.gov (United States)

    Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

    2014-01-01

    A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.

  15. Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines.

    Directory of Open Access Journals (Sweden)

    Suvi Sutela

    Full Text Available A small multigene family encodes 4-coumarate:CoA ligases (4CLs catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1 to the lignin and soluble phenolic compound composition of silver birch (Betula pendula Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S and guaiacyl (G lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1 were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.

  16. Molecular cloning, bioinformatics analysis, and transcriptional profiling of JAZ1 and JAZ2 from Salvia miltiorrhiza.

    Science.gov (United States)

    Zhou, Yangyun; Zhou, Xun; Li, Qing; Chen, Junfeng; Xiao, Ying; Zhang, Lei; Chen, Wansheng

    2017-01-01

    Production of major effective metabolites, tanshinones and lithospermic acid B (LAB), was dramatically enhanced by exogenous jasmonate (JA) treatment in Salvia miltiorrhiza. However, the molecular mechanism of such metabolic activation in S. miltiorrhiza has not been elucidated yet. Here, we focused on jasmonate ZIM-domain (JAZ) proteins that act as repressors of JA signaling. Open reading frames of two novel genes, SmJAZ1 and SmJAZ2, from S. miltiorrhiza were amplified according to the annotation of S. miltiorrhiza transcriptome. Compared to plant JAZs, SmJAZ1 and SmJAZ2 were clustered into different groups by phylogenetic analysis. Organ expression pattern was studied by real-time quantitative PCR (RT-qPCR), showing higher transcription level of both genes in stems than roots and leaves. The two SmJAZs responded to methyl jasmonate at early stage and the transcriptional level significantly increased at 4 H. Our experimental results indicate that SmJAZ1 and SmJAZ2 are JA responsive and presented similar expression trend in JA response. The whole research will certainly facilitate further characterization of JAs effect on effective metabolites and help to ultimately achieve high yield of target compounds (tanshinones and LAB).

  17. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes

    OpenAIRE

    Jonathan L Schmid-Burgk; Tobias Schmidt; Vera Kaiser; Klara Höning; Veit Hornung

    2013-01-01

    Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DN...

  18. Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

    Science.gov (United States)

    Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

    2016-04-01

    H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.

  19. The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

    Science.gov (United States)

    Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

    1992-07-01

    We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

  20. Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity

    Science.gov (United States)

    2004-01-01

    The first IMPI (inhibitor of metalloproteinases from insects) was identified in the greater wax moth, Galleria mellonella [Wedde, Weise, Kopacek, Franke and Vilcinskas (1998) Eur. J. Biochem. 255, 535–543]. Here we report cloning and expression of a cDNA coding for this IMPI. The IMPI mRNA was identified among the induced transcripts from a subtractive and suppressive PCR analysis after bacterial challenge of G. mellonella larvae. Induced expression of the IMPI during a humoral immune response was confirmed by real-time PCR, which documented up to 500 times higher amounts of IMPI mRNA in immunized larvae in comparison with untreated ones. The IMPI sequence shares no similarity with those of tissue inhibitors of metalloproteinases or other natural inhibitors of metalloproteinases, and the recombinant IMPI specifically inhibits thermolysin-like metalloproteinases, but not matrix metalloproteinases. These results support the hypothesis that the IMPI represents a novel type of immune-related protein which is induced and processed during the G. mellonella humoral immune response to inactivate pathogen-associated thermolysin-like metalloproteinases. PMID:15115439

  1. Molecular cloning and expression of nanos in the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae).

    Science.gov (United States)

    Ogaugwu, Christian E; Wimmer, Ernst A

    2013-01-01

    The gene nanos (nos) is a maternal-effect gene that plays an important role in posterior patterning and germ cell development in early stage embryos. nos is known from several diverse insect species, but has so far not been described for any Tephritid fruit fly. Here, we report the molecular cloning and expression pattern of the nos orthologous gene, Ccnos, in the Mediterranean fruit fly Ceratitis capitata, which is a destructive pest of high agricultural importance. CcNOS contains 398 amino acids and has a C-terminal region with two conserved CCHC zinc-binding motifs known to be essential for NOS function. Transcripts of Ccnos were confirmed by in situ hybridization to be maternally-derived and localized to the posterior pole of early stage embryos. Regulatory regions of nos have been employed in genetic engineering in some dipterans such as Drosophila and mosquitoes. Given the similarity in spatial and temporal expression between Ccnos and nos orthologs from other dipterans, its regulatory regions will be valuable to generate additional genetic tools that can be applied for engineering purposes to improve the fight against this devastating pest. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Antisense transcription as a tool to tune gene expression.

    Science.gov (United States)

    Brophy, Jennifer A N; Voigt, Christopher A

    2016-01-14

    A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

  3. Transcriptional Modulation of Heat-Shock Protein Gene Expression

    OpenAIRE

    Anastasis Stephanou; Latchman, David S.

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex sig...

  4. Transcriptional modulation of heat-shock protein gene expression.

    OpenAIRE

    A. Stephanou; Latchman, D S

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex sig...

  5. Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

    Science.gov (United States)

    Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

    2015-01-01

    Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

  6. Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

    Directory of Open Access Journals (Sweden)

    Ravi Chandra Yandamuri

    2014-10-01

    Full Text Available eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

  7. Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

    Directory of Open Access Journals (Sweden)

    Chung Jongkyeong

    2008-06-01

    Full Text Available Abstract Background Signal transducer and activator of transcription 1 (STAT1 is a critical component of interferon (IFN-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus. Results A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%. Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development. Conclusion Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish.

  8. Expression of Drosophila forkhead transcription factors during kidney development.

    Science.gov (United States)

    Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F; Zuo, Xiaofeng; Lipschutz, Joshua H

    2014-03-28

    The Drosophila forkhead (Dfkh) family of transcription factors has over 40 family members. One Dfkh family member, BF2 (aka FoxD1), has been shown, by targeted disruption, to be essential for kidney development. In order to determine if other Dfkh family members were involved in kidney development and to search for new members of this family, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using degenerate primers of the consensus sequence of the DNA binding domain of this family and developing rat kidney RNA. The RT-PCR product was used to probe RNA from a developing rat kidney (neonatal), from a 20-day old kidney, and from an adult kidney. The RT-PCR product hybridized only to a developing kidney RNA transcript of ∼2.3 kb (the size of BF2). A lambda gt10 mouse neonatal kidney library was then screened, using the above-described RT-PCR product as a probe. Three lambda phage clones were isolated that strongly hybridized to the RT-PCR probe. Sequencing of the RT-PCR product and the lambda phage clones isolated from the developing kidney library revealed Dfkh BF2. In summary, only Dfkh family member BF2, which has already been shown to be essential for nephrogenesis, was identified in our screen and no other candidate Dfkh family members were identified.

  9. Iron bioavailability in larvae yellow snapper (Lutjanus argentiventris): cloning and expression analysis of ferritin-H.

    Science.gov (United States)

    Reyes-Becerril, Martha; Angulo-Valadez, Carlos; Macias, Ma Esther; Angulo, Miriam; Ascencio-Valle, Felipe

    2014-04-01

    Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, ferritin H subunit was cloned from the larvae of yellow snapper, Lutjanus argentiventris, by rapid amplification of cDNA ends (RACE) following in silico transcriptome analysis. The full-length cDNAs of the LaFeH was 1231 bp in length encoding 177 amino acids with a predicted molecular mass (MW) about 20.82 kDa and theoretical isoelectric point (pI) of 5.79. Amino acid alignment revealed that LaFeH shared high similarity with other known ferritins. It shared high degree identity to the ferritin H subunits of Lates calcarifer (99%), Takifugu rubripes (97%) and Dicentrarchus labrax (97%), and low identity to that of human (82%) and mouse (84%). By real-time PCR assays, the mRNA transcripts of LaFeH was found to be higher expressed in head-kidney, eye, heart and brain. Moreover, mRNA expression levels of LaFeH was measured by real-time PCR in larvae exposed with graded levels of iron (6.8 μg/ml and 13.6 μg/ml (Fe2x and Fe4x, respectively) and an iron chelation assay. Results showed that the expression of the LaFeH mRNA increased gradually with Fe2x in water. The LaFeH gene expression declined with increasing iron exposure levels at Fe4x. Finally, we can observe a high expression of LaFeH gene in larvae exposed to iron chelation therapy at 2 h; however this increase was gradually decreasing over time. In summary, the LaFeH gene expression for larvae yellow snapper showed a dose-depend increase following the iron treatment. These data indicated that iron bioavailability regulates LaFeH at transcriptional level in larvae yellow snapper. Further studies are necessary to ascertain their role in the immune response in teleost fish.

  10. Involvement of transcriptional enhancers in the regulation of developmental expression of yellow gene

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Upstream regulatory region and flanking DNA of yellow gene wereisolated and cloned from a Drosophila genomic library. A vector containing yellow gene and regulatory elements was constructed using the recombinant DNA technique. Then this vector was integrated into Drosophila genome by genetic transformation. Using both FLP/FRT and Cre/LoxP site-specific recombination systems, two new yellow alleles were created at the same position in the genome of transgenic flies. Results from genetic and molecular analysis indicated that transcriptional enhancers regulate the developmental expression of the transgene. Furthermore, interactions between new-created yellow alleles were observed. Such interactions can influence markedly the expression of yellow gene during development. This effect may also be a form of enhancer-mediated gene expression.

  11. Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3

    Institute of Scientific and Technical Information of China (English)

    Rong Long Jiang; Qiao Sheng Lu; Kang Xian Luo

    2000-01-01

    AIM To clone core gene cDNA of Chinese hepatitis C virus ( HCV ) into eukaryotic expression vector cosmid pTM3 and to express HCV core antigen in HepG2 cells. METHODS Core gene cDNA of HCV was introduced into eukaryotic expression vector cosmid pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with the recombinant plasmid pTM3-Q534 by lipofectin. RESULTS From the transfected bacteria Top10F′, 2 pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10 ampicillin-resistant colonies. By reverse transcription PCR and indirect immunofluorescence technique, HCV RNA and core protein was identified in HepG2 cells transfected with the recombinant plasmid.CONCLUSION The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 was successful.

  12. Sequential logic model deciphers dynamic transcriptional control of gene expressions.

    Directory of Open Access Journals (Sweden)

    Zhen Xuan Yeo

    Full Text Available BACKGROUND: Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented here. METHODOLOGY: Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM is developed to decipher control mechanisms of dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. PRINCIPAL FINDINGS: SLM is verified by a set of well studied expression data on endo16 of Strongylocentrotus purpuratus (sea urchin during the embryonic midgut development. A dynamic regulatory mechanism for endo16 expression controlled by three binding sites, UI, R and Otx is identified and demonstrated to be consistent with experimental findings. Furthermore, we show that during transition from specification to differentiation in wild type endo16 expression profile, SLM reveals three binary activities are not sufficient to explain the transcriptional regulation of endo16 expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. CONCLUSIONS/SIGNIFICANCE: The sequential logic formalism allows for a simplification of regulation network dynamics going from a continuous to a discrete representation of gene activation in time. In effect our SLM is non-parametric and model-independent, yet

  13. 受青枯菌诱导表达的马铃薯转录因子类StWRKY8的克隆与表达分析%Cloning and Expression Analysis of the Potato Transcription Factor StWRKY8 Like Gene Induced byRalstonia solanacearum

    Institute of Scientific and Technical Information of China (English)

    薛珍; 李卉; 孔超越; 段婷婷; 郜刚

    2015-01-01

    Objective]Clone the partial cDNA of theStWRKY8-like gene from a cultivated Solanum tuberosum Zhongshu 3 after inoculation with Ralstonia solanacearum. Analyze theStWRKY8-like gene coding sequence, and study the differential expression pattern ofStWRKY8-like between susceptible and resistant S. tuberosum towardR. solanacearum and the tissue-specific expression ofStWRKY8-like.[Method]R. solanacearum-resistant ED13 andR.solanacearum-susceptible Zhongshu 3S. tuberosum were root-inoculated withR. solanacearum strain PO41. RNA were extracted from the leaves and reserve-transcribed into cDNA which was subjected for the construction of subtractive cDNA bank with the PCR Select cDNA Subtraction Kit. 384 positive clones were obtained as SMART cDNA bank and used as a template for 5′-RACE with SMART-RACE cDNA Amplification Kit to PCR amplify theStWRKY8-like gene. TheStWRKY8-like gene sequence and DNA sequence similarity were analyzed by BioEdit and BLAST tools. Phylogenetic trees were established by MEGA 5.0. The biochemical feature, tertiary structure, phosphorylation sites and sub-cellular location of the StWRKY8-like protein were predicted by ProtParam/ProtScale, SWISS-MODEL, NetPhos2.0 Server, WOLF PSORT/TargetP1.1 Server, respectively. The RNA were extracted from the leaves of ED13 and Zhongshu 3S. tuberosumat 6 h, 12 h, 1 d, 2 d, 3 d, 4 d, and 6 d post inoculation of theR. solanacearum strain PO41 for RT-PCR and real-time PCR analysis of the StWRKY8-like gene expression. Digoxin-labeledStWRKY8-like specific probes werein situ hybridized withS. tuberosum stem and leaf sections to determine its expression in different tissues.[Result] The partial cDNA of theStWRKY8-like gene (563 bp) was obtained, which contained a 258 bp open reading frame coding a peptide with 85 amino acids. The StWRKY8-like protein has a classical conserved WRKY domain with a zinc finger motif of C2H2and belongs to the subgroup II of WRKY family. The amino acid sequence of StWRKY8-like protein was

  14. [Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bge.f.alba].

    Science.gov (United States)

    Jiang, Dan; Rong, Qi-Xian; Yuan, Qing-Jun; Zhang, Wen-Jing; Zhang, Yong-Qing; Huang, Lu-Qi

    2014-11-01

    According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.

  15. In vitro transcription of a cloned vaccinia virus gene by a soluble extract prepared from vaccinia virus-infected HeLa cells.

    OpenAIRE

    Foglesong, P D

    1985-01-01

    Faithful transcription of a vaccinia virus gene was accomplished in vitro by using a soluble extract prepared from vaccinia virus-infected HeLa cells. Specific transcription of the cloned vaccinia virus gene was detected by using template DNA restricted within the transcribed region. The vaccinia virus gene was not transcribed by extracts prepared from uninfected HeLa cells even with supplementation by purified vaccinia virus RNA polymerase, nor was a clone of adenovirus 2 DNA bearing the maj...

  16. Transcriptional Modulation of Heat-Shock Protein Gene Expression

    Directory of Open Access Journals (Sweden)

    Anastasis Stephanou

    2011-01-01

    Full Text Available Heat-shock proteins (Hsps are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

  17. Transcriptional modulation of heat-shock protein gene expression.

    Science.gov (United States)

    Stephanou, Anastasis; Latchman, David S

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

  18. cDNA cloning, Phylogenic Analysis and Gene Expression Pattern of Phenylalanine ammonia-lyase in Sugarcane (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    Mahmoud Hashemitabar

    2014-08-01

    Full Text Available The aim of the present study was to clone and characterize a full length cDNA of sugarcane (Saccharum officinarum phenylalanine ammonia-lyase (SoPAL. Differential tissue expression pattern of the SoPAL transcript and its enzyme activity was also analyzed during the tillering stage of growth. The full-length of SoPAL cDNA was 2118 bp long and contained a protein with 706 amino acids, determined by encoding technique. The amino acid sequence and phylogenic analysis of the cloned SoPAL showed high similarity to PAL from other monocotyledonous such as sorghum (96%, maize (93% and Bamboos (87.12%. The highest levels of SoPAL transcript were observed in the root and stem, while its minimal gene expression levels were in the leaves and sheath, respectively. The highest level of SoPAL enzyme activity was in the leaves. These results helped to understanding the characteristics of PAL biosynthesis and its regulation at the molecular level in sugarcane. This information could be critical for the manipulation of phenylpropanoid biosynthesis in the plant using biotechnological processes.

  19. Identification and Cloning of Differentially Expressed SOUL and ELIP Genes in Saffron Stigmas Using a Subtractive Hybridization Approach

    Science.gov (United States)

    Ahrazem, Oussama; Argandoña, Javier; Castillo, Raquel; Rubio-Moraga, Ángela

    2016-01-01

    Using a subtractive hybridization approach, differentially expressed genes involved in the light response in saffron stigmas were identified. Twenty-two differentially expressed transcript-derived fragments were cloned and sequenced. Two of them were highly induced by light and had sequence similarity to early inducible proteins (ELIP) and SOUL heme-binding proteins. Using these sequences, we searched for other family members expressed in saffron stigma. ELIP and SOUL are represented by small gene families in saffron, with four and five members, respectively. The expression of these genes was analyzed during the development of the stigma and in light and dark conditions. ELIP transcripts were detected in all the developmental stages showing much higher expression levels in the developed stigmas of saffron and all were up-regulated by light but at different levels. By contrast, only one SOUL gene was up-regulated by light and was highly expressed in the stigma at anthesis. Both the ELIP and SOUL genes induced by light in saffron stigmas might be associated with the structural changes affecting the chromoplast of the stigma, as a result of light exposure, which promotes the development and increases the number of plastoglobules, specialized in the recruitment of specific proteins, which enables them to act in metabolite synthesis and disposal under changing environmental conditions and developmental stages. PMID:28030614

  20. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  1. Molecular cloning and transcriptional analysis of a NPY receptor-like in common Chinese cuttlefish Sepiella japonica

    Science.gov (United States)

    Yang, Jingwen; Xu, Yuchao; Xu, Ke; Ping, Hongling; Shi, Huilai; Lü, Zhenming; Wu, Changwen; Wang, Tianming

    2017-08-01

    Neuropeptide Y (NPY) has a pivotal role in the regulation of many physiological processes. In this study, the gene encoding a NPY receptor-like from the common Chinese cuttlefish Sepiella japonica (SjNPYR-like) was identified and characterized. The full-length SjNPYR-like cDNA was cloned containing a 492-bp of 5' untranslated region (UTR), 1 182 bp open reading frame (ORF) encoding a protein of 393 amino acid residues, and 228 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 45.54 kDa and an isoelectric point (pI) of 8.13. By informatic analyses, SjNPYR-like was identified as belonging to the class A G protein coupled receptor (GPCR) family (the rhodopsin-type). The amino acid sequence contained 12 potential phosphorylation sites and five predicted N-linked glycosylation sites. Multiple sequence alignment and 3D structure modeling were conducted to clarify SjNPYR bioinformatics characteristics. Phylogenetic analysis identifies it as an NPYR with identity of 33% to Lymnaea stagnalis NPFR. Transmembrane properties of SjNPYR-like were demonstrated in vitro using HEK293 cells and the pEGFP-N1 plasmid. Relative quantification of SjNPYR-like mRNA level confirmed a high level expression and broad distribution of SjNPYR - like in various tissues of female S. japonica. In addition, the transcriptional profile of SjNPYR - like in the brain, liver, and ovary during gonadal development was analyzed. The results provide basic understanding on the molecular characteristics of SjNPYR-like and its potentially physical functions.

  2. Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

    Directory of Open Access Journals (Sweden)

    Zhao Dingsheng

    2008-02-01

    Full Text Available Abstract Background Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (β-actin, VEGF, oct4, TERT, H19 and Igf2 and a repetitive sequence (art2 in five organs (heart, liver, spleen, lung and kidney from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3, the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3 died after the perinatal period. Normally reproduced cattle served as a control group (n = 3. Results Aberrant DNA methylation, histone H4 acetylation and gene expression were observed in both cloned groups. The ED group showed relatively fewer severe DNA methylation abnormalities (p Conclusion Deaths of clones may be ascribed to abnormal expression of a very limited number of genes.

  3. Expression of a novel dipeptidyl peptidase 8 (DPP8) transcript variant, DPP8-v3, in human testis

    Institute of Scientific and Technical Information of China (English)

    Hui Zhu; Zuo-Min Zhou; Li Lu; Min Xu; Hui Wang; Jian-Min Li; Jia-Hao Sha

    2005-01-01

    Aim: To investigate the role of a novel dipeptidyl peptidase 8 transcript variant (DPP8-v3) gene in testis development and/or spermatogenesis. Methods: A human testis cDNA microarray was hybridized with mRNA of human adult and fetal testes. Differentially expressed clones were sequenced and characterized and their expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Southern-blot analysis. Results: A new transcript variant of the human dipeptidyl peptidase (DPP8), exhibiting a 5-fold higher expression level in human adult than that in fetal testes, was cloned and was named DPP8 variant 3 (DPP8-v3). The full-length sequence of DPP8-v3was 3,030 bp, encoding a protein of 898 amino acids. Conclusion: DPP8-v3 is a novel human DPP8 transcript variant highly expressed in the adult testis. Similar to DPPⅣ, DPP8-v3 may play a key role in the immunoregulation of testes and accordingly may influence spermatogenesis and male fertility.

  4. Transcriptional effects of CRP* expression in Escherichia coli

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    Ghosh Debashis

    2009-08-01

    Full Text Available Abstract Background Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp* enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP to that of mutant strain PC05 (expressing CRP* in the presence and absence of glucose. Results The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression. We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of

  5. The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yun-Ling; Li, Li; Wu, Keqiang [Michigan State Univ., East Lansing, MI (United States)] [and others

    1995-07-03

    The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6 figs.

  6. Expression of a novel alternative transcript of the novel retinal pigment epithelial cell gene NORPEG in human testes

    Institute of Scientific and Technical Information of China (English)

    Wa Yuan; Ying Zheng; Ran Huo; Li Lu; Xiao-Yan Huang; Lan-Lan Yin; Jian-Min Li; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis. Methods: A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR.Results: The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues. Conclusion: sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis.

  7. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  8. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    Science.gov (United States)

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  9. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. (Univ. of California, San Diego, La Jolla (United States)); McPherson, J.P. (Univ. of California, Irvine (United States)); Evans, G.A. (Salk Inst. for Biological Studies, La Jolla, CA (United States))

    1992-02-15

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCr analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

  10. Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor.

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

    2013-10-15

    CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

  11. Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

    Directory of Open Access Journals (Sweden)

    Yeon Soo Han

    2013-10-01

    Full Text Available CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63 was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

  12. Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse.

    Science.gov (United States)

    Kaneko, K J; Cullinan, E B; Latham, K E; DePamphilis, M L

    1997-05-01

    mTEF-1 is the prototype of a family of mouse transcription factors that share the same TEA DNA binding domain (mTEAD genes) and are widely expressed in adult tissues. At least one member of this family is expressed at the beginning of mouse development, because mTEAD transcription factor activity was not detected in oocytes, but first appeared at the 2-cell stage in development, concomitant with the onset of zygotic gene expression. Since embryos survive until day 11 in the absence of mTEAD-1 (TEF-1), another family member likely accounts for this activity. Screening an EC cell cDNA library yielded mTEAD-1, 2 and 3 genes. RT-PCR detected RNA from all three of these genes in oocytes, but upon fertilization, mTEAD-1 and 3 mRNAs disappeared. mTEAD-2 mRNA, initially present at approx. 5,000 copies per egg, decreased to approx. 2,000 copies in 2-cell embryos before accumulating to approx. 100,000 copies in blastocysts, consistent with degradation of maternal mTEAD mRNAs followed by selective transcription of mTEAD-2 from the zygotic genome. In situ hybridization did not detect mTEAD RNA in oocytes, and only mTEAD-2 was detected in day-7 embryos. Northern analysis detected all three RNAs at varying levels in day-9 embryos and in various adult tissues. A fourth mTEAD gene, recently cloned from a myotube cDNA library, was not detected by RT-PCR in either oocytes or preimplantation embryos. Together, these results reveal that mTEAD-2 is selectively expressed for the first 7 days of embryonic development, and is therefore most likely responsible for the mTEAD transcription factor activity that appears upon zygotic gene activation.

  13. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    Science.gov (United States)

    Kula, Anna; Marcello, Alessandro

    2012-01-01

    Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. PMID:24832221

  14. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    Directory of Open Access Journals (Sweden)

    Alessandro Marcello

    2012-07-01

    Full Text Available Gene expression of the human immunodeficiency virus type 1 (HIV-1 is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE. These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function.

  15. Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.

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    Jin-Song Gong

    Full Text Available BACKGROUND: Nitrilase is an important member of the nitrilase superfamiliy. It has attracted substantial interest from academia and industry for its function of converting nitriles directly into the corresponding carboxylic acids in recent years. Thus nitrilase has played a crucial role in production of commercial carboxylic acids in chemical industry and detoxification of nitrile-contaminated wastes. However, conventional studies mainly focused on the bacterial nitrilase and the potential of fungal nitrilase has been far from being fully explored. Research on fungal nitrilase gene expression will advance our understanding for its biological function of fungal nitrilase in nitrile hydrolysis. METHODOLOGY/PRINCIPAL FINDINGS: A fungal nitrilase gene from Gibberella intermedia was cloned through reverse transcription-PCR. The open reading frame consisted of 963 bp and potentially encoded a protein of 320 amino acid residues with a theoretical molecular mass of 35.94 kDa. Furthermore, the catalytic triad (Glu-45, Lys-127, and Cys-162 was proposed and confirmed by site-directed mutagenesis. The encoding gene was expressed in Escherichia coli Rosetta-gami (DE3 and the recombinant protein with His(6-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity at 45°C and pH 7.8. This nitrilase was specific towards aliphatic and aromatic nitriles. The kinetic parameters V(max and K(m for 3-cyanopyridine were determined to be 0.81 µmol/min·mg and 12.11 mM through Hanes-Woolf plot, respectively. 3-Cyanopyridine (100 mM could be thoroughly hydrolyzed into nicotinic acid within 10 min using the recombinant strain with the release of about 3% nicotinamide and no substrate was detected. CONCLUSIONS/SIGNIFICANCE: In the present study, a fungal nitrilase was cloned from the cDNA sequence of G. intermedia and successfully expressed in E. coli Rosetta-gami (DE3. The recombinant strain displayed good 3-cyanopyridine

  16. Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide.Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed using cDNAs as templates from melonHuangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named mp-19was cloned and sequenced. The Open Reading Frame (ORF) of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of mp-19. The obvious expression differences detected by semi-quantitative RTPCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

  17. Cloning, Expression, and Characterization of Prophenoloxidases from Asian Corn Borer, Ostrinia furnacalis (Gunée

    Directory of Open Access Journals (Sweden)

    Shasha Zhang

    2016-01-01

    Full Text Available Insect phenoloxidase (PO belongs to the type 3 copper protein family and possesses oxidoreductase activities. PO is typically synthesized as a zymogen called prophenoloxidase (PPO and requires the proteolytic activation to function. We here cloned full-length cDNA for 3 previously unidentified PPOs, which we named OfPPO1a, OfPPO1b, and OfPPO3, from Asian corn borer, Ostrinia furnacalis (Gunée, in addition to the previously known OfPPO2. These conceptual PPOs and OfPPO2 all contain two common copper-binding regions, two potential proteolytic activation sites, a plausible thiol-ester site, and a conserved C-terminal region but lack a secretion signal peptide sequence at the N-terminus. O. furnacalis PPOs were highly similar to other insect PPOs (42% to 79% identity and clustered well with other lepidopteran PPOs. RT-PCR assay showed the transcripts of the 4 OfPPOs were all detected at the highest level in hemocytes and at the increased amounts after exposure to infection by bacteria and fungi. Additionally, we established an Escherichia coli (E. coli expression system to produce recombinant O. furnacalis PPO proteins for future use in investigating their functions. These insights could provide valuable information for better understanding the activation and functioning mechanisms of O. furnacalis PPOs.

  18. Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

    Science.gov (United States)

    Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

    2016-09-01

    The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems ( Pcloned from a halophyte, and it might contain a diff erent metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  19. The Role of Multiple Transcription Factors In Archaeal Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Charles J. Daniels

    2008-09-23

    Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcanii was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of

  20. Cloning, expression, purification and refolding of microtubule affinity-regulating kinase 4 expressed in Escherichia coli.

    Science.gov (United States)

    Naz, Farha; Asad, Mohd; Malhotra, Pawan; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2014-03-01

    Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a member of the family Ser/Thr kinase and involved in numerous biological functions including microtubule bundle formation, nervous system development, positive regulation of programmed cell death, cell cycle control, cell polarity determination, cell shape alterations, cell division etc. For various biophysical and structural studies, we need this protein in adequate quantity. In this paper, we report a novel cloning strategy for MARK4. We have cloned MARK4 catalytic domain including 59 N-terminal extra residues with unknown function and catalytic domain alone in PQE30 vector. The recombinant MARK4 was expressed in the inclusion bodies in M15 cells. The inclusion bodies were solubilized effectively with 1.5% N-lauroylsarcosine in alkaline buffer and subsequently purified using Ni-NTA affinity chromatography in a single step with high purity and good concentration. Purity of protein was checked on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by using mass spectrometry immunoblotting. Refolding of the recombinant protein was validated by ATPase assay. Our purification procedure is quick, simple and produces adequate quantity of proteins with high purity in a limited step.

  1. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jingwen; Danehower, S.; Besterman, J.M.; Husain, I. [Glaxo Research Inst., Research Triangle Park, NC (United States)] [and others

    1995-10-01

    Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating supermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replications. In contrast, elevated levels of DNA ligase III mRNA were observed in primary supermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells. 62 refs., 7 figs.

  2. Three slow skeletal muscle troponin genes in small-tailed Han sheep (Ovis aries): molecular cloning, characterization and expression analysis.

    Science.gov (United States)

    Sun, Yan; Wang, Guizhi; Ji, Zhibin; Chao, Tianle; Liu, Zhaohua; Wang, Xiaolong; Liu, Guanqing; Wu, Changhao; Wang, Jianmin

    2016-09-01

    To explore the basic characteristics and expressing profile of the three slow skeletal muscle troponin genes TNNC1 (Troponin C type 1), TNNI1 (troponin I type 1) and TNNT1 (troponin T type 1). Three purebred Dorper sheep and another three purebred small-tailed Han sheep were selected. The sequence of the genes from the small-tailed Han sheep was cloned using rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction; The characteristics of the predicted amino acids sequences were analyzed using bioinformatics analysis software; Gene expression analyses were performed using quantitative reverse transcription PCR. The full-length cDNA sequences of the genes were 707, 898, and 1001 bp, respectively, and were submitted to GenBank under accession numbers KR153938, KT218688 and KT218690. The three predicted proteins were predicted to be hydrophilic, non-secretory proteins and contain several phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted proteins were relatively conserved in mammals. The expression results of the three genes in eight tissues of Dorper and small-tailed Han sheep revealed that the three genes had a similar mRNA expression pattern, whereas distinct differences were observed among the eight tissues of the two sheep species. We cloned the full-length cDNA of the three genes, analyzed the amino acid sequences, and determined the expression levels of the genes. These results might play important roles in facilitating the future research of the three genes.

  3. Cloning and expression of follistatin gene in half-smooth tongue sole Cynoglossus semilaevis during the reproduction cycle

    Science.gov (United States)

    Wen, Haishen; Si, Yufeng; Zhang, Yuanqing; He, Feng; Li, Jifang

    2015-03-01

    Follistatin (FST) is a monomeric glycoprotein highly enriched in cysteines and belongs to TGF-β superfamily. FST can suppress the secretion of follicle-stimulating hormone and plays a vital role in the reproduction of vertebrates. We used rapid amplification of cDNA ends technology to clone the FST gene of half-smooth tongue sole, Cynoglossus semilaevis. We characterized its phylogenetic context and expression patterns to elucidate its function in the breeding season. The full-length sequence of FST is 1 455 bp and encodes a protein of 321 amino acids. We investigated the expression pattern of FST in C. semilaevis at different stages of reproduction using reverse transcription-polymerase chain reaction (RTPCR). FST mRNA was expressed in all 13 tissues analyzed, and was expressed at high levels in gonad and at slightly lower levels in gill and brain. During the reproductive cycle of C. semilaevis, the transcript level of FST was the highest in the perinucleolus stage, decreased in the primary yolk stage, slightly increased in the tertiary yolk stage, and then reduced to a minimal level in the atretic follicles stage of the ovary. We concluded that FST suppressed follicle-stimulating hormone, which stimulated oocyte development. However, no significant variation was observed across all stages of testis development, although the expression level in the spermatogenesis stage was relatively low, which may result from the regulation of FST by aromatase.

  4. Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

    Science.gov (United States)

    2013-01-01

    Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens

  5. Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

    Science.gov (United States)

    Zhao, Tian-Tian; Zhang, Jin; Liang, Li-Song; Ma, Qing-Hua; Chen, Xin; Zong, Jian-Wei; Wang, Gui-Xi

    2015-01-01

    Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.

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

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2001-09-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes 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 of H. 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.

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

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2013-03-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes 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 of H. 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.

  8. Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome

    2013-01-01

    auxotrophic and dominant markers for convenience of use. Our vector set also contains both integrating and multicopy vectors for stability of protein expression and high expression level. We will make the new vector system available to the yeast community and provide a comprehensive protocol for cloning...

  9. A practical approach to T-cell receptor cloning and expression.

    Directory of Open Access Journals (Sweden)

    Sébastien Wälchli

    Full Text Available Although cloning and expression of T-cell Receptors (TcRs has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5'-RACE amplification. We here present an improved 5'-RACE protocol that represents a fast and reliable way to identify a TcR from 10(5 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality.

  10. Cloning of the homeotic Sex combs reduced gene in Drosophila and in situ localization of its transcripts.

    Science.gov (United States)

    Kuroiwa, A; Kloter, U; Baumgartner, P; Gehring, W J

    1985-12-30

    We have extended our ;chromosomal walk' in the Antennapedia-complex (ANT-C) by isolating overlapping DNA sequences spanning the chromosomal segment between Antennapedia (Antp) and Deformed (Dfd). The transcription units, homeoboxes and M-repeats were mapped within this region. Four transcription units Antp, fushi tarazu (ftz), Sex combs reduced (Scr) and Dfd contain both a homeobox and an M repeat, whereas at least two additional transcription units, x and z, were found to lack these elements. The Scr locus was identified by deletion mapping. It consists of at least two exonic regions separated by a large intron. The homeobox is located in the 3' exon and is 82% homologous to the one in Antp. Scr encodes a major 3.9-kb RNA. A corresponding cDNA clone was used as a probe for in situ hybridization to sections of various embryonic stages. At gastrula stages Scr transcripts accumulate in the posterior head and the anterior thoracic region of the germ band. At later stages a strong accumulation of transcripts is observed in the suboesophageal and the prothoracic ganglion of the ventral nervous system.

  11. Cloning of the coat protein gene from beet necrotic yellow vein virus and its expression in sugar beet hairy roots.

    Science.gov (United States)

    Ehlers, U; Commandeur, U; Frank, R; Landsmann, J; Koenig, R; Burgermeister, W

    1991-06-01

    Expression of the beet necrotic yellow vein virus (BNYVV) coat protein (CP) gene in transgenic sugar beet hairy roots was accomplished as a step towards CP-mediated virus resistance. A cDNA for the CP gene and its 5' terminal untranslated leader sequence was prepared from BNYVV RNA, using two oligodeoxynucleotides to prime the synthesis of both strands. Second-strand synthesis and amplification of the cDNA were done by Taq DNA polymerase chain reactions. Run-off transcripts of the cloned cDNA sequence were obtained and translated in vitro, yielding immunoreactive CP. A binary vector construction containing the CP gene under the control of the 35S promoter of cauliflower mosaic virus was prepared and used for Agrobacterium rhizogenes-mediated transformation of sugar beet tissue. Stable integration and expression of the CP gene in sugar beet hairy roots was demonstrated by Southern, Northern, and Western blot analysis, respectively.

  12. The ubiquitin extension protein S27a is differentially expressed in developing flower organs of Thompson seedless versus Thompson seeded grape isogenic clones.

    Science.gov (United States)

    Hanania, Uri; Velcheva, Margarita; Sahar, Nachman; Flaishman, Moshe; Or, Etti; Degani, Oded; Perl, Avihai

    2009-07-01

    In Vitis vinifera L. cv. Thompson Seedless, fertilization occurs but seeds abort, a type of stenospermocarpy. To clone transcripts with differential expression during flower development, suppressive subtractive hybridization was carried out using two isogenic clones 'Thompson seedless' and 'Thompson seeded', at three stages of inflorescence development (from bud break to ~20 days prior to anthesis). Differential screening and sequencing of a forward and reverse subtractive cDNA library yielded several singleton ESTs. One differentially expressed clone in 'Thompson' seeded versus seedless isogenic clones was the ubiquitin extension protein S27a. In situ hybridization demonstrated its significantly higher expression in the carpel and ovaries of 'Thompson' seedless versus seeded isogenic clones during flower development. Overexpression of this gene resulted in abnormal plant regeneration and inhibited shoot development compared to controls; its silencing in embryogenic callus induced cell necrosis and callus death, evidencing tight regulation of this gene in developing organs of grape. S27a overexpression in carpels and integuments of the seedless flower may interfere with normal development of these organs, leading to embryo abortion and seedlessness.

  13. pBaSysBioll : an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

    NARCIS (Netherlands)

    Botella, Eric; Fogg, Mark; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette; Denham, Emma. L.; Le Chat, Ludovic; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; van Dijl, Jan Maarten; Aymerich, Stephane; Wilkinson, Anthony J.; Devine, Kevin M.

    Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type

  14. pBaSysBioll : an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

    NARCIS (Netherlands)

    Botella, Eric; Fogg, Mark; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette; Denham, Emma. L.; Le Chat, Ludovic; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; van Dijl, Jan Maarten; Aymerich, Stephane; Wilkinson, Anthony J.; Devine, Kevin M.

    2010-01-01

    Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type

  15. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

    Science.gov (United States)

    Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

  16. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    Science.gov (United States)

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  17. Cloning of zebrafish nkx6.2 and a comprehensive analysis of the conserved transcriptional response to Hedgehog/Gli signaling in the zebrafish neural tube.

    Science.gov (United States)

    Guner, Burcu; Karlstrom, Rolf O

    2007-04-01

    Sonic Hedgehog (Shh) signaling helps pattern the vertebrate neural tube, in part by regulating the dorsal/ventral expression of a number of homeodomain containing transcription factors. These Hh responsive genes have been divided into two classes, with Class II genes being activated by Hh signaling and Class I genes being repressed by Hh signaling. While the transcriptional response to varying Hh levels is well defined in chick and mouse, it is only partially described in zebrafish, despite the fact that zebrafish has emerged as a powerful genetic system for the study of neural patterning. To better characterize the Hh response in the zebrafish neural tube, we cloned the zebrafish Class II Hh target genes nkx2.9 and nkx6.2. We then analyzed the expression of a number of Class I and Class II Hh responsive genes in wild type, Hh mutant, and Hh over-expressing zebrafish embryos. We show that expression of Class I and Class II genes is highly conserved in the vertebrate neural tube. Further, ventral-most Class II gene expression was completely lost in all Hh pathway mutants analyzed, indicating high levels of Hh signaling are blocked in all of these mutants. In contrast, more dorsally expressed genes were variably affected in different Hh pathway mutants, indicating mid-levels of Hh signaling are differentially affected. This comprehensive expression study provides an important tool for the characterization of Hh signaling in zebrafish and provides a sensitive assay for determining the degree to which newly identified zebrafish mutants affect Hh signaling.

  18. A transcriptional cofactor YAP regulates IFNT expression via transcription factor TEAD in bovine conceptuses.

    Science.gov (United States)

    Kusama, K; Bai, R; Sakurai, T; Bai, H; Ideta, A; Aoyagi, Y; Imakawa, K

    2016-10-01

    Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These

  19. Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases.

    OpenAIRE

    Anderberg, R J; Walker-Simmons, M K

    1992-01-01

    Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA...

  20. Melanoma antigen expression and metastatic ability of mutant B16 melanoma clones.

    Science.gov (United States)

    Nozue, M; Sakiyama, H; Tsuchiya, K; Hirabayashi, Y; Taniguchi, M

    1988-11-15

    The biological functions of murine melanoma-associated antigens recognized by monoclonal antibodies (MAbs) (M562, M622 and M2590) were examined by using mutant clones which differed in their degree of expression of these antigens. Four clones of high expressors of 3 types of antigen (MEA group), 5 clones of low or non-expressors of M562- and M622-recognizing antigens (MEB group) and 4 clones of non-expressor of GM3 recognized by M2590 (MEC group) were used. Attachment of these clones to components of extracellular matrix was different between the groups. Two clones of the MEA group showed the highest ability to adhere to laminin and type-IV collagen, whereas the clones of the MEB and MEC groups significantly lost their ability to attach to laminin and type-IV collagen. In experimental lung metastasis, metastasizing ability of MEA-group cells was higher than that of MEB- and MEC-group cells. Our results suggest that these antigens play some functional role in metastasis mediated by increasing capacity for attachment to laminin and type-IV collagen.

  1. Expression analysis of TALE family transcription factors during avian development.

    Science.gov (United States)

    Coy, Sarah E; Borycki, Anne-Gaëlle

    2010-04-01

    The TALE family of homeodomain containing transcription factors consists of the Meis, Prep and Tgif, and the Pbx subfamily of proteins. Several TALE orthologues have been identified in amniotes, but no comprehensive analysis of their expression pattern during embryogenesis has been performed. Here, we report on TALE gene expression in the avian embryo. During embryonic development, Pbx genes are predominantly expressed in the neural ectoderm and paraxial mesoderm, although Pbx3 is restricted to the intermediate and lateral mesoderm, and anterior central nervous system. Members of the Meis, Prep, and Tgif subfamilies are expressed at high levels in the paraxial mesoderm, and display differential expression along the anterior-posterior and dorsoventral axes of the developing neural tube. Overall the expression patterns reported in this study are consistent with the known function of the TALE gene family in controlling early patterning of limb, neural tube and paraxial mesoderm tissues during embryogenesis.

  2. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    Science.gov (United States)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  3. Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression.

    Science.gov (United States)

    Mourtada-Maarabouni, Mirna; Kirkham, Lucy; Farzaneh, Farzin; Williams, Gwyn T

    2004-12-16

    Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways independently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone' by ultraviolet radiation, and by the anticancer drug cisplatin. In all cases, colony-forming ability is protected, indicating that fau affects the critical events prior to commitment to cell death. Overexpression of fau in the sense orientation induces cell death, which is inhibited both by Bcl-2 and by inhibition of caspases, in line with its proposed role in apoptosis.

  4. Cloning and expression of acetylcholinesterase from Electrophorus. Splicing pattern of the 3' exons in vivo and in transfected mammalian cells.

    Science.gov (United States)

    Simon, S; Massoulié, J

    1997-12-26

    We cloned and expressed a cDNA encoding acetylcholinesterase (AChE) of type T from Electrophorus electricus organs. When expressed in COS, HEK, and Chinese hamster ovary cells, the AChET subunits generated dimers and tetramers. The cells produced more activity at 27 than at 37 degrees C. The kinetic parameters of a recombinant enzyme, produced in the yeast Pichia pastoris, were close to those of the natural AChE. Analysis of genomic clones showed that the coding sequence is interrupted by an intron that does not exist in Torpedo and differs in its location from that observed in the mouse. This intron is preceded by a sequence encoding a non-conserved 29-amino acid peptide, which does not exist in Torpedo or mammalian AChEs. According to a three-dimensional model, this non-conserved peptide is located at the surface of the protein, opposite from the entry of the catalytic gorge; its deletion did not modify the catalytic parameters. Sequence analyses and expression of various constructs showed that the gene does not contain any H exon. We also found that splicing of transcripts in mammalian cells reveals cryptic donor sites in exons and acceptor sites in introns, which do not appear to be used in vivo.

  5. Construction and characteristics of a transformed lepidopteran cell clone expressing baculovirus p35

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guiling; LI Changyou; LI Guoxun; WANG Ping; Robert R. Granados

    2005-01-01

    A transformed cell line was constructed from Mythimna separata cells Ms7311 by lipofection method. TMs7311 cells were generated using a double selection technique involving a selection in the antibiotic Zeocin, followed by a second round of selection by exhibiting cell characterization. A cell clone expressing p35 was obtained with high level of AcMNPV and recombinant proteins. Compared with wild type Ms7311 cells, the cell clone showed increased resistance to Actinamycin D-induced apoptosis and a profound resistance to nutrient development (PBS). When the cell clone was infected with recombinant baculoviruses expressing secreted alkaline phosphatase (SEAP) and β-galactosi- dase, expression of the recombinant proteins from TMs7311 cells exceeded that from parental Ms7311 cells. Production of budded virus and occlusion body was significantly higher than that from parental cells Ms7311.

  6. Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

    Institute of Scientific and Technical Information of China (English)

    Xin Li; Ying-Nian Yu; Ge-Jian Zhu; Yu-Li Qian

    2001-01-01

    AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9. METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC. RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells. CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.

  7. Molecular Cloning, Expression Pattern and Genotypic Effects on Glucoraphanin Biosynthetic Related Genes in Chinese Kale (Brassica oleracea var. alboglabra Bailey).

    Science.gov (United States)

    Yin, Ling; Chen, Changming; Chen, Guoju; Cao, Bihao; Lei, Jianjun

    2015-11-11

    Glucoraphanin is a plant secondary metabolite that is involved in plant defense and imparts health-promoting properties to cruciferous vegetables. In this study, three genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4 (BCAT4), methylthioalkylmalate synthase 1 (MAM1) and dihomomethionine N-hydroxylase (CYP79F1), were cloned from Chinese kale (Brassica oleracea var. alboglabra Bailey). Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The transcript levels of BCAT4, MAM1 and CYP79F1 were higher in cotyledon, leaf and stem compared with flower and silique. BCAT4, MAM1 and CYP79F1 were expressed throughout leaf development with lower transcript levels during the younger stages. Glucoraphanin content varied extensively among different varieties, which ranged from 0.25 to 2.73 µmol·g(-1) DW (dry weight). Expression levels of BCAT4 and MAM1 were high at vegetative-reproductive transition phase, while CYP79F1 was expressed high at reproductive phase. BCAT4, MAM1 and CYP79F1 were expressed significantly high in genotypes with high glucoraphanin content. All the results provided a better understanding of the roles of BCAT4, MAM1 and CYP79F1 in the glucoraphanin biosynthesis of Chinese kale.

  8. Insights into alpha-hemolysin (Hla) evolution and expression among Staphylococcus aureus clones with hospital and community origin

    DEFF Research Database (Denmark)

    Tavares, Ana; Nielsen, Jesper B; Boye, Kit;

    2014-01-01

    in the RNAIII binding site were not associated to hla expression. Although expression rates of hla were in general strain-specific, we observed CA clones showed significantly higher hla expression (p = 0.003) when compared with HA clones. CONCLUSION: We propose that the hla gene has evolved together...

  9. Trypanosoma cruzi: expression of antigenic component 5 among 35 laboratory clones obtained from 18 different isozymic variants

    Directory of Open Access Journals (Sweden)

    Simone F. Breniere

    1987-04-01

    Full Text Available Two monoclonal antibodies anti-component 5 of Trypanosoma cruzi (I-35/115 and II-190/30 were tested in IFA and ELISA respectively against 35 T. cruzi laboratory clones. Among the 35 clones tested, 18 different isozyme patterns were detected. All clones were recognized by both monoclonal antibodies except one clone which did not react with II-190/30. These results support the universal expression of specific component 5 within the taxon T. cruzi.

  10. Cloning, tissue expression pattern, and chromosome localization of human protein kinase Bγ gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkb? was used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3'-terminal of this fragment was extended to 2788 bp by 'electronic walking' screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBγ and γ, respectively, and was designated as human PKB?. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the highest expression level in brain, and lower levels with variation in the other tissues. By RH mapping, the PKBγ was placed on chromosome 1q43, between markers D1S304 and D1S2693. It is a valuable clue for cloning the candidate genes related to prostate cancer; Arrhythmogenic Right Ventricular Dysplasia (ARVD); Chediak-Higashi, NK cell Deficiency (CHS); and Hypoparathyrodism with Short Stature, Mental Retardation and Seizures which have already been mapped in this chromosomal region.

  11. Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.

    Science.gov (United States)

    Zhang, Min; Jiang, Shao-tong; Zheng, Zhi; Li, Xing-jiang; Luo, Shui-zhong; Wu, Xue-feng

    2015-07-01

    Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226)  → Glu(226) and Val(274)  → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification.

  12. Molecular cloning of Reteplase and its expression in E. coli using tac promoter

    Directory of Open Access Journals (Sweden)

    Safieh Aghaabdollahian

    2014-01-01

    Full Text Available Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression. Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG, expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.

  13. The transcriptional interactome: gene expression in 3D.

    Science.gov (United States)

    Schoenfelder, Stefan; Clay, Ieuan; Fraser, Peter

    2010-04-01

    Transcription in the eukaryotic nucleus has long been thought of as conforming to a model in which RNA polymerase complexes are recruited to and track along isolated templates. However, a more dynamic role for chromatin in transcriptional regulation is materializing: enhancer elements interact with promoters forming loops that often bridge considerable distances and genomic loci, even located on different chromosomes, undergo chromosomal associations. These associations amass to form an extensive 'transcriptional interactome', enacted at functional subnuclear compartments, to which genes dynamically relocate. The emerging view is that long-range chromosomal associations between genomic regions, and their repositioning in the three-dimensional space of the nucleus, are key contributors to the regulation of gene expression. 2010 Elsevier Ltd. All rights reserved.

  14. Cloning and expression of a rat cardiac delayed rectifier potassium channel.

    OpenAIRE

    Paulmichl, M.; Nasmith, P; Hellmiss, R; Reed, K.; Boyle, W A; Nerbonne, J M; Peralta, E G; Clapham, D.E.

    1991-01-01

    We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the p...

  15. Spatial expression of transcription factors in Drosophila embryonic organ development.

    Science.gov (United States)

    Hammonds, Ann S; Bristow, Christopher A; Fisher, William W; Weiszmann, Richard; Wu, Siqi; Hartenstein, Volker; Kellis, Manolis; Yu, Bin; Frise, Erwin; Celniker, Susan E

    2013-12-20

    Site-specific transcription factors (TFs) bind DNA regulatory elements to control expression of target genes, forming the core of gene regulatory networks. Despite decades of research, most studies focus on only a small number of TFs and the roles of many remain unknown. We present a systematic characterization of spatiotemporal gene expression patterns for all known or predicted Drosophila TFs throughout embryogenesis, the first such comprehensive study for any metazoan animal. We generated RNA expression patterns for all 708 TFs by in situ hybridization, annotated the patterns using an anatomical controlled vocabulary, and analyzed TF expression in the context of organ system development. Nearly all TFs are expressed during embryogenesis and more than half are specifically expressed in the central nervous system. Compared to other genes, TFs are enriched early in the development of most organ systems, and throughout the development of the nervous system. Of the 535 TFs with spatially restricted expression, 79% are dynamically expressed in multiple organ systems while 21% show single-organ specificity. Of those expressed in multiple organ systems, 77 TFs are restricted to a single organ system either early or late in development. Expression patterns for 354 TFs are characterized for the first time in this study. We produced a reference TF dataset for the investigation of gene regulatory networks in embryogenesis, and gained insight into the expression dynamics of the full complement of TFs controlling the development of each organ system.

  16. Transcription factors expressed in soybean roots under drought stress.

    Science.gov (United States)

    Pereira, S S; Guimarães, F C M; Carvalho, J F C; Stolf-Moreira, R; Oliveira, M C N; Rolla, A A P; Farias, J R B; Neumaier, N; Nepomuceno, A L

    2011-10-21

    To gain insight into stress-responsive gene regulation in soybean plants, we identified consensus sequences that could categorize the transcription factors MYBJ7, BZIP50, C2H2, and NAC2 as members of the gene families myb, bzip, c2h2, and nac, respectively. We also investigated the evolutionary relationship of these transcription factors and analyzed their expression levels under drought stress. The NCBI software was used to find the predicted amino acid sequences of the transcription factors, and the Clustal X software was used to align soybean and other plant species sequences. Phylogenetic trees were built using the Mega 4.1 software by neighbor joining and the degree of confidence test by Bootstrap. Expression level studies were carried out using hydroponic culture; the experiments were designed in completely randomized blocks with three repetitions. The blocks consisted of two genotypes, MG/BR46 Conquista (drought-tolerant) and BR16 (drought-sensitive) and the treatments consisted of increasingly long dehydration periods (0, 25, 50, 75, and 100 min). The transcription factors presented domains and/or conserved regions that characterized them as belonging to the bzip, c2h2, myb, and nac families. Based on the phylogenetic trees, it was found that the myb, bzip and nac genes are closely related to myb78, bzip48 and nac2 of soybean and that c2h2 is closely related to c2h2 of Brassica napus. Expression of all genes was in general increased under drought stress in both genotypes. Major differences between genotypes were due to the lowering of the expression of the mybj7 and c2h2 genes in the drought-tolerant variety at some times. Over-expression or silencing of some of these genes has the potential to increase stress tolerance.

  17. A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

    OpenAIRE

    Scholz, J; Besir, H.; Strasser, C.; Suppmann, S.

    2013-01-01

    Background: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. Results: Here we describe a method to tailor selected expression vectors for para...

  18. Molecular Cloning of phd1 and Comparative Analysis of phd1, 2, and 3 Expression in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Dandan Han

    2012-01-01

    Full Text Available Intensive gene targeting studies in mice have revealed that prolyl hydroxylase domain proteins (PHDs play important roles in murine embryonic development; however, the expression patterns and function of these genes during embryogenesis of other vertebrates remain largely unknown. Here we report the molecular cloning of phd1 and systematic analysis of phd1, phd2, and phd3 expression in embryos as well as adult tissues of Xenopus laevis. All three phds are maternally provided during Xenopus early development. The spatial expression patterns of phds genes in Xenopus embryos appear to define a distinct synexpression group. Frog phd2 and phd3 showed complementary expression in adult tissues with phd2 transcription levels being high in the eye, brain, and intestine, but low in the liver, pancreas, and kidney. On the contrary, expression levels of phd3 are high in the liver, pancreas, and kidney, but low in the eye, brain, and intestine. All three phds are highly expressed in testes, ovary, gall bladder, and spleen. Among three phds, phd3 showed strongest expression in heart.

  19. Cloning and Identification of Porcine HSPC117 Gene Differentially Expressed in F1 Crossbreds and Their Parents

    Institute of Scientific and Technical Information of China (English)

    XIE Hong-tao; LEI Ming-gang; XIONG Yuan-zhu; DENG Chang-yan; JIANG Si-wen; LI Feng-e; ZUO Bo; XU De-quan

    2007-01-01

    To investigate the molecular basis of porcine heterosis, suppression subtractive hybridization (SSH) was performed to detect the differences in gene expression between porcine longissimus dorsi of Meishan × Large White (MS × LW) F1hybrids and their parents Meishan pigs. An expression sequence tag (EST) differentially expressed was found, designated as ML556, which was homologous to a hypothetical protein HSPC117, from human hematopoietic stem/progenitor cells(HSPCs), and the full-length cDNA of porcine HSPC117 was cloned using rapid amplification of cDNA ends (RACE)method. Translation of the mRNA transcript revealed an open reading frame (ORF) of 505 amino acid residues encoding a peroxisomal targeting signal (PTS) with theoretical molecular weight of 55 kDa. Alignment analysis revealed that the deduced protein sequence exhibit 98, 98, 98, 97, and 97% identity with that of cattle, human, dog, rat, and mouse,respectively. The tissue expression analysis indicated that the porcine HSPC117 gene is highly expressed in muscle,spleen, lung, kidney, uterus, ovary and testis, moderately expressed in fat, heart, and liver, and not expressed in stomach and small intestine. The possible role of porcine HSPC117 and its relationship with porcine heterosis were discussed.

  20. Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression.

    Science.gov (United States)

    Lu, Shan; Xu, Ran; Jia, Jun-Wei; Pang, Jihai; Matsuda, Seiichi P T; Chen, Xiao-Ya

    2002-09-01

    Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern.

  1. Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones.

    Science.gov (United States)

    Xiu, Hao; Nuruzzaman, Mohammed; Guo, Xiangqian; Cao, Hongzhe; Huang, Jingjia; Chen, Xianghui; Wu, Kunlu; Zhang, Ru; Huang, Yuzhao; Luo, Junli; Luo, Zhiyong

    2016-03-04

    Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C₂H₂-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress-inducible genes responding to both salt and hormones.

  2. Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones

    Directory of Open Access Journals (Sweden)

    Hao Xiu

    2016-03-01

    Full Text Available Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C2H2-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA, abscisic acid (ABA, and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress–inducible genes responding to both salt and hormones.

  3. Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

    Science.gov (United States)

    Knight, C; Simmons, D; Gu, T T; Gluhak-Heinrich, J; Pavlin, D; Zeichner-David, M; MacDougall, M

    2001-10-01

    Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

  4. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characte

  5. Post-transcriptional regulation of gene expression in Yersinia species

    Directory of Open Access Journals (Sweden)

    Chelsea A Schiano

    2012-11-01

    Full Text Available Proper regulation of gene expression is required by bacterial pathogens to respond to continually changing environmental conditions and the host response during the infectious process. While transcriptional regulation is perhaps the most well understood form of controlling gene expression, recent studies have demonstrated the importance of post-transcriptional mechanisms of gene regulation that allow for more refined management of the bacterial response to host conditions. Yersinia species of bacteria are known to use various forms of post-transcriptional regulation for control of many virulence-associated genes. These include regulation by cis- and trans-acting small non-coding RNAs, RNA-binding proteins, RNases, and thermoswitches. The effects of these and other regulatory mechanisms on Yersinia physiology can be profound and have been shown to influence type III secretion, motility, biofilm formation, host cell invasion, intracellular survival and replication, and more. In this review, we will discuss these and other post-transcriptional mechanisms and their influence on virulence gene regulation, with a particular emphasis on how these processes influence the virulence of Yersinia in the host.

  6. Using Transcriptomics to Identify Differential Gene Expression in Response to Salinity among Australian Phragmites australis Clones.

    Science.gov (United States)

    Holmes, Gareth D; Hall, Nathan E; Gendall, Anthony R; Boon, Paul I; James, Elizabeth A

    2016-01-01

    Common Reed (Phragmites australis) is a frequent component of inland and coastal wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically diverse range of salt-tolerant P. australis plants. This has prompted an interest in examining the variation in salinity tolerance among clones and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L(-1) TDS) and three from low salinity sites (<6 g L(-1)) were grown in containers irrigated with either fresh (<0.1 g L(-1)) or saline water (16 g L(-1)). An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the higher relative expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of certain clones to

  7. Analysis of gene expression during flowering in apomeiotic mutants of Medicago spp.: cloning of ESTs and candidate genes for 2n eggs.

    Science.gov (United States)

    Barcaccia, G; Varotto, S; Meneghetti, S; Albertini, E; Porceddu, A; Parrini, P; Lucchin, M

    2001-12-01

    Mutants showing features of apomixis have been documented in alfalfa (Medicago sativa L.), a natural outcrossing sexual species. A differential display of mRNAs that combines cDNA-AFLP markers and bulked segregant analysis was carried out with the aim of selecting expressed sequence tags (ESTs) and cloning candidate genes for apomeiosis in mutants of alfalfa characterized by 2n egg formation at high frequencies. The approach enabled us to select either mutant- or wild type-specific transcript derived-fragments and to detect transcriptional changes potentially related to 2n eggs. Sequence alignments of a subset of 40 polymorphic clones showed significant homologies to genes of known function. An EST with identity to a β-tubulin gene, highly expressed in the wild type and poorly expressed in the apomeiotic mutants, and an EST with identity to a Mob1-like gene, qualitatively polymorphic between pre- and post-meiotic stages, were selected as candidate genes for apomeiosis because of their putative roles in the cell cycle. A number of clone-specific primers were designed for performing both 5' and 3' rapid amplification of cDNA ends to obtain the full-length clones. Southern blot hybridization revealed that both clones belong to a multi-gene family with a minimum of three genomic DNA members each. Northern blot hybridization of total RNA samples and in situ hybridization of whole buds enabled the definition of their temporal and spatial expression patterns in reproductive organs. Experimental achievements towards the elucidation of apomeiotic megasporogenesis in alfalfa are presented and discussed.

  8. Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases.

    Science.gov (United States)

    Goyer, Aymeric; Illarionova, Victoria; Roje, Sanja; Fischer, Markus; Bacher, Adelbert; Hanson, Andrew D

    2004-05-01

    Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.

  9. Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus.

    Science.gov (United States)

    Marsolier, M C; Debrosses, G; Hirel, B

    1995-01-01

    A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

  10. Molecular cloning, expression and characterization of a bovine serotonin transporter

    DEFF Research Database (Denmark)

    Mortensen, O V; Kristensen, A S; Rudnick, G

    1999-01-01

    -methylenedioxymethamphetamine (MDMA) was mainly unchanged. RT-PCR amplification of RNA from different tissues demonstrated expression of SERT in placenta, brain stem, bone marrow, kidney, lung, heart, adrenal gland, liver, parathyroid gland, thyroid gland, small intestine and pancreas....

  11. Transcription and Splicing Factor TDP-43: Role in Regulation of Gene Expression in Testis.

    Science.gov (United States)

    Reddi, Prabhakara P

    2017-03-01

    TDP-43 (TAR DNA binding Protein of 43 kD) is a transcription factor and RNA-binding protein with diverse functions. We cloned TDP-43 from the mouse testis in a screen for promoter-binding proteins and showed that it functions as a transcriptional repressor. TDP-43 plays a role in maintaining the precise pattern of spatiotemporal expression of the spermatid-specific Acrv1 gene during spermatogenesis by facilitating RNA polymerase II pausing at the promoter. We also showed that TDP-43 plays a partial role in preventing somatic cell expression of the Acrv1 gene by acting as an insulator-binding protein. Since the discovery of a causative link to several neurodegenerative diseases 10 years ago, TDP-43 has emerged as a protein of major human health relevance. Aberrant posttranslational modifications, nuclear exit, and cytoplasmic aggregate formation contribute to loss of neuronal function in patients. Interestingly, aberrant TDP-43 expression has also been reported in the testis and sperm of infertile men. Finally, our unpublished work shows that TDP-43 is indispensable for sperm formation and male fertility. The potential role of TDP-43 in male germ cells and fertility is discussed in this review. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  12. Hydra constitutively expresses transcripts involved in vertebrate neural differentiation

    Indian Academy of Sciences (India)

    Sandipan Chatterjee; Shweta Lahudkar; N N Godbole; Surendra Ghaskadbi

    2001-06-01

    The diploblastic Hydra is among the most primitive multicellular organisms. Using cross-hybridization with Xenopus probes, noggin-like transcripts were detected in the hypostome and basal disc of adult Hydra (Pelmatohydra oligactis), regions with properties similar to that of the amphibian organizer. This points to the possibility of a close molecular similarity between the Xenopus and Hydra organizers. The constitutive expression of a noggin-like gene in Hydra may be responsible for its regenerative capacity.

  13. Cloning and expression of nucleocapsid protein gene of TGEV HB06 strain

    Institute of Scientific and Technical Information of China (English)

    FAN Jinghui; ZUO Yuzhu; ZHAO Yuelan; LI Tanqing; ZHANG Xiaobo

    2007-01-01

    The nucleocapsid protein gene of transmissible gastroenteritis virus,1 149 bp in length,was amplified by RT-PCR from isolated strain HB06 and cloned into pMD 18T.Sequence comparison with other transmissible gastroenteritis virus (TGEV) strains selected from the Gene Bank revealed that the homology of N gene complete sequence shares more than 97% in nucleotide.N gene was cloned into BamHI and EcoRI multiple cloning sites of the prokaryotic expression vector pET 20 b,and named pETN.After being induced by isopropyl-β-D-thiogalactopyranoside (IPTG),the recombinant nucleocapsid protein was expressed.The result of SDS-PAGE and Western-blot showed that the recombinant nucleocapsid protein was 47 kDa and had strong positive reactions with TGEV-specific antibody.

  14. cDNA cloning, expression and antibacterial activity of lysozyme C in the blue shrimp (Litopenaeus stylirostris)

    Institute of Scientific and Technical Information of China (English)

    Weijun Mai; Chaoqun Hu

    2009-01-01

    The gene coding for lysozyme in blue shrimp (Litopenaeus stylirostris) was cloned, sequenced and expressed in pET-32a vector. The deduced amino acid sequence of F. Merguiensi lysozyme showed 37-93% similarity with the mouse, human, chicken, and tiger prawn counterparts. The lysozyme was purified to homogeneity and observed as a band of approximately 15 kDa in 15% SDS-PAGE. Semi-quantitative RT-PCR analysis demonstrated that mRNA transcripts of lysozyme could be mainly detected in the tissues of haemocytes, gill, gonad and the lymphoid organ of unchallenged shrimps, whereas the expression of lysozyme transcripts was increased in all the tested tissues after the heat-killed Vibrio alginolyticus challenge. The temporal expression of lysozyme mRNA in haemolymph challenged by Micrococcus luteus and V. Alginolyticus was both up-regulated and reached the maximum level at 8 and 16 h post-stimulation, respec-tively, and then dropped back to the original level. Bacteriolytic activity of the lysozyme against different bacterial cultures was deter-mined by the solid phase and turbidimetric assays. The results demonstrated that the lysozyme we obtained was not only against Gram-positive and Gram-negative bacteria but also against shrimp pathogens V. Alginolyticus and V. Parahemolyticus. In addition, the study of the inhibition mechanism revealed that the antibacterial activity of the lysozyme was a result of the bactericidal effect.

  15. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

    Science.gov (United States)

    Ringer, Kerry L; Davis, Edward M; Croteau, Rodney

    2005-03-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene

  16. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N

    Energy Technology Data Exchange (ETDEWEB)

    McAllister, G.; Amara, S.G.; Lerner, M.R. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1988-07-01

    Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules (small nuclear ribonucleoprotein (snRNP) particles). In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified M{sub r} 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as N, is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone ({lambda}rb91) from a rat brain phage {lambda}gt11 cDNA expression library. A longer cDNA clone was obtained by rescreening the library with {lambda}rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.

  17. A circadian neuropeptide PDF in the honeybee, Apis mellifera: cDNA cloning and expression of mRNA.

    Science.gov (United States)

    Sumiyoshi, Miho; Sato, Seiji; Takeda, Yukimasa; Sumida, Kazunori; Koga, Keita; Itoh, Tsunao; Nakagawa, Hiroyuki; Shimohigashi, Yasuyuki; Shimohigashi, Miki

    2011-12-01

    Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5'UTR and 3'UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5'UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5'UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain.

  18. Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

    Institute of Scientific and Technical Information of China (English)

    SONG Jin-zhu; YANG Xiao-xue; WANG Yun; YANG Qian

    2009-01-01

    To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microor-ganisms, the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction (PCR), and was ligated into the yeast expression vector pYES2. The expression vector plasmid was transformed into Saccharomyces cerevisiae H158. Gene expression took place upon induction with 2% galac-tose. The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium. The enzyme activity approaches the peak of 0. 63 U/mL when the culture time is 36 h.

  19. Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

    Directory of Open Access Journals (Sweden)

    Wang Ya-Ping

    2008-03-01

    Full Text Available Abstract Background Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp in length, with a 342 bp open reading frame (ORF encoding a putative protein of 113 amino acids (aa. Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

  20. Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

    Science.gov (United States)

    Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

    2016-12-01

    Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Cloning, characterization, and mRNA expression analysis of novel human fetal cochlear cDNAs.

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Pol, T.J.R. van de; Duijnhoven, G.C.F. van; Hollander, A.I. den; Caat, J. ten; Limpt, V. van; Brunner, H.G.; Kremer, J.M.J.; Cremers, F.P.M.

    2003-01-01

    To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fift

  2. Cloning and expression of a beta-xylosidase from the fungus Fusarium verticillioides

    Science.gov (United States)

    In silico analysis of the genome of Fusarium verticillioides, an endophyte and pathogen of maize, revealed several genes with potential use in the hydrolysis of hemicelluloses. We have cloned a gene, FVEG_05677.3, with putative xylosidase and arabinofuranosidase activities. The gene was expressed ...

  3. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    Science.gov (United States)

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASEStephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

  4. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    Science.gov (United States)

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  5. Cloning, expression, and chromosome mapping of human galectin-7

    DEFF Research Database (Denmark)

    Madsen, Peder; Rasmussen, H H; Flint, T

    1995-01-01

    comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta...

  6. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    Science.gov (United States)

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  7. Molecular cloning and regulation of expression of the genes for initiation factor 3 and two aminoacyl-tRNA synthetases.

    Science.gov (United States)

    Elseviers, D; Gallagher, P; Hoffman, A; Weinberg, B; Schwartz, I

    1982-10-01

    A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184. The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E. coli NP37. pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase. Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells. The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms.

  8. Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

    Directory of Open Access Journals (Sweden)

    Zeinab Soleimanifar

    2016-10-01

    Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

  9. Effects of transcriptional pausing on gene expression dynamics.

    Directory of Open Access Journals (Sweden)

    Tiina Rajala

    2010-03-01

    Full Text Available Stochasticity in gene expression affects many cellular processes and is a source of phenotypic diversity between genetically identical individuals. Events in elongation, particularly RNA polymerase pausing, are a source of this noise. Since the rate and duration of pausing are sequence-dependent, this regulatory mechanism of transcriptional dynamics is evolvable. The dependency of pause propensity on regulatory molecules makes pausing a response mechanism to external stress. Using a delayed stochastic model of bacterial transcription at the single nucleotide level that includes the promoter open complex formation, pausing, arrest, misincorporation and editing, pyrophosphorolysis, and premature termination, we investigate how RNA polymerase pausing affects a gene's transcriptional dynamics and gene networks. We show that pauses' duration and rate of occurrence affect the bursting in RNA production, transcriptional and translational noise, and the transient to reach mean RNA and protein levels. In a genetic repressilator, increasing the pausing rate and the duration of pausing events increases the period length but does not affect the robustness of the periodicity. We conclude that RNA polymerase pausing might be an important evolvable feature of genetic networks.

  10. Using transcriptomics to identify differential gene expression in response to salinity among Australian Phragmites australis clones

    Directory of Open Access Journals (Sweden)

    Gareth Donald Holmes

    2016-04-01

    Full Text Available Common Reed (Phragmites australis is a frequent component of inland, and coastal, wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically-diverse range of salt-tolerant P. australis lineages. This has prompted an interest in examining the variation in salinity tolerance among lineages and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L-1 TDS and three from low salinity sites (<6 g L-1 were grown in containers irrigated with either fresh (<0.1 g L-1 or saline water (16 g L-1. An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the relative higher expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of

  11. Molecular cloning and expression of a larval immunogenic protein from the cattle tick Boophilus annulatus.

    Science.gov (United States)

    Shahein, Yasser Ezzat

    2008-02-15

    A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936bp cloned fragment was sequenced and showed an open reading frame of 516bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.

  12. Cloning and characterization of the nitrate reductase-encoding gene from Chlorella vulgaris: structure and identification of transcription start points and initiator sequences.

    Science.gov (United States)

    Dawson, H N; Pendleton, L C; Solomonson, L P; Cannons, A C

    1996-06-01

    The reduction of nitrate to nitrite catalyzed by nitrate reductase (NR) is considered to be the rate-limiting and regulated step of nitrate assimilation, a major metabolic pathway occurring in a wide range of organisms which in turn supply the nutritional nitrogen requirements for other forms of life. Chlorella vulgaris NR mRNA levels are very responsive to changes in nitrogen source. In the presence of ammonia as the sole nitrogen source, under repressed conditions, NR mRNA is undetectable. Under inducing conditions, the removal of ammonia and addition of nitrate, rapid NR mRNA synthesis occurs. We are studying the elements involved in regulating the expression of this important gene. Two overlapping genomic clones (NRS1 and NR5') were isolated from a cosmid library. The two clones were sequenced and their sequences were aligned with that of a full-length NR cDNA. The gene is approximately 8 kb long and consists of 19 exons and 18 introns. Unlike NR isolated from other species, the exons which code for the functional domains of C. vulgaris are separated by introns. Two transcription start points (tsp) were identified and each is surrounded by potential initiator sequences. No TATA, CAAT or GC-rich promoter elements were located. A time course of NR induction revealed that while transcription initiation from one tsp remains at a constant level from the point of induction through steady state, the level of initiation from another tsp is high upon induction, but decreases as steady state is attained.

  13. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  14. Cloning and Expression of Helicobacter pylori HpaA Gene

    Directory of Open Access Journals (Sweden)

    Moein Farshchian

    2009-01-01

    Full Text Available Objective: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, gastric adenocarcinomaand gastric mucosa-associated lymphoid tissue (MALT lymphoma. Antibiotictherapies do not protect from potential re-infection and have a risk for development of drugresistance. Therefore, prophylactic vaccine mediated protection against H. pylori is an attractiveclinical interest. H. pylori adhesin A (HpaA is a conserved surface lipoprotein and playsimportant roles in the pathogenesis of infection. In this study the recombinant protein (rHpaAwas over-expressed in E.coli.Materials and Methods: The hpaA gene was amplified by PCR. Prokaryote expression vectorpET28a-hpaA was constructed, and used to transform E.coli BL21DE3. The expressionof recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot wereused to determine immunoreactivity of rHpaA by a rabbit polyclonal antibodies against wholecell of H. pylori.Results: The hpaA gene nucleotide sequence in the recombinant plasmid vector of pET-28-a-hpaA was consistent with that of H.pylori hpaA as published in the GenBank. SDS-PAGEdemonstrated that the constructed prokaryotic expression efficiently produced rHpaA at the1.5 mmol/L of IPTG. HpaA fusion protein was able to react with the rabbit polyclonal antibodyagainst whole cells of H. pylori.Conclusion: A prokaryotic expression system pET-28a-hpaA-BL21 with high efficiency of H.pylori hpaA gene was successfully established and the HpaA fusion protein showed satisfactoryimmunoreactivity. These results indicate that production of a specific recombinant proteinis an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

  15. Calcium regulates caveolin-1 expression at the transcriptional level

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Li, Yan [Experimental Animal Center, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Liu, Dan; Zhang, Xue-Cheng [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Sato, Toshinori [Department of Biosciences and Informatics, Keio University, Hiyoshi, Yokohama 223-8522 (Japan); Yamagata, Sadako [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Yamagata, Tatsuya, E-mail: tcyamagata@gmail.com [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China)

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  16. Differential gene expression regulated by oscillatory transcription factors.

    Directory of Open Access Journals (Sweden)

    Luca Cerone

    Full Text Available Cells respond to changes in the internal and external environment by a complex regulatory system whose end-point is the activation of transcription factors controlling the expression of a pool of ad-hoc genes. Recent experiments have shown that certain stimuli may trigger oscillations in the concentration of transcription factors such as NF-κB and p53 influencing the final outcome of the genetic response. In this study we investigate the role of oscillations in the case of three different well known gene regulatory mechanisms using mathematical models based on ordinary differential equations and numerical simulations. We considered the cases of direct regulation, two-step regulation and feed-forward loops, and characterized their response to oscillatory input signals both analytically and numerically. We show that in the case of indirect two-step regulation the expression of genes can be turned on or off in a frequency dependent manner, and that feed-forward loops are also able to selectively respond to the temporal profile of oscillating transcription factors.

  17. Transcript expression profiling for adventitious roots of Panax ginseng Meyer.

    Science.gov (United States)

    Subramaniyam, Sathiyamoorthy; Mathiyalagan, Ramya; Natarajan, Sathishkumar; Kim, Yu-Jin; Jang, Moon-Gi; Park, Jun-Hyung; Yang, Deok Chun

    2014-08-01

    Panax ginseng Meyer is one of the major medicinal plants in oriental countries belonging to the Araliaceae family which are the primary source for ginsenosides. However, very few genes were characterized for ginsenoside pathway, due to the limited genome information. Through this study, we obtained a comprehensive transcriptome from adventitious roots, which were treated with methyl jasmonic acids for different time points (control, 2h, 6h, 12h, and 24h) and sequenced by RNA 454 pyrosequencing technology. Reference transcriptome 39,304,529 (0.04GB) was obtained from 5,724,987,880 bases (5.7GB) of 22 libraries by de novo assembly and 35,266 (58.5%) transcripts were annotated with biological schemas (GO and KEGG). The digital gene expression patterns were obtained from in vitro grown adventitious root sequences which mapped to reference, from that, 3813 (6.3%) unique transcripts were involved in ≥2 fold up and downregulations. Finally, candidates for ginsenoside pathway genes were predicted from observed expression patterns. Among them, 30 transcription factors, 20 cytochromes, and 11 glycosyl transferases were predicted as ginsenoside candidates. These data can remarkably expand the existing transcriptome resources of Panax, especially to predict existence of gene networks in P. ginseng. The entity of the data provides a valuable platform to reveal more on secondary metabolism and abiotic stresses from P. ginseng in vitro grown adventitious roots. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Neurotoxocarosis alters myelin protein gene transcription and expression.

    Science.gov (United States)

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  19. Cloning and expression of the HpaI restriction-modification genes.

    OpenAIRE

    Ito, H; Shimato, H; Sadaoka, A; Kotani, H; Kimizuka, F; Kato, I

    1992-01-01

    The genes from Haemophilus parainfluenzae encoding the HpaI restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the HpaI endonuclease (R.HpaI) to have 254 amino acid residues (Mr 29,630) and the HpaI methyltransferase (M.HpaI) to have 314 amino acid residues (37,390). The R.HpaI and M.HpaI genes overlapped by 16 base pairs on the chromosomal DNA. The genes had the same orientation. The clone, named E. coli HB101-HPA2, overproduced...

  20. Molecular cloning, construction of the expression vector and detection of transcription activity of vasa regulatory regions in half-smooth tongue sole (Cynoglossus semilaevis)%半滑舌鳎vasa调控区的克隆、表达载体构建及其驱动活性检测

    Institute of Scientific and Technical Information of China (English)

    黄进强; 陈松林; 邵长伟; 刘洋; 林帆; 李亚亚; 王娜

    2015-01-01

    . However, the function of vasa gene (Csvasa) regulatory regions in tongue sole remains unknown. We aimed to isolate the regulatory regions of the Csvasa gene, examine its transcriptional activity in transgenic medaka fish, and identify the conditions required for manipulation of PGCs. Two DNA fragments, including a 5166-bp 5′region and a 1655-bp 3′region, were obtained by genome walking and polymerase chain reaction of genomic DNA. The 5′ region of Csvasa contains the major promoter region, exon 1, intron 1 and exon 2. The transcription initiation site and a putative TATA box were identified and potential transcription factor binding sites were bioinformatically analyzed. Several potential transcription factor binding sites that may perform important function relating to gene transcription, such as SRY, Oct-1, Sox-5, CREB, GATA, AP-1, C/EBP, Sp-1, c-Myc, HNF, NKX2-5, and V-Myb were predicted. Based on the above information, the Csvasa regulatory regions were inserted into a pEGFP-N3 vector lacking the CMV promoter. This vector expresses the green fluorescent protein(GFP). The recombinant pCsvasa-GFP-T plasmid was injected into medaka fertilized eggs by microinjection. Microfluoroscopy revealed that the level of GFP expression was 81% in the injected embryos. GFP-fluorescent medaka embryos were cul-tured to adult fish and the integration efficiency of pCsvasa-GFP-T was 11.5% in these samples. These results show that the Csvasa promoter can efficiently drive GFP expression in medaka embryos, and lay the foundations for identifying, labeling and tracing PGCs, investigating germ cell biology, and manipulating the sex of half-smooth tongue sole.

  1. Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

    Directory of Open Access Journals (Sweden)

    Stahl Stefanie

    2002-02-01

    Full Text Available Abstract Background Mucolipidosis type IV (MLIV is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. Results The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. Conclusions Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

  2. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

    Directory of Open Access Journals (Sweden)

    Hui eYuan

    2015-09-01

    Full Text Available Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3pLysS. In summary, this recombinant expression library of Pyrococcus furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.

  3. Cloning and Prokaryotic Expression of Transcriptional Co-activator Gene of Clonorchis sinensis and Functional Analysis of the Expressed Protein%华支睾吸虫成虫转录辅激活因子基因在原核细胞中的克隆、表达及其生物功能分析

    Institute of Scientific and Technical Information of China (English)

    张咏莉; 余新炳; 吴德; 吴忠道; 毕惠祥

    2005-01-01

    目的构建华支睾吸虫成虫转录辅激活因子(transcriptional coactivator, TC)基因(TC基因)原核重组质粒, 进行原核表达、鉴定及生物功能分析. 方法根据TC基因已知序列设计1 对引物, 从华支睾吸虫成虫 cDNA 文库中扩增TC基因片段;将目的基因进行聚合酶链反应(PCR), 其产物和空质粒pGEX-4T-1、pET30a(+)同时用限制性内切酶 BamHⅠ、 SalⅠ双酶切, 纯化回收后连接并转化大肠埃希菌( E.coli BL21).将构建的重组质粒pGEX-4T-1-TC和pET30a(+)-TC分别经双酶切、PCR及测序鉴定后在大肠埃希菌BL21中诱导表达.用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)鉴定其表达效果, 用亲和层析法纯化重组质粒pET30a(+)-TC表达产生的组氨酸重组蛋白(His-TC). 结果构建了TC基因原核重组质粒pGEX-4T-1-TC和pET30a(+)-TC.SDS-PAGE 结果表明TC基因在大肠埃希菌BL21 系统获得高效表达, 其重组蛋白分子量与理论值相符. Western blotting 结果表明重组质粒pGEX-4T-1-TC表达的谷胱甘肽硫转移酶(GST)重组蛋白(GST-TC)可被华支睾吸虫免疫兔血清识别, 具有免疫反应性. 纯化的TC基因的组氨酸(His)重组蛋白(His-TC)经SDS-PAGE显示单一条带. 结论通过生物信息学方法由华支睾吸虫成虫cDNA文库中筛选出TC基因, 并成功予以克隆、表达及纯化.

  4. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  5. Cloning and expression of Xenopus Prickle, an orthologue of a Drosophila planar cell polarity gene.

    Science.gov (United States)

    Wallingford, John B; Goto, Toshiyasu; Keller, Ray; Harland, Richard M

    2002-08-01

    We have cloned Xenopus orthologues of the Drosophila planar cell polarity (PCP) gene Prickle. Xenopus Prickle (XPk) is expressed in tissues at the dorsal midline during gastrulation and early neurulation. XPk is later expressed in a segmental pattern in the presomitic mesoderm and then in recently formed somites. XPk is also expressed in the tailbud, pronephric duct, retina, and the otic vesicle. The complex expression pattern of XPk suggests that PCP signaling is used in a diverse array of developmental processes in vertebrate embryos.

  6. Comparative transcriptional analysis reveals differential gene expression between Sand Daffodil tissues.

    Science.gov (United States)

    De Felice, Bruna; Manfellotto, Francesco; D'Alessandro, Raffaella; De Castro, Olga; Di Maio, Antonietta; Trifuoggi, Marco

    2013-12-01

    Sand Daffodil (Pancratium maritimum) is a world-wide endangered Amayllidaceae species and represents an important anti-cancer medicinal resource due to alkaloids production. Despite its increasing pharmaceutical importance, there are not molecular resources that can be utilized toward improving genetic traits. In our research, the suppression subtractive hybridization (SSH) method conducted to generate large-scale expressed sequence tags (EST), was designed to identify gene candidates related to the morphological and physiological differences between the two tissues, leaves and bulbs, since lycorine, the main anti-cancer compound, is there synthesized. We focused on identification of transcripts in different tissues from Sand Daffodil using PCR-based suppression SSH to identify genes involved in global pathway control. Sequencing of 2,000 differentially screened clones from the SSH libraries resulted in 136 unigenes. Functional annotation and gene ontology analysis of up-regulated EST libraries showed several known biosynthetic genes and novel transcripts that may be involved in signaling, cellular transport, or metabolism. Real time RT-PCR analysis of a set of 8 candidate genes further confirmed the differential gene expression.

  7. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

    2016-01-01

    TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator...... translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application...... of contact uncoupling and TGFβ. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses...

  8. Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2.

    Science.gov (United States)

    Iversen, Annette; Cazzamali, Giuseppe; Williamson, Michael; Hauser, Frank; Grimmelikhuijzen, Cornelis J P

    2002-12-13

    The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.

  9. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  10. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  11. Molecular cloning and its expression of trachealess gene (As-trh) during development in brine shrimp, Artemia sinica.

    Science.gov (United States)

    Wang, Jia-Qing; Hou, Lin; Yi, Nan; Zhang, Riu-Feng; Zou, Xiang-Yang; Xiao, Qin; Guo, Ran

    2012-02-01

    Basic helix-loop-helix-PAS (bHLH-PAS) family transcription factors are implicated in multiple developmental and physiological regulatory processes. Herein, a full-length cDNA encoding a bHLH-PAS domain transcription factor trachealess gene (designated as As-trh) was cloned and characterized from brine shrimp (Artemia sinica) for the first time. The full-length cDNA of As-trh was 2,698 bp with a 2,319 bp open reading frame encoding a deduced protein of 772 amino acid polypeptide with a calculated molecular mass of 86.02 kDa and an isoelectric point of 5.87. Sequence alignment revealed that As-trh had high homology with other species trh gene, including the D-trh gene in Drosophila melanogaster and Bm-trh in Bombyx mori. The early and persistent expression of As-trh in the naupliar stages by whole-mount embryonic in situ hybridization and immunohistochemistry suggest that As-trh functions very early in the salt gland and may be required continuously in this tissue. Later in development, expression of As-trh begins to decrease and disappear in salt gland of the older nauplius and appears in the thoracic epipods of the sub-adult Artemia. These results indicated that As-trh might play an important role in osmoregulatiory organ development from the larvae stages through adult stages.

  12. Molecular cloning of two structure variants of crustacean hyperglycemic hormone (CHH) from the swimming crab (Portunus trituberculatus), and their gene expression during molting and ovarian development.

    Science.gov (United States)

    Xie, Xi; Zhu, Dongfa; Yang, Jifen; Qiu, Xier; Cui, Xiaoyu; Tang, Jie

    2014-12-01

    Two full-length cDNA (Pt-CHH1 and Pt-CHH2) sequences encoding crustacean hyperglycemic hormone (CHH) were cloned from tissues of the swimming crab (Portunus trituberculatus) using RACE. Pt-CHH1 was cloned from eyestalk, whereas Pt-CHH2 was cloned from thoracic ganglia. Sequence and structure analyses of Pt-CHH1 and Pt-CHH2 suggest that they may be generated from alternative splicing. Tissue distribution showed that transcript of Pt-CHH1 was only detected in eyestalk, while transcript of Pt-CHH2 was observed in several extra-eyestalk tissues. The transcript levels of Pt-CHH1 and Pt-CHH2 during molting and ovarian development were determined using qPCR. In molting process, level of Pt-CHH1 in eyestalk increased from stage A (postmolt), and to significant higher at stage C (intermolt), then decreased during premolt (D0-D4). In ovarian development, level of Pt-CHH1 in eyestalk decreased from previtellogenic stage (II), and to significant lower at mature stage (IV). The expression patterns of Pt-CHH2 in thoracic ganglia and Y-organ were distinct from that of Pt-CHH1 in eyestalk. The combined results suggest that Pt-CHH1 may be involved in inhibition of molting and ovarian development, whereas Pt-CHH2 may have other physiological functions.

  13. In silico cloning and characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in Carica papaya.

    Science.gov (United States)

    Idrovo Espín, Fabio Marcelo; Peraza-Echeverria, Santy; Fuentes, Gabriela; Santamaría, Jorge M

    2012-05-01

    The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya.

  14. Molecular cloning, characterization and expression analysis of TGF-β and receptor genes in the woodchuck model.

    Science.gov (United States)

    Wang, Lu; Wang, Junzhong; Liu, Yana; Wang, Baoju; Yang, Shangqing; Yu, Qing; Roggendorf, Michael; Lu, Mengji; Liu, Jia; Yang, Dongliang

    2016-12-20

    Transforming growth factor beta (TGF-β) is an important cytokine with pleiotropic regulatory functions in the immune system and in the responses against viral infections. TGF-β acts on a variety of immune cells through the cell surface TGF-β receptor (University of Duisburg-EssenTGFBR). The woodchuck has been used as a biomedical model for studies of obesity and energy balance, endocrine and metabolic function, cardiovascular, cerebrovascular and neoplastic disease. Woodchucks infected with woodchuck hepatitis virus (WHV) represent an informative animal model to study hepatitis B virus (HBV) infection. In this study, the cDNA sequences of woodchuck TGF-β1, TGF-β2, TGFBR1 and TGFBR2 were cloned, sequenced and characterized. The full-length TGFBR1 cDNA sequence consisted of 1305bp coding sequence (CDS) that encoded 434 amino acids with a molecular weight of 48.9kDa. The phylogenetic tree analysis revealed that the woodchuck TGF-β family genes had a closer genetic relationship with Ictidomys tridecemlineatus. One antibody with cross-reactivity to woodchuck TGFBR1 was identified by flow cytometry. Moreover, the expression of these genes were analyzed at the transcriptional level. The quantitative PCR analysis showed that the TGF-β family transcripts were constitutively expressed in many tissues tested. Altered expression levels of the TGF-β family transcripts in the liver of WHV infected woodchucks were observed. These results serve as a foundation for further insight into the role of the TGF-β family in viral hepatitis in woodchuck model. Our work also possesses the potential value for characterizing the TGF-β family in other related diseases, such as obesity-related diseases, metabolic disorder, cardiovascular disease and cancer.

  15. Molecular cloning, in vitro expression and bioactivity of quail BAFF.

    Science.gov (United States)

    Chen, Chuan-mei; Ren, Wen-hua; Yang, Guang; Zhang, Chuan-song; Zhang, Shuang-quan

    2009-07-15

    B cell activating factor (BAFF), belonging to the TNF (tumor necrosis factor) family, is critical for B cell survival and maturation. In the present study, a quail BAFF cDNA, named qBAFF, was amplified from quail spleen by RT-PCR and RACE (rapid amplification of cDNA ends) strategies. The open reading frame (ORF) of qBAFF cDNA encodes a protein consisting of 288-amino acid. The deduced amino acid sequence contains a predicted transmembrane domain and a putative furin protease cleavage site like other identified BAFF homologues. The qBAFF shows 96, 93, 93, 53 and 51% amino acid sequence identity with chicken (cBAFF), goose (gBAFF), duck (dBAFF), human (hBAFF) and mouse BAFF (mBAFF), respectively, with the functional soluble parts of qBAFF is 98, 99, 98, 78 and 71%, respectively. RT-PCR showed that BAFF is expressed in many tissues in the quail, including bursa, spleen, liver, brain, heart, intestine, kidney, thymus and muscle. Recombinant soluble qBAFF (qsBAFF) fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and its molecular weight of approximately 19kDa was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified qsBAFF was able to promote the survival of quail bursa B cells. Our results suggest that qBAFF plays an important role in survival of quail B cells cultured in vitro.

  16. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F. William (Stony Brook, NY); Dubendorff, John W. (Sound Beach, NY)

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  17. Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F. William (Stony Brook, NY); Dubendorff, John W. (Sound Beach, NY)

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  18. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F.W.; Dubendorff, J.W.

    1998-10-20

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  19. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F.W.; Dubendorff, J.W.

    1998-11-03

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  20. Roots Stimulate Expression of Decomposition Transcripts in the Soil Microbiome

    Science.gov (United States)

    Nuccio, E. E.; Karaoz, U.; Zhou, J.; Brodie, E.; Firestone, M.; Pett-Ridge, J.

    2016-12-01

    The soil surrounding plant roots, the rhizosphere, has long been recognized as a zone of great functional importance in terrestrial ecosystems. Rhizosphere microorganisms can affect the breakdown of plant tissues and root litter, and can accelerate the decomposition of detrital plant biomass, which is a process commonly described as "priming." However, the molecular mechanisms underlying rhizosphere C cycling are poorly understood, and the carbohydrate and lignolytic gene transcripts mediating the decomposition of root litter in soil are largely unidentified. We hypothesized that root exudates stimulate the expression of enzymes that are involved in decomposition of macromolecular C compounds. To assess how the abundance and diversity of decomposition enzymes differs in the rhizosphere relative to the surrounding bulk soil, we sequenced the community gene expression (metatranscriptomes) and single cell genomes of rhizosphere and bulk soil associated with wild oat (Avena fatua) over time (3, 6, 12, and 22 days). To isolate roots of a defined age in a mature plant, we used microcosms with a transparent experimental sidecar to track roots as they grew. Our results showed that a large number of C decomposition enzymes were more highly expressed in the rhizosphere compared to bulk soil, and that overall, transcripts tended to be elevated in younger roots than older roots. Genes relevant to aromatic C breakdown (nitroreductase, 4-hydroxybenzoate degradation, pectin methylesterase) and organic N cycling (ammonification) were elevated in the rhizosphere. This work identifies the potential molecular mechanisms that underpin priming in rhizosphere soil.

  1. Cloning and expression of the ureC geneo f H.pylori

    Institute of Scientific and Technical Information of China (English)

    JunRongXu; LiZhang; XiaoJunYan; DaXiangCui; HongJiang; FengChanHan

    2001-01-01

    AIM: To express Hp ureC gene in E.coli, a fragment from Hp ureC was amplified by the polymerase chain reaction and was cloned into E.coli expressing plasmid vector--- pBV220. This study lay the foundation for further studies of its functions. METHODS : An 1173bp gene fragment was amplified by the polymerase chain reaction. The template DNA was purified from Hp clinical strain. The gene fragment was digested by BamH I and EcoR I and cloned into pGEM-3Zf (-) plasmid. Its sequence was determined by autosequencing instrument from two directions. Then the gene fragment was cloned into pBV220. Subsequently the DH5 a was transformed by pBV220/ureC and was induced by heat when temperature reached 42"C. The form of expressed protein was analyzed to learn that in which form the recombinant protein was expressed. The thin layer chromatogram was used to scan thegel to learn that the quantity of the expressed protein. Antigenicity of the expressed protein wasconfirmed by ELISA and DIGFA. RESULTS z 95% of the DNA sequence we got is the same as that ofHp strain M60398. A Mr 44000 recombinant protein was induced by heat when temperature reached 42℃. The molecular mass of the expressed ureC gene is coincide with expected. The expressed proteinaccounts for 24.8% of the whole protein of the host bactria. Although most of the recombinant proteinsare inclusion bodies in host bactria after translation, there does exist certain quantity of solvable proteins in the supernatant of bacteria lysate. CONCLUSION:Recombinant H.pylori urease C protein is expressed in E.coli and the expressed protein has the antigenicity ofureC. Our study lays the foundation for further studies of the functions of lip ureC.

  2. Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

    Science.gov (United States)

    Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

    2015-01-01

    Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

  3. Molecular cloning and expression analysis of fushi tarazu factor 1 in the brain of air-breathing catfish, Clarias gariepinus.

    Directory of Open Access Journals (Sweden)

    Parikipandla Sridevi

    Full Text Available BACKGROUND: Fushi tarazu factor 1 (FTZ-F1 encodes an orphan nuclear receptor belonging to the nuclear receptor family 5A (NR5A which includes adrenal 4-binding protein or steroidogenic factor-1 (Ad4BP/SF-1 and liver receptor homologue 1 (LRH-1 and plays a pivotal role in the regulation of aromatases. METHODOLOGY/PRINCIPAL FINDINGS: Present study was aimed to understand the importance of FTZ-F1 in relation to brain aromatase (cyp19a1b during development, recrudescence and after human chorionic gonadotropin (hCG induction. Initially, we cloned FTZ-F1 from the brain of air-breathing catfish, Clarias gariepinus through degenerate primer RT-PCR and RACE. Its sequence analysis revealed high homology with other NR5A1 group members Ad4BP/SF-1 and LRH-1, and also analogous to the spatial expression pattern of the latter. In order to draw functional correlation of cyp19a1b and FTZ-F1, we analyzed the expression pattern of the latter in brain during gonadal ontogeny, which revealed early expression during gonadal differentiation. The tissue distribution both at transcript and protein levels revealed its prominent expression in brain along with liver, kidney and testis. The expression pattern of brain FTZ-F1 during reproductive cycle and after hCG induction, in vivo was analogous to that of cyp19a1b shown in our earlier study indicating its involvement in recrudescence. CONCLUSIONS/SIGNIFICANCE: Based on our previous results on cyp19a1b and the present data, it is plausible to implicate potential roles for brain FTZ-F1 in ovarian differentiation and recrudescence process probably through regulation of cyp19a1b in teleosts. Nevertheless, these interactions would require primary coordinated response from ovarian aromatase and its related transcription factors.

  4. Isolation, Cloning, Expression and Purification of Recombinant RhD Antigen from Cord Blood

    Directory of Open Access Journals (Sweden)

    M Habibi Roudkenar

    2008-09-01

    Full Text Available "nBackground: Rh (Rhesus is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blod, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study."nMethods: Total RNAs were extracted from cord blood (O+.  The quality of RNA was determined by electrophoresis. In or­der to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The iso­lated RhD gene was   cloned to pUC18 vector and transformed to DH5α. The confirmed construct was sub cloned into expres­sion vector, pBADgIII/A, and expressed in Top10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available hu­man anti-RhD polyclonal   antibody with   peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. "nRe­sults: RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane."nConclusion: The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applica­ble for production anti- D antibody in an animal model.

  5. Transcriptional, post-transcriptional and post-translational regulations of gene expression during leaf polarity formation

    Institute of Scientific and Technical Information of China (English)

    Lin Xu; Li Yang; Hai Huang

    2007-01-01

    Leaf morphogenesis requires the establishment of adaxial-abaxial polarity after primordium initiation from the shoot apical meristem (SAM). Several families of transcription factors are known to play critical roles in promoting adaxial or abaxial leaf fate. Recently, post-transcriptional gene silencing pathways have been shown to regulate the establishment of leaf polarity, providing novel and exciting insights into leaf development. For example, microRNAs (miR165/166)and a trans-acting siRNA (TAS3-derived tasiR-ARF) have been shown to repress the expression of several key transcription factor genes. In addition, yet another level of regulation, post-translational regulation, has been revealed recently by studies on the role of the 26S proteasome in leaf polarity. Although our understanding regarding the molecular mechanisms underlying establishment of adaxial-abaxial polarity has greatly improved, there is still much that remains elusive.This review aims to discuss recent progress, as well as the remaining questions, regarding the molecular mechanisms underlying leaf polarity formation.

  6. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    ). The main goal was to purify full-length hTPH1. Based on earlier results, hTPH1 was purified using detergent in the purification methods. After incubation of the hTPH1 sample with 0.1 % of n-dodecyl-β-D-maltopyranoside (DDM) the protein binds to the anion exchange column and elutes over a large area...... in the anion exchange, indicating that the protein still exists in different oligomer forms. This was also observed in the gel filtration. Variants of both hTPH1 and hTPH2 containing the regulatory domain or parts of it were constructed and tested for expression in Escherichia coli as well as solubility...... was determined and compared with parameters of chTPH2. Large differences were observed between the two isoforms and tryptophan inhibition was observed for chTPH1 but not for chTPH2. Mass spectrometric analysis of chTPH1 shows that the sample contains two species: chTPH1 and another species, which could...

  7. Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages.

    Science.gov (United States)

    Liu, Kuan-Fu; Yeh, Maw-Sheng; Kou, Guang-Hsiung; Cheng, Winton; Lo, Chu-Fang

    2010-09-01

    Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

  8. Identification and cloning of the second type transglutaminase from Litopenaeus vannamei, and its transcription following pathogen infection and in relation to the haemolymph coagulation.

    Science.gov (United States)

    Yeh, Maw-Sheng; Tsai, Wan-Lin; Cheng, Winton

    2013-11-01

    Complementary (c)DNA encoding transglutaminaseII (TGII) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134), tiger shrimp, Penaeus monodon (AAV49005; AAO33455), kuruma shrimp, Marsupenaeus japonicus (BAD36808) and Pacifastacus leniusculus (AAK69205) TG. The 2405-bp cDNA contained an open reading frame (ORF) of 2292 bp, a 31-bp 5'-untranslated region (UTR), and an 82-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (764 aa) was 85.9 kDa with an estimated pI of 5.32. The L. vannamei TGII (abbreviated LvTGII) contains a typical TG-like homologue, two putative integrin binding motif (RGD and KGD), and five calcium-binding sites; three catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two groups, STGI and STGII, and LvTGII is more closely related to STGII than to STGI. LvTGII mRNA was detected in all tested tissues of L. vannamei, and was highly expressed in haemocytes. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant increase of LvTGI and LvTGII mRNA expression at 6 h followed by a notable decrease at 24 h in LvTGI and a continually increase in LvTGII indicating a complementary effect, which implied that both LvTGs involved in the immune response of shrimp, and LvTGII was more important in the later defense response. The gene silencing of LvTGII in shrimp significantly decreased LvTGII expression and TG activity of haemocytes, and significantly increased clotting time of haemolymph, suggests that the cloned LvTGII is a clotting enzyme involved in haemolymph coagulation of L. vannamei. In conclusion, the cloned LvTGII is a clotting enzyme

  9. Model transcriptional networks with continuously varying expression levels

    Directory of Open Access Journals (Sweden)

    Carneiro Mauricio O

    2011-12-01

    Full Text Available Abstract Background At a time when genomes are being sequenced by the hundreds, much attention has shifted from identifying genes and phenotypes to understanding the networks of interactions among genes. We developed a gene network developmental model expanding on previous models of transcription regulatory networks. In our model, each network is described by a matrix representing the interactions between transcription factors, and a vector of continuous values representing the transcription factor expression in an individual. Results In this work we used the gene network model to look at the impact of mating as well as insertions and deletions of genes in the evolution of complexity of these networks. We found that the natural process of diploid mating increases the likelihood of maintaining complexity, especially in higher order networks (more than 10 genes. We also show that gene insertion is a very efficient way to add more genes to a network as it provides a much higher chance of developmental stability. Conclusions The continuous model affords a more complete view of the evolution of interacting genes. The notion of a continuous output vector also incorporates the reality of gene networks and graded concentrations of gene products.

  10. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Induction and differential expression of beta-1,3-glucanase mRNAs in tolerant and susceptible Hevea clones in response to infection by Phytophthora meadii.

    Science.gov (United States)

    Thanseem, I; Joseph, A; Thulaseedharan, A

    2005-11-01

    Most cultivated rubber tree (Hevea brasiliensis Willd. ex A. Juss.) clones in India are susceptible to abnormal leaf fall disease (ALF), which is caused by various Phytophthora species and results in yield losses of up to 40%. Because the conventional breeding programs for this perennial tree crop are complex and time consuming, we attempted to find a molecular solution to increase the tolerance of rubber trees to ALF. The expression patterns of the gene coding for the pathogenesis-related beta-1,3-glucanase (beta-glu) enzyme in a tolerant (RRII 105) and a highly susceptible (RRIM 600) clone of rubber tree were examined, following infection with ALF-causing Phytophthora meadii McRae. Infected leaf samples were collected at different times after inoculation, and RNA was extracted and subjected to Northern blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). On hybridization with a 1.25 kb beta-glu probe, Northern blots showed a marked increase in beta-glu transcript levels in both clones 48 h after inoculation. However, compared with the susceptible RRIM 600 clone, the tolerant RRII 105 clone had a higher rate of increase and a more prolonged induction, with beta-glu transcript levels remaining high for 4 days after inoculation. In RRIM 600, the mRNA levels decreased significantly 48 h after inoculation. On re-hybridization with an 18S rRNA probe, uniform signals were detected in all the lanes, indicating that an equal amount of total RNA was present in all samples. Similar results were obtained in relative quantitative RT-PCR experiments with the housekeeping actin gene as an internal control. Thus, although induction of the beta-glu gene occurred in both tolerant and susceptible clones, the predominant difference between clones was in the intensity and duration of the response. The tolerance of clone RRII 105 may be associated with the prolonged expression of the gene following infection. The antifungal activity of these hydrolase

  12. Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells

    Directory of Open Access Journals (Sweden)

    Koike Chieko

    2006-03-01

    Full Text Available Abstract Background Sterile alpha motif (SAM domains are ~70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein. Results mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3–6 (P3-6, when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. Conclusion We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis

  13. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    Directory of Open Access Journals (Sweden)

    Anne Mathilde Lund

    Full Text Available A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

  14. Molecular cloning and expression of a new gene, GON-SJTU1 in the rat testis

    Directory of Open Access Journals (Sweden)

    Tian Geng G

    2010-05-01

    Full Text Available Abstract Background Spermatogenesis is a complex process involving cell development, differentiation and apoptosis. This process is governed by a series of genes whose expressions are highly regulated. Male infertility can be attributed to multiple genetic defects or alterations that are related to spermatogenesis. The discovery, cloning and further functional study of genes related to spermatogenesis is of great importance to the elucidation of the molecular mechanism of spermatogenesis. It is also physiologically and pathologically significant to the therapy of male infertility. Methods GON-SJTU1 was identified and cloned from rat testis by cDNA library screening and 3'-and 5'-RACE. The products of GON-SJTU1 were assessed by Northern and Western blotting. The expression of GON-SJTU1 was also examined by In situ hybridization and immunohistochemistry. Results Here we identified and cloned a new gene, GON-SJTU1, with the biological process of spermatogenesis. GON-SJTU1 is highly expressed in the testis from day 1 to 15 and then decreased, suggesting that GON-SJTU1 might be a time-related gene and involved in the early stage of spermatogenesis. And the expression of GON-SJTU1 in the testis occurred in some male germ cells, particularly in gonocytes and spermatogonial stem cells. Conclusion GON-SJTU1 may play a role in the biological process of spermatogenesis.

  15. Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

    Directory of Open Access Journals (Sweden)

    Ali Dina

    2016-01-01

    Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

  16. Molecular cloning and expression of rat prostaglandin E receptor EP2 subtype.

    Science.gov (United States)

    Sando, T; Usui, T; Tanaka, I; Mori, K; Sasaki, Y; Fukuda, Y; Namba, T; Sugimoto, Y; Ichikawa, A; Narumiya, S

    1994-05-16

    A cDNA clone encoding the rat prostaglandin (PG) E receptor EP2 subtype was cloned from a rat lung cDNA library. It encodes 488 amino acid residues with putative seven-transmembrane domains. Specific binding of [3H]PGE2 was found in COS-7 cells transfected with the cDNA and was displaced with unlabeled prostaglandins in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the significant expression being observed in the thymus, lung, spleen, heart stomach, and pancreas.

  17. cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Molecular cloning of the porcine leukemia inhibitor factor(pLIF) has not been reported. A full-length cDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLIFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.

  18. Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Collins, M T; Høiby, N;

    1989-01-01

    To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly...... will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function....

  19. CLONING SEGMENT SPIKE PROTEIN GENE OF SARS-COV AND ITS EXPRESSION IN ESCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    刘中华; 许文波; 毛乃颖; 张燕; 朱贞; 崔爱利; 杨建国; 胡海涛

    2004-01-01

    Objective Expressing and purifying the segment of SARS-CoV spike protein in E.Coli. Methods The target gene was obtained by RT-PCR. The PCR product was cloned into pEGM- T Easy Vector, sequencing and double restriction digestion ( BamHⅠ,PstⅠ) were performed. The target gene was subcloned into PQE30 expression vector. The gene was expressed in the E.coli strain M15 cells induced by IPTG. The protein was purified with a nickel HiTrap chelating metal affinity column. Results The recombinant expression plasmid was successfully constructed and the protein was well expressed in E. coli strain M15 cells. The ideal pure protein was obtained by purification. Western blotting analysis suggested the protein could act with the convalescent sera of lab confirmed SARS patients. Conclusion The segment of SARS-CoV spike protein was well expressed and purified, and can be applied in diagnosis and immunological research of SARS.

  20. Regulation of tissue-specific expression of alternative peripheral myelin protein-22 (PMP22) gene transcripts by two promoters

    Energy Technology Data Exchange (ETDEWEB)

    Patel, P.I.; Schoener-Scott, R.; Lupski, J.R. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Mutations affecting the peripheral myelin protein-22 (PMP22) gene have been shown to be associated with inherited peripheral neuropathies. We have cloned and characterized the human PMP22 gene which spans approximately 40 kilobases and contains four coding exons. Towards developing gene therapy regimens for the associated peripheral neuropathies, we have initiated detailed analysis of the 5{prime} flanking region of the PMP22 gene and identified two alternatively transcribed, but untranslated exons. Mapping of separate PMP22 mRNA transcription initiation sites to each of these exons indicates that PMP22 expression is regulated by two alternatively used promoters. Both putative promoter sequences demonstrated the ability to drive expression of reporter genes in transfection experiments. Furthermore, the structure of the 5{prime} portion of the PMP22 gene appears to be identical in rat and human, supporting the biological significance of the observed arrangement of regulatory regions. The relative expression of the alternative PMP22 transcripts is tissue-specific and high levels of the exon 1A-containing transcript are tightly coupled to myelin formation. In contrast, exon 1B-containing transcripts are predominant in non-neural tissues and in growth-arrested primary fibroblasts. The observed regulation of the PMP22 by a complex molecular mechanism is consistent with the proposed dual role of PMP22 in neural and non-neural tissue.

  1. Molecular Cloning and Characterization of Different Expression of MYOZ2 and MYOZ3 in Tianfu Goat

    OpenAIRE

    Lu Wan; Jisi Ma; Nianlu Wang; Daihua Wang; Gangyi Xu

    2013-01-01

    The myozenin family of proteins binds calcineurin, which is involved in myocyte differentiation of skeletal muscle. Moreover, gene expression of myozenin is closely related to meat quality. To further understand the functions and effects of myozenin2 (MYOZ2) and myozenin3 (MYOZ3) genes in goat, we cloned them from Tianfu goat longissimus dorsi muscle. Sequence analyses revealed that full-length coding sequence of MYOZ2 consisted of 795 bp and encoded 264 amino acids, and full-length coding se...

  2. Cloning and expression of Mycobacterium bovis secreted protein MPB51 in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    JIANG Xiuyun; WANG Chunfeng; WANG Chunfang; HE Zhaoyang

    2007-01-01

    The purpose of this study is to clone,identify,and express the mature secreted protein MPB51 from Mycobacterium bovis and to lay a good foundation for the diagnosis of M.bovis,for applying M.bovis vaccine into clinical practice,and for detection of immunity effectiveness.The gene encoding MPB51 was amplified from M.bovis Valleel 11 chromosomal DNA by using PCR technique,PCR product was approximately 800 bp DNA segment.Clone vector pGEM-T-51 was successfully constructed by the PCR product that was cloned into pGEM-T vector by using T-A clone technique,pGEM-T-51 and pET28a(+)were digested by BamH I and EcoR I double enzymes.The prokaryotic expression vector pET28a-51 was constructed by using the purified MPB51 gene that was subcloned into the expression vector pET28a(+).Plasmid containing pET28a-51 was transformed into competence E.coli BL21(DE3).The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE.An approximately 30 kDa exogenous protein was observed on the SDS-PAGE.The protein was analyzed by using Western-blotting and it had the antigenic activity ofM.bovis.These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.

  3. Cloning of a Putative Pectate Lyase Gene Expressed in the Subventral Esophageal Glands of Heterodera glycines.

    Science.gov (United States)

    De Boer, J M; Davis, E L; Hussey, R S; Popeijus, H; Smant, G; Baum, T J

    2002-03-01

    We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

  4. Retinoic acid regulates the expression of photoreceptor transcription factor NRL.

    Science.gov (United States)

    Khanna, Hemant; Akimoto, Masayuki; Siffroi-Fernandez, Sandrine; Friedman, James S; Hicks, David; Swaroop, Anand

    2006-09-15

    NRL (neural retina leucine zipper) is a key basic motif-leucine zipper (bZIP) transcription factor, which orchestrates rod photoreceptor differentiation by activating the expression of rod-specific genes. The deletion of Nrl in mice results in functional cones that are derived from rod precursors. However, signaling pathways modulating the expression or activity of NRL have not been elucidated. Here, we show that retinoic acid (RA), a diffusible factor implicated in rod development, activates the expression of NRL in serum-deprived Y79 human retinoblastoma cells and in primary cultures of rat and porcine photoreceptors. The effect of RA is mimicked by TTNPB, a RA receptor agonist, and requires new protein synthesis. DNaseI footprinting and electrophoretic mobility shift assays (EMSA) using bovine retinal nuclear extract demonstrate that RA response elements (RAREs) identified within the Nrl promoter bind to RA receptors. Furthermore, in transiently transfected Y79 and HEK293 cells the activity of Nrl-promoter driving a luciferase reporter gene is induced by RA, and this activation is mediated by RAREs. Our data suggest that signaling by RA via RA receptors regulates the expression of NRL, providing a framework for delineating early steps in photoreceptor cell fate determination.

  5. cDNA cloning of the murine PEX gene implicated in X-linked hypophosphatemia and evidence for expression in bone

    Energy Technology Data Exchange (ETDEWEB)

    Du, L.; Desbarats, M.; Viel, J. [McGill Univ., Montreal, Quebec (Canada)] [and others

    1996-08-15

    The recently identified human PEX g ene apparently encodes for a neutral endopeptidase that is mutated in patients with X-linked hypophosphatemia. The 3{prime} and 5{prime} ends of the coding region of PEX have not been cloned, nor has the tissue expression of the gene been identified. Here we report the isolation and characterization of the complete open reading frame of the mouse Pex gene and the demonstration of its expression in bone. Mouse Pex cDNA is predicted to encode a protein of 749 amino acids with 95% identity to the available human PEX sequence and significant homology to members of the membrane-bound metalloendopeptidase family. Northern blot analysis revealed a 6.6-kb transcript in bone and in cultured osteoblasts from normal mice that was not detectable in samples from the Hyp mouse, the murine homolog of human X-linked hypophosphatemia. Pex transcripts were, however, detectable in Hyp bone by RT-PCR amplification. Of particular interest, a cDNA clone from rat incisor shows 93% sequence identity to the 5{prime} end of Pex cDNA, suggesting that Pex may be expressed in another calcified tissue, the tooth. The association of impaired mineralization of bone and teeth and disturbed renal phosphate reabsorption with altered expression of Pex suggests that the Pex gene product may play a critical role in these processes. 47 refs., 2 figs., 1 tab.

  6. Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos.

    Science.gov (United States)

    Park, Mi-Ryung; Im, Gi-Sun; Kim, Sung Woo; Hwang, Seongsoo; Park, Jae-Hong; Kim, Hyun; Do, Yoon Jung; Park, Soo Bon; Yang, Bo-Suck; Song, Young Min; Cho, Jae-Hyeon; Ko, Yeoung-Gyu

    2013-06-01

    The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

  7. Expression of Innate Immune Response Genes in Liver and Three Types of Adipose Tissue in Cloned Pigs

    DEFF Research Database (Denmark)

    Højbøge, Tina Rødgaard; Skovgaard, Kerstin; Stagsted, Jan;

    2012-01-01

    remain unaffected by the cloning process. We investigated the expression of 40 innate immune factors by high-throughput quantitative real-time PCR in samples from liver, abdominal subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and neck SAT in cloned pigs compared to normal outbred pigs...

  8. Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates

    Science.gov (United States)

    A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZaA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included ...

  9. Residual expression of reprogramming factors affects the transcriptional program and epigenetic signatures of induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Cesar A Sommer

    Full Text Available Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON vs Gtl2(OFF iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

  10. Cloning and Expression Analysis of a Prion Protein Encoding Gene in Guppy (Poecilia reticulata)

    Institute of Scientific and Technical Information of China (English)

    WU Suihan; WEI Qiwei; YANG Guanpin; WANG Dengqiang; ZOU Guiwei; CHEN Daqing

    2008-01-01

    The full length eDNA of a prion protein (PrP) encoding gene of guppy (Poecilia reticulata) and the corresponding ge-nomic DNA were cloned.The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a pro-tein of 515 amino acids,which held all typical structural characteristics of the functional PrP.The cloned genomic DNA fragmentcorresponding to the eDNA was 3720 bp in length,consisting of 2 introns and 2 exons.The 5' untranslated region of eDNA origi-nated from the 2 exons,while the ORF originated from the second exon.Although the gene was transcribed in diverse tissues in-cluding brain,eye,liver,intestine,muscle and tail,its transcript was most abundant in the brain.In addition,the transcription of thegene was enhanced by 5 salinity,implying that it was associated with the response of guppy to saline stress.

  11. Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls.

    Directory of Open Access Journals (Sweden)

    Fei Gao

    Full Text Available Animal breeding via Somatic Cell Nuclear Transfer (SCNT has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF. Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver, using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.

  12. Molecular cloning and expression analysis of the ethylene insensitive3 (EIN3) gene in cucumber (Cucumis sativus).

    Science.gov (United States)

    Bie, B B; Pan, J S; He, H L; Yang, X Q; Zhao, J L; Cai, R

    2013-10-07

    The plant gaseous hormone ethylene regulates many aspects of plant growth, development, and responses to the environment. Ethylene insensitive3 (EIN3) is a key transcription factor involved in the ethylene signal transduction pathway. To gain a better understanding of this particular pathway in cucumber, the full-length cDNA encoding EIN3 (designated as CsEIN3) was cloned from cucumber for the first time by rapid amplification of cDNA ends. The full length of CsEIN3 was 2560 bp, with an open reading frame of 1908 bp encoding 635 amino acids. Sequence alignment and phylogenetic analyses revealed that CsEIN3 has high homology with other plant EIN3/EIL proteins that were derived from a common ancestor during evolution, and CsEIN3 was grouped into a cluster along with melon. Homology modeling demonstrated that CsEIN3 has a highly similar structure to the specific DNA-binding domain contained in EIN3/EIL proteins. Based on quantitative reverse transcription-polymerase chain reaction analysis, we found that CsEIN3 was constitutively expressed in all organs examined, and was increased during flower development and maturation in both male and female flowers. Our results suggest that CsEIN3 is involved in processes of flower development. In conclusion, this study will provide the basis for further study on the role of EIN3 in relevant biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

  13. Cloning, expression and localization of a trypsin-like serine protease in the spruce budworm, Choristoneura fumiferana

    Institute of Scientific and Technical Information of China (English)

    Wen-Ying He; Yi-Ping Zheng; Lin Tang; Si-Chun Zheng; Catherine Béliveau; Daniel Doucet; Michel Cusson; Qi-Li Feng

    2009-01-01

    A trypsin-like molting-related serine protease cDNA (CfMRSP) was cloned from the spruce budworm, Choristoneura fumiferana. The full-length CfMRSP comple-mentary DNA (cDNA) encoded a 43 kDa protein that contained a trypsin-like serine protease catalytic domain, but no clip domain. The C-terminal extension contained five cystein residues, which may allow the protein to form a homodimer through interchain disulfide bonds and regulate the activity of CfMRSP. Phylogenetic tree analysis showed that CfMRSP clusters with lepidopteran homologues such as serine protease 1 of Lonomia obliqua, hemolymph proteinase 20 (HP20), pattern recognition serine proteinase precursor (ProHP14) and a trypsin-like protein of Manduca sexta. Northern blot analysis of devel-opmental expression of CfMRSP indicated that its transcripts were found primarily in the epidermis and were produced during all of the tested stadia, from 4th instar larvae to pupae, but increased levels of C/MRSP transcripts were always found after each molt. A high level of the protein was found in the epidermis by immunohistochemistry analysis. Altogether these data suggest that CfMRSP plays a role in the epidermis during molting and metamorphosis.

  14. Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases.

    Science.gov (United States)

    Nakaya, N; Nishibori, M; Kawabata, M; Saeki, K

    1996-12-01

    A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.

  15. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    Science.gov (United States)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  16. Cloning and Expression Analysis of p26 Gene in Artemia sinica

    Institute of Scientific and Technical Information of China (English)

    Lijuan JIANG; Lin HOU; Xiangyang ZOU; Ruifeng ZHANG; Jiaqing WANG; Wenjing SUN; Xintao ZHAO; Jialu AN

    2007-01-01

    The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the α-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expression of the p26 gene at different developmental stages of A. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development.

  17. Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts.

    Science.gov (United States)

    Wan, Yongjie; Deng, Mingtian; Zhang, Guomin; Ren, Caifang; Zhang, Hao; Zhang, Yanli; Wang, Lizhong; Wang, Feng

    2016-01-01

    Somatic cell nuclear transfer (SCNT) is a useful way to produce cloned animals. However, SCNT animals exhibit DNA methylation and genomic imprinting abnormalities. These abnormalities may be due to the faulty epigenetic reprogramming of donor cells. To investigate the consequence of SCNT on the genomic imprinting and global methylation in the donor cells, growth patterns and apoptosis of cloned goat fibroblast cells (CGFCs) at passage 7 were determined. Growth patterns in CGFCs were similar to the controls; however, the growth rate in log phase was lower and apoptosis in CGFCs were significantly higher (P < 0.01). In addition, quantitative expression analysis of three DNA methyltransferases (Dnmt) and two imprinted genes (H19, IGF2R) was conducted in CGFCs: Dnmt1 and Dnmt3b expression was significantly reduced (P < 0.01), and H19 expression was decreased sixfold (P < 0.01); however, the expression of Dnmt3a was unaltered and IGF2R expression was significantly increased (P < 0.05). Finally, we used bisulfite sequencing PCR to compare the DNA methylation patterns in differentially methylated regions (DMRs) of H19 and IGF2R. The DMRs of H19 (P < 0.01) and IGF2R (P < 0.01) were both highly methylated in CGFCs. These results indicate that the global genome might be hypomethylated. Moreover, there is an aberrant expression of imprinted genes and DMR methylation in CGFCs.

  18. Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

    Science.gov (United States)

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

    2017-08-30

    Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (Prock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these

  19. [Cloning, expression and identification of hpaA gene from a clinical isolate of Helicobacter pylori].

    Science.gov (United States)

    Mao, Ya-Fei; Yan, Jie; Li, Li-Wei

    2003-02-01

    To clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity of the fusion protein. The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25% approximate, equals 97.32%, while the homology of its putative amino acid sequence was as high as 95.38% approximate, equals 98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein. An expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

  20. cDNA cloning, prokaryotic expression and purification of rat α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Xin LI; Yao-Hua LI; Jun-Yan HAN; Shun YU; Biao CHEN

    2006-01-01

    Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat α-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.

  1. Cloning and Expression of Nano Body Gene against Enterotoxin B of Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    Zahra Tavassoli

    2017-02-01

    Full Text Available Background & Objectives: Staphylococcus aureus bacteria causes many different diseases by secretion of various enterotoxins. Therefore, it is necessary to develop ways that facilitate the detection of enterotoxins. Nowadays, immunochemical methods which are based on monoclonal antibody technology are used. The heavy chain antibodies that are called VHH or Nano body were found in blood serum of the Camelidae family. The unique properties of this antibody such as their binding to small molecules like toxins make them attractive candidates for the development of immunodiagnostic tests. The present study was done to achieve a VHH molecules against Staphylococcus enterotoxin B. Materials & Methods: Freighting phage library for isolate private Nano bodies against enterotoxin B was done in previous works. Next, pCANTAB 5E vector that consists VHH, extracted from E.coli bacteria strain xl1blue, and after doing PCR process with relative primers, sub cloning in pET21a(+ as an expression vector with cut sites NdeI and XhoI was done. Transformation in E.coli bacteria strain BL21(DE3 was done. Then, the cells effected with IPTG and producing time, and other terms were optimized. Finally, the expression of the protein with SDS-PAGE and western blot techniques was evaluated. Result: For proving cloning of nano body gene in pET21a (+ vector, nucleotide sequence of gene was analyzed, and transforming to E.coli bacteria strain BL21(DE3 was successful. After inspiration, active protein in cell was seen by SDS-PAGE technique and proved by western blot. Conclusion: cloning, sub cloning, and nonabody expression were surveyed in this research. Production of this protein can help to develop new therapeutic methods and produce vaccine against enterotoxin B of Staphylococcus aureus

  2. Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Kovalenko O. P.

    2011-12-01

    Full Text Available Aim. Cloning and sequencing of the T. thermophilus leucyl-tRNA synthetase (LeuRSTT followed by the creation of genetically engineered construct for protein expression in E.coli cells and its purification. Methods. Searching for the LeuRSTT gene was performed by Southern blot hybridization with chromosomal DNA, where digoxigenin-labeled PCR fragments of DNA were used as probes. Results. The gene of T. thermophilus HB27 leucyl-tRNA synthetase was cloned and sequenced. The open reading frame encodes a polypeptide chain of 878 amino acid residues in length (molecular mass 101 kDa. Comparison of the amino acid sequence of T. thermophilus LeuRS with that of the enzymes from other organisms showed that LeuRSTT was a part of the group of similar enzymes of prokaryotes, formed by the proteins of protobacteriae, rickettsia and mitochondria of eukaryotes. The resulting phylogenetic tree of LeuRSs reveals dichotomous branching into two lines: prokaryotic/eukaryotic mitochondrial and arhaeal/eukaryotic cytosolic proteins. Differences between prokaryotic and arhaeal branches of the LeuRSs phylogenetic tree are primarily due to the structure of two domains of the enzyme – the editing and the C-terminal. T. thermophilus LeuRS was expressed in E. coli cells by cloning the corresponding gene into pET29b vector. Conclusions. The cloned T. thermophilus leuS gene and expressed recombinant protein will be used for structural and functional studies on LeuRSTT, including X-ray analysis of the enzyme and its mutant forms in complex with different substrates

  3. Cloning and expression of a novel human HCUTA cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Copper is one of the most important trace elements to life. Human HCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 of E. coli. The whole opening reading frame of HCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed in E. coli BL21 (DE3).

  4. Molecular cloning, expression and in situ hybridization of rat brain glutamic acid decarboxylase messenger RNA.

    Science.gov (United States)

    Julien, J F; Legay, F; Dumas, S; Tappaz, M; Mallet, J

    1987-01-14

    A cDNA library was generated in the expression vector lambda GT11 from rat brain poly(A)+ RNAs and screened with a GAD antiserum. Two clones reacted positively. One of them was shown to express a GAD activity which was specifically trapped on anti-GAD immunogel and was inhibited by gamma-acetylenic-GABA. Blot hybridization analysis of RNAs from rat brain revealed a single 4 kilobases band. Preliminary in situ hybridizations showed numerous cells labelled by the GAD probe such as the Purkinje and stellate cells in the cerebellar cortex and the cells of the reticular thalamic nucleus.

  5. Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

    Science.gov (United States)

    He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

    2015-10-01

    An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories.

  6. Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon.

    Science.gov (United States)

    Blatny, J M; Brautaset, T; Winther-Larsen, H C; Haugan, K; Valla, S

    1997-01-01

    The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment. PMID:9023917

  7. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

    Science.gov (United States)

    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene.

  8. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  9. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish.

    Science.gov (United States)

    Leong, Ivone U S; Skinner, Jonathan R; Shelling, Andrew N; Love, Donald R

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients.

  10. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    Institute of Scientific and Technical Information of China (English)

    Ge-Jian Zhu; Ying-Nian Yu; Xin Li; Yu-Li Qian

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 ( CYP2 C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2 C9 eDNA was cloned and expressed stably in CHL cellsMETHODS: After extraction of total RNA from human livertissue, the human CYP2C9 eDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR),and cloned into cloning vector pGEM-T. The cDNA fragmentwas identified by DNA sequencing and subcloned into amammalian expression vector pREP9. A transgenic cell linewas established by transfecting the recombinant vector ofpREP9-CYP2C9 into CHL cells. The enzyme activity ofCYP2C9 catalyzing oxidation of tolbutamide to hydroxytolbutamide in S9 fraction of the cell was determined by highperformance liquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from theeDNA segment was identical to that of CYP2 C9 * 1, the wildtype CYP2 C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acidsequence was the same, L7, P382. The S9 fraction of theestablished cell line metabolizes tolbutamide to hydroxytolbutamide; tolbutamide hydroxylass activity was found to be0.465 ± 0.109 μmol@ min-1 . g1 S9 protein or 8.62 ± 2.02 mol@ min 1 ~mol-1 CYP, but was undetectable in parental CHL cell.CONCLUSION: The cDNA of human CYP2C9 was successfullycloned and a cell line of CHL- CYP2C9, efficiently expressingthe protein of CYP2C9, was established.

  11. Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer.

    Science.gov (United States)

    Everts, Robin E; Chavatte-Palmer, Pascale; Razzak, Anthony; Hue, Isabelle; Green, Cheryl A; Oliveira, Rosane; Vignon, Xavier; Rodriguez-Zas, Sandra L; Tian, X Cindy; Yang, Xiangzhong; Renard, Jean-Paul; Lewin, Harris A

    2008-03-14

    Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT, n = 20), in vitro fertilization (IVF, n = 9), and artificial insemination (AI, n = 9) at or near term development was performed to better understand why SCNT and IVF often result in placental defects, hydrops, and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF, and AI on gene expression, taking into account the effects of parturition (term or preterm), sex of fetus, breed of dam, breed of fetus, and pathological finding in the offspring (hydrops, normal, or other abnormalities). Differential expression of 20 physiologically important genes was confirmed with quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentas were collected at term or preterm (i.e., whether the collection was because of disease or to obtain stage-matched controls) followed by placentome source (AI, IVF, or SCNT). Gene expression in SCNT placentomes was dramatically different from AI (n = 336 genes; 276 >2-fold) and from IVF (n = 733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, and gene expression. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS.

  12. The chemosensory protein of Chinese honeybee, Apis cerana cerana: Molecular cloning of cDNA, immunocytochemical localization and expression

    Institute of Scientific and Technical Information of China (English)

    LI HongLiang; LOU BingGan; CHENG Jia'An; GAO QiKang

    2007-01-01

    Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs thought to be involved in chemical communication.Here we report the first cDNA of CSPs,called Ac-ASP3,cloned and characterized from antennas of adult worker bees in Chinese honeybee,Apis cerana cerana.The Ac-ASP3 cDNA comprises 2 exons,with an ORF of 393-bp encoding 130 aa.Protein signature analyses show that the protein consists of four conserved cysteines and a signal peptide with 19 aa in the N-terminal sequence.The deduced protein sequence shares high homology with Am-ASP3 of Apis mellifera and low similarity with other species of insects.Immunocytochemical localization shows that Ac-ASP3 is only specifically expressed on the antenna contact chemosensilla such as sensilla trichodea B and sensilla basiconica,whereas Ac-ASP3 is scarcely expressed on olfactory chemosensilla such as sensilla placodea.Real-time PCR of Ac-ASP3 transcripts shows that Ac-ASP3 is highly expressed on wings and legs,but expression is lower on antenna.Temporal expression patterns suggest that Ac-ASP3 is expressed during the period of pupa and adults from 1-d to 6-d stages when bees act as house bees,cleaning the comb and taking care of the queen and larvae in comb.The above evidence suggests that Ac-ASP3 is unique in species and is generally not involved in olfaction during searching for honey and pollen.Rather,the protein seems to function in recognition of chemosensory substances on bees' cuticle and mechanical movement of antenna.

  13. Cloning and Expression of Bile Salt Hydrolase Gene from Lactobacillus plantarum M1-UVS29

    Institute of Scientific and Technical Information of China (English)

    Yu Chang-qing; Li Rong

    2015-01-01

    We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 inLactococcus lactis NZ9000 successfully. Gene-specific primers for amplification ofL. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH. pNZ8148-BSH was transferred intoLactococcus lactis NZ9000. Sequencing indicated that the clonedbsh fragment contained 995 nucleotides, and shared 99.3% sequence homology withbsh gene fromL. plantarum MBUL10. Clonedbsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 µmol• min-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.

  14. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  15. Molecular cloning and expression of the Leishmania tropica KMP-11 gene.

    Science.gov (United States)

    Meriee, Mouayad; Soukkarieh, Chadi; Abbady, Abdul Qader A

    2014-08-01

    Kinetoplastid membrane protein-11 (KMP-11) is a small protein of 11 kDa present in all kinetoplastid protozoa studded so far. This protein which is highly expressed in all stages of the Leishmania life cycle is considered a potential candidate for a leishmaniasis vaccine against many leishmania species. KMP-11 has been recently described in Leishmania tropica. In the present study, the KMP-11 gene was extracted from L. tropica by PCR using two oligonucleotide primers designed to amplify the entire coding region of this gene. Then, the purified PCR products were successfully ligated into a high expression vector the pRSET-GFP. This expression vector provides the opportunity to clone the desired insert as a fusion protein with a GFP and a tag, polyhistidine region. The GFP use as a carrier to improve immune response and the polyhistidine tag facilitates detection of the expressed protein with anti-His antibodies and also purification of the protein using affinity purification. After wards KMP-11 coding region was sequenced and the recombinant protein was induced and purified from Escherichia coli cultures. The results of the present study will increase our knowledge about molecular cloning and expression of the L. tropica KMP-11 gene, and this may be used as an effective target for controlling cutenous leishmaniasis.

  16. Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls

    DEFF Research Database (Denmark)

    Fei, Gao; Luo, Yonglun; Li, Shengting

    2011-01-01

    Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural...... breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation......-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls...

  17. Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI.

    Science.gov (United States)

    de Souza, Adriana F; Torquato, Ricardo J S; Tanaka, Aparecida S; Sampaio, Claudio A M

    2005-11-01

    A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. The clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. The deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. The BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.

  18. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    Directory of Open Access Journals (Sweden)

    Yvonne D Trigoso

    Full Text Available The enzyme dihydrodipicolinate reductase (DHDPR is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(PH dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3. The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  19. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    Science.gov (United States)

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  20. Comparative analysis of a BAC contig of the porcine RN region and the human transcript map: implications for the cloning of trait loci.

    Science.gov (United States)

    Jeon, J T; Amarger, V; Rogel-Gaillard, C; Robic, A; Bongcam-Rudloff, E; Paul, S; Looft, C; Milan, D; Chardon, P; Andersson, L

    2001-03-15

    The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.

  1. Sequence determination of cDNA clones of transcripts from the tumor-associated region of the Marek's disease virus genome.

    Science.gov (United States)

    Iwata, A; Ueda, S; Ishihama, A; Hirai, K

    1992-04-01

    The number of 132-bp tandem direct repeats within the long inverted repeat region of the Marek's disease virus type 1 (MDV1) genome increases concomitantly with the loss of oncogenicity during serial passages in cultured cells. Twelve clones carrying the 132-bp sequence were isolated from a cDNA library constructed from chicken embryo fibroblasts infected with the MDV1 Md5 strain. Through sequence analysis of a cDNA clone and primer extension analysis, the corresponding mRNA was found to be a linear transcript which included the two 132-bp tandem direct repeats. Two open reading frames were found in this transcript. One had a week homology with v-fms. The other should increase its size concomitantly with expansion of the 132-bp tandem direct repeat. PCR analysis of both cDNA clones and RNA gave amplified products which were as large as that produced from the genomic clone, indicating that a majority of mRNA from this region is composed of unspliced transcripts.

  2. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C......-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed...

  3. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

    Directory of Open Access Journals (Sweden)

    Claire L. Anderson

    2003-01-01

    Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

  4. Functional cDNA expression cloning: Pushing it to the limit

    Science.gov (United States)

    OKAYAMA, Hiroto

    2012-01-01

    The 1970s and the following decade are the era of the birth and early development of recombinant DNA technologies, which have entirely revolutionized the modern life science by providing tools that enable us to know the structures of genes and genomes and to dissect their components and understand their functions at the molecular and submolecular levels. One major objective of the life sciences is to achieve molecular and chemical understandings of the functions of genes and their encoded proteins, which are responsible for the manifestation of all biological phenomena in organisms. In the early 1980s, I developed, together with Paul Berg, a new technique that enables the cloning of full-length complementary DNAs (cDNAs) on the basis of their functional expression in a given cell of interest. I review the development, application and future implications in the life sciences of this gene-cloning technique. PMID:22450538

  5. Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

    Science.gov (United States)

    Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

    2015-09-01

    The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

  6. A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript

    Directory of Open Access Journals (Sweden)

    H.C.S. Silveira

    2004-06-01

    Full Text Available The Down's syndrome candidate region 1 (DSCR1 protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD. Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT. UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATaexpressing AD-UXT with the strain Y187 (MATalpha expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

  7. Glucagon and Insulin Cooperatively Stimulate Fibroblast Growth Factor 21 Gene Transcription by Increasing the Expression of Activating Transcription Factor 4.

    Science.gov (United States)

    Alonge, Kimberly M; Meares, Gordon P; Hillgartner, F Bradley

    2017-03-31

    Previous studies have shown that glucagon cooperatively interacts with insulin to stimulate hepatic FGF21 gene expression. Here we investigated the mechanism by which glucagon and insulin increased FGF21 gene transcription in primary hepatocyte cultures. Transfection analyses demonstrated that glucagon plus insulin induction of FGF21 transcription was conferred by two activating transcription factor 4 (ATF4) binding sites in the FGF21 gene. Glucagon plus insulin stimulated a 5-fold increase in ATF4 protein abundance, and knockdown of ATF4 expression suppressed the ability of glucagon plus insulin to increase FGF21 expression. In hepatocytes incubated in the presence of insulin, treatment with a PKA-selective agonist mimicked the ability of glucagon to stimulate ATF4 and FGF21 expression. Inhibition of PKA, PI3K, Akt, and mammalian target of rapamycin complex 1 (mTORC1) suppressed the ability of glucagon plus insulin to stimulate ATF4 and FGF21 expression. Additional analyses demonstrated that chenodeoxycholic acid (CDCA) induced a 6-fold increase in ATF4 expression and that knockdown of ATF4 expression suppressed the ability of CDCA to increase FGF21 gene expression. CDCA increased the phosphorylation of eIF2α, and inhibition of eIF2α signaling activity suppressed CDCA regulation of ATF4 and FGF21 expression. These results demonstrate that glucagon plus insulin increases FGF21 transcription by stimulating ATF4 expression and that activation of cAMP/PKA and PI3K/Akt/mTORC1 mediates the effect of glucagon plus insulin on ATF4 expression. These results also demonstrate that CDCA regulation of FGF21 transcription is mediated at least partially by an eIF2α-dependent increase in ATF4 expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

    Science.gov (United States)

    Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

    1985-12-01

    A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

  9. Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L

    Institute of Scientific and Technical Information of China (English)

    Ai-lin TAO; Shao-heng HE

    2005-01-01

    Aim: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LüCao in Chinese) and short ragweed (Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. Methods: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3'cDNA ends were identified by using the 3'-RACE method. The gene function conferred by the full-length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET-44 RosettaBlue cells were subsequently characterized by N-terminal end sequencing, IgE binding, and crossreactivity. Results: Three full-length cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%-90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in nonfusion forms and were confirmed by using the N-terminal end sequence identity.Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia, but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross-reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full-length cDNA demonstrated that alternative nucleotide degeneracy occurred. Conclusion: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little-studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.

  10. Cloning, Expression of apxI Gene of Actinobacillus pleuropneumoniae and Development of ELISA

    Institute of Scientific and Technical Information of China (English)

    LIU Jian-jie; HE Qi-gai; CHEN Huan-chun; WU Bin; XU Xiao-juan; LIU Jun-fa; TANG Xian-chun; BEI Wei-cheng

    2003-01-01

    Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniaein Genbank(S4074), a pair of primers were designed. A 3 640 bp(4 687 -8 326 bp)gene fragment was ampli-fied by PCR from the isolated strain of A. pleuropneumoniae serovar 1. Then, it was cloned into pMD18-T,identified by both restriction endonuclease and sequence analysis, and inserted into pET-28a expression vectorto yield the expression plasmid. SDS-PAGE result indicated expression of apxICA in BL21 (DE3), Westernblot analysis showed the protein's immunogenicity. Using the expressed protein, ELISA was established to de-tect serum antibody against ApxI. The feature of ELISA to detect highly virulent A. pleuropneumoniae strainsinfection was proved by primary clinical application.

  11. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments...... efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells......, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors....

  12. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    Science.gov (United States)

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  13. Cloning and expression analysis of an E-class MADS-box gene from ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... As shown by relative–quantitative real-time polymerase chain reaction ... mation of higher-order transcription factor complexes with .... synthesized from 2.0 µg RNA using Superscript II RNase- Reverse ..... program (2006BAD01A15), the National Basic Research ... Structure and expression of duplicate.

  14. Cloning, Characterization, and Expression Patterns of Three Sarco/Endoplasmic Reticulum Ca2+-ATPase Isoforms from Pearl Oyster (Pinctada fucata)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A large amount of calcium is required for mollusk biomineralization. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a well-known protein with the function of sustaining the calcium homeostasis. How does it possibly function in the process of pearl oyster biomineralization? Three SERCA isoforms, namely PSERA, PSERB, and PSERC were cloned from the pearl oyster, Pinctada fucata. The cDNAs of the three isoforms were isolated by reverse transcription-polymerase chain reaction (RT-PCR)and rapid amplification of cDNA ends. PSERA consisted of 3568 bp encoding 1007 amino acids, PSERB included 3953 bp encoding 1024 amino acids, and PSERC comprised of 3450 bp encoding 1000 amino acids.The three isoforms showed high homology (65%-87%) with SERCAs from other species. Consistent with the results from other invertebrates, Southern blot analysis revealed that the three isoforms originated from a single gene that was also related to SERCA1, SERCA2, and SERCA3 of vertebrates. The splicing mechanism of the three isoforms was similar to that of isoforms of vertebrate SERCA3. Semiquantitative RT-PCR was carried out to study the expression patterns of the three isoforms. The results showed that PSERB was ubiquitously expressed in all tested tissues and was a potential "housekeeping" SERCA isoform; PSERA was expressed in the adductor muscle and foot and was likely to be a muscle-specific isoform, and PSERC was expressed in the other tissues except the adductor muscle or foot with the highest expression levels in the gill and mantle, indicating that it was a non-muscle-specific isoform and might be involved in calcium homeostasis during pearl oyster biomineralization.

  15. Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Kandasamy Eniyan; Urmi Bajpai

    2015-01-01

    Objective:To clone, express and purify2-methylcitrate synthase(Rv1131) gene of Mycobacterium tuberculosis(M. tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 gene was amplified by polymerase chain reaction usingM. tuberculosisH37Rv genomicDNA and cloned into pGEM-T easy vector and sequenced. The gene was sub-cloned in pET28c vector, expressed inEscherichia coliBL21(E. coliBL21) (DE3) cells and the recombinant protein was identified byWestern blotting.The protein was purified usingNickel affinity chromatography and the structural characteristics like sub-cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools.Tertiary structure of the protein and phylogenetic analysis was also established byin silico analysis.Results:The expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti-HIS antibodies and the protein was purified from the soluble fraction.In silicoanalysis showed that the protein contains no signal peptide and transmembrane helices.Active site prediction showed that the protein has histidine and aspartic acid residues at242,281 &332 positions respectively.Phylogenetic analysis showed 100% homology withmajor mycobacterial species.Secondary structure predicts2-methylcitrate synthase contain51.9% alpha-helix,8.7% extended strand and39.4% random coils.Tertiary structure of the protein was also established.Conclusions:The enzyme2-methylcitrate synthase from M. tuberculosisH37Rv has been successfully expressed and purified.The purified protein will further be utilized to develop assay methods for screening new inhibitors.

  16. Cloning, expression, functional validation and modeling of cinnamyl alcohol dehydrogenase isolated from xylem of Leucaena leucocephala.

    Science.gov (United States)

    Pandey, Brijesh; Pandey, Veda Prakash; Dwivedi, Upendra Nath

    2011-10-01

    A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.

  17. Identification and cloning of GOLDEN2-LIKE1 (GLK1), a transcription factor associated with chloroplast development in Brassica napus L.

    Science.gov (United States)

    Pan, Y L; Pan, Y; Qu, C M; Su, C G; Li, J H; Zhang, X G

    2017-02-16

    Photosynthesis is the process by which dry matter accumulates, which affects rapeseed yield. In this study, we identified GOLDEN2-LIKE1 (GLK1), located on chromosome A07 and 59.2 kb away from the single nucleotide polymorphism marker SNP16353A07, which encodes a transcription factor associated with the rate of photosynthesis in leaves. We then identified 96 GLK1 family members from 53 species using a hidden Markov model (HMM) search and found 24 of these genes, which were derived from 17 Brassicaceae species. Phylogenetic analysis showed that 24 Brassicaceae proteins were classified into three subgroups, named the Brassica family, Adenium arabicum, and Arabidopsis. Using homologous cloning methods, we identified four BnaGLK1 copies; however, the coding sequences were shorter than the putative sequences from the reference genome, probably due to splicing errors among the reference genome sequence or different gene copies being present in the different B. napus lines. In addition, we found that BnaGLK1 genes were expressed at higher levels in leaves with more chloroplasts than were present in other leaves. Overexpression of BnaGLK1a resulted in darker leaves and siliques than observed in the control, suggesting that BnaGLK1 might promote chloroplast development to affect the rate of photosynthesis in leaves. These results will help to elucidate the mechanism of chloroplast biogenesis by GLK1 in B. napus.

  18. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

    Directory of Open Access Journals (Sweden)

    Zhengyi Liu

    2014-01-01

    Full Text Available Heat shock protein 90 (HSP90, a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90 by combining homology cloning with rapid amplification of cDNA ends (RACE. Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰ was almost fourfold of that at 25 in salinity (‰ for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes.

  19. Construction, cloning, and expression of synthetic genes encoding spider dragline silk.

    Science.gov (United States)

    Prince, J T; McGrath, K P; DiGirolamo, C M; Kaplan, D L

    1995-08-29

    Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.

  20. Map-based cloning and expression analysis of BMR-6 in sorghum

    Indian Academy of Sciences (India)

    Jieqin Li; Lihua Wang; Qiuwen Zhan; Yanlong Liu

    2015-09-01

    Brown midrib mutants in sorghum are associated with reduced lignin content and increased cell wall digestibility. In this study, we characterized a bmr-6 sorghum mutant, which shows reddish pigment in the midrib and stem after the fifth-leaf stage. Compared to wild type, Kalson lignin content of bmr-6 is decreased significantly. We used histological analysis to determine that the mutant exhibited a modified pattern of lignin staining and found an increased polysaccharide content. We cloned BMR-6 gene, a gene encoded a cinnamyl alcohol dehydrogenase (CAD), using a map-based cloning approach. Genetic complementation confirmed that CAD is responsible for the BMR-6 phenotype. BMR-6 gene was expressed in all tested sorghum tissues, with the highest being in midrib and stem. Transient expression assays in Nicotiana benthamiana leaves demonstrated cytomplasmic localization of BMR-6. We found that the expression level of bmr-6 was significantly decreased in the mutant but expression of SbCAD3 and SbCAD5 were significantly increased. Our results indicate that BMR-6 not only affects the distribution of lignin but also the biosynthesis of lignin in sorghum.

  1. [Cloning, expression and identification of Der f7 gene from Dermatophagoides farinae and its immunological characteristics].

    Science.gov (United States)

    Zheng, Man-Yin; Wu, Yu-Lan; Yan, Hao; Ji, Kun-Mei; Liu, Zhi-Gang

    2013-10-01

    To clone and express Der f7 gene of Dermatophagoides farinae, and identify its immunogenicity. Total RNA was extracted from D. farinae mites. A reference sequence (Accession No. AY283292) was used to design specific primers. The Der f7 gene fragment was amplified by RT-PCR, and cloned into pET-32a vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced with IPTG for protein expression. The recombinant protein was purified by Ni2+ chelating affinity chromatography and analyzed by SDS-PAGE and Western blotting. The Der f7 gene fragment was about 650 bp, and shared 99% homology with the published one (Accession No. FJ436108). SDS-PAGE result showed its relative molecular weight (M(r)) of 23 000. The recombinant protein showed appropriate combination ability with IgE in sera of mite allergic patients. Der f 7 gene has been expressed in prokaryotic expression system and shows allergenicity.

  2. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    Science.gov (United States)

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  3. cDNA cloning and expression analysis of a mannose-binding lectin from Pinellia pedatisecta

    Indian Academy of Sciences (India)

    Juan Lin; Xuanwei Zhou; Shi Gao; Xiaojun Liu; Weisheng Wu; Xiaofen Sun; Kexuan Tang

    2007-03-01

    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.

  4. Genetic mapping and comparative expression analysis of transcription factors in cotton.

    Science.gov (United States)

    Chen, Xuemei; Jin, Xin; Li, Ximei; Lin, Zhongxu

    2015-01-01

    Transcription factors (TFs) play an important role in the regulation of plant growth and development. The study of the structure and function of TFs represents a research frontier in plant molecular biology. The findings of these studies will provide significant information regarding genetic improvement traits in crops. Currently, a large number of TFs have been cloned, and their function has been verified. However, relatively few studies that genetically map TFs in cotton are available. To genetically map TFs in cotton in this study, specific primers were designed for TF genes that were published in the Plant Transcription Factor Database. A total of 977 TF primers were obtained, and 31 TF polymorphic loci were mapped on 15 cotton chromosomes. These polymorphic loci were clearly preferentially distributed on chromosomes 5, 11, 19 and 20; and TFs from the same family mapped to homologous cotton chromosomes. In-silico mapping verified that many mapped TFs were mapped on their corresponding chromosomes or their homologous chromosomes' corresponding chromosomes in the diploid genomes. QTL mapping for fiber quality revealed that TF-Ghi005602-2 mapped on Chr19 was associated with fiber length. Eighty-five TF genes were selected for RT-PCR analysis, and 4 TFs were selected for qRT-PCR analysis, revealing unique expression patterns across different stages of fiber development between the mapping parents. Our data offer an overview of the chromosomal distribution of TFs in cotton, and the comparative expression analysis between Gossypium hirsutum and G. barbadense provides a rough understanding of the regulation of TFs during cotton fiber development.

  5. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes.

    Science.gov (United States)

    Schmid-Burgk, Jonathan L; Schmidt, Tobias; Kaiser, Vera; Höning, Klara; Hornung, Veit

    2013-01-01

    Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

  6. Cloning and expression analysis of GhDET3, a vacuolar H+-ATPase subunit C gene, from cotton

    Institute of Scientific and Technical Information of China (English)

    Zhongyi Xiao; Kunling Tan; Mingyu Hu; Peng Liao; Kuijun Chen; Ming Luo

    2008-01-01

    Vacuolar H+-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgot-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an impor-tant role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To ana-lyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high ho-mology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expres-sion pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.

  7. Infectious in vitro transcripts from cloned cDNA of beet necrotic yellow vein virus RNA3 and RNA4and their functional study

    Institute of Scientific and Technical Information of China (English)

    李毅; 魏春红; 田波; 潘乃穟; 陈章良

    1995-01-01

    The full-length double-stranded cDNAs of beet necrotie yellow vein virus RNA3 and RNA 4were synthesized by using oligo(dT) 15 as well as RNA3 and RNA4 specific primers, and cloned downstream of the bacteriophage Sp6 RNA polymerase promoter of the transcription vector pGEM3Zf(+). The in vitro "run-off" transcription products obtained in the presence of Sp6 RNA polymerase and template DNA have high biological activities. In the 2 transcription systems, the transcription and capping in 2 separate reactions are more efficient. The simultaneous transcription and capping are not so efficient, but the transcripts are more infectious. Although there are a number of nonviral nucleotide sequences at the 5- and 3’-ends of RNA3 and RNA4 transcripts, the biological activities of both transcripts were not affected. The mechanical coinoculation of sugarbeet roots with the infectious transcripts of the prepared RNA3 and RNA4 and BNYW Rgl isolate has confirmed that RNA3 is the main cause of sugarbeet rhizomania.

  8. Alternative Splicing and Differential Expression of Two Transcripts of Nicotine Adenine Dinucleotide Phosphate Oxidase B Gene from Zea mays

    Institute of Scientific and Technical Information of China (English)

    Fan Lin; Yun Zhang; Ming-Yi Jiang

    2009-01-01

    With the exception of rice, little is known about the existence of respiratory burst oxidase homolog (rboh) gene in cereals. The present study reports the cloning and analysis of a novel rboh gene, termed ZmrbohB, from maize (Zea mays L.). The full-length cDNA of ZmrbohB encodes a 942 amino acid protein containing all of the respiratory burst oxidase homolog catalytically critical motifs.Altemative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-α and -β. Spliced transcript ZmrbohB-β retains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. Expression analysis showed that two splice isoforms were differentially expressed in various tissues and at different developmental stages, and the major product was ZmrbohB-α. The transcripts of ZmrbohB-α accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold (4℃), heat (40℃), UV and salinity stress. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB except wounding. These results provide new insight into roles in the expression regulation of plant rboh genes and suggest that ZmrbohB gene may play a role in response to environmental stresses.

  9. Expression analysis of OsbZIP transcription factors in resistance ...

    African Journals Online (AJOL)

    zino

    2013-08-21

    Aug 21, 2013 ... Plant basic leucine zipper (bZIP) proteins play an essential role in the genes ... Key words: OsbZIP transcription factors, rice blast, resistance ... quantitative reverse transcriptions polymerase chain reaction. ... eukaryotes, which shared two common structures: a .... RNA extration and reverse transcription.

  10. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    Science.gov (United States)

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  11. Molecular cloning and in silico analysis of the duck (Anas platyrhynchos MEF2A gene cDNA and its expression profile in muscle tissues during fetal development

    Directory of Open Access Journals (Sweden)

    Hehe Liu

    2012-01-01

    Full Text Available The role of myogenic enhancer transcription factor 2a (MEF2A in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752 and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF -serum response factor, MEF2 and mitogen-activated protein kinase (MAPK transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues.

  12. Molecular cloning, expression, purification and crystallographic analysis of PRRSV 3CL protease

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Xinsheng; Feng, Youjun [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Zhao, Tiezhu [China Animal Disease Control Center, Beijing 100094 (China); Peng, Hao; Yan, Jinghua [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Jiang, Fan [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Tian, Kegong, E-mail: tiankg@263.net [China Animal Disease Control Center, Beijing 100094 (China); Gao, Feng, E-mail: tiankg@263.net [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); College of Biological Sciences, China Agricultural University, Beijing 100094 (China)

    2007-08-01

    Recombinant PRRSV 3CL protease was crystallized and the crystals diffracted to 2.1 Å resolution. 3CL protease, a viral chymotrypsin-like proteolytic enzyme, plays a pivotal role in the transcription and replication machinery of many RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the full-length 3CL protease from PRRSV was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that diffracted to 2.1 Å resolution and belong to space group C2, with unit-cell parameters a = 112.31, b = 48.34, c = 42.88 Å, β = 109.83°. The Matthews coefficient and the solvent content were calculated to be 2.49 Å{sup 3} Da{sup −1} and 50.61%, respectively, for one molecule in the asymmetric unit.

  13. Cloning, expression and mo-lecular characterization of promoter elements from Ba-cillus pumilus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Promoter elements from random chromosomal DNA of a rice epiphytic Bacillus pumilus were cloned into promoter probe shuttle vector ECE7 and sequenced. The results showed that these elements were all new DNA sequences. Six strong promoter elements were obtained by determination of CAT enzyme activity in both E. coli and B. pumilus. Transcription start sites of the cat mRNA were located by primer extension using total RNA. Comparison of the promoter sequences indicated that three of them contain -10 and -35 regions like B. pumilus s43 consensus sequence and another one is similar to B. pumilus s29. The other two have no typical consensus sequences of known sigma factors so far.

  14. Determination of protein expression and plasmid copy number from cloned genes in Escherichia coli by flow injection analysis using an enzyme indicator vector.

    Science.gov (United States)

    Schendel, F J; Baude, E J; Flickinger, M C

    1989-10-20

    On-line determination of expression rates from cloned genes in Escherichia coli and of plasmid copy number would be useful for monitoring accumulation of non-secreted proteins. As an initial model for monitoring gene expression in intact cells, a non-gene-fusion enzyme-based indicator plasmid has been constructed containing the phoA gene coding for alkaline phosphatase (AP) in pUCIS and pACYC184. The activity of AP can be rapidly determined in permeabilized cells. A flow injection analysis (FIA) assay has been developed which allows the direct real-time measurement of the AP activity during cell growth. A model target gene coding for E. coli cyanase (cynS) has been inserted in order to determine the ratio between the expression of the target and indicator, AP. A linear relationship has been found between plasmid copy number and AP activity for the high-copy pUC vector. To minimize indicator expression, transcription terminators have been inserted between the cynS and phoA genes, altering the target-to-indicator ratio by 10- to 40-fold. These vectors may be useful for the rapid continuous determination of plasmid copy number and target gene expression for nonsecreted proteins and would overcome the limitations of in situ probe biosensors for real-time determination of the accumulation of proteins from cloned genes in E. coli.

  15. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Davanloo, P.; Rosenberg, A.H.; Dunn, J.J.; Studier, F.W.

    1984-04-01

    The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNAs in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase. 25 references, 3 figures.

  16. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    Energy Technology Data Exchange (ETDEWEB)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. (F. Hoffmann-La Roche Ltd., Basel (Switzerland))

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  17. UII and UT in grouper: cloning and effects on the transcription of hormones related to growth control.

    Science.gov (United States)

    Sun, Caiyun; Duan, Da; Li, Bo; Qin, Chaobin; Jia, Jirong; Wang, Bin; Dong, Haiyan; Li, Wensheng

    2014-01-01

    Urotensin II (UII) is a cyclic peptide that was originally extracted from the caudal neurosecretory system (CNSS) of fish. UII is well known to exhibit cardiovascular, ventilatory, and motor effects in vertebrates. Studies have reported that UII exerts mitogenic effects and can act as an autocrine/paracrine growth factor in mammals. However, similar information in fish is limited. In this study, the full-length cDNAs of UII and its receptor (UT) were cloned and characterized in the orange-spotted grouper. UII and UT were expressed ubiquitously in various tissues in grouper, and particularly high levels were observed in the CNSS, CNS, and ovary. A functional study showed that UT was coupled with intracellular Ca2+ mobilization in HEK293 cells. Studies carried out using i.p. injections of UII in grouper showed the following: i) in the hypothalamus, UII can significantly stimulate the mRNA expression of ghrh and simultaneously inhibit the mRNA expression of somatostatin 1 (ss1) and ss2 3 h after injection; ii) in the pituitary, UII also significantly induced the mRNA expression of gh 6 and 12  h after injection; and iii) in the liver, the mRNA expression levels of ghr1/ghr2 and igf1/igf2 were markedly increased 12 and 3  h after the i.p. injection of UII respectively. These results collectively indicate that the UII/UT system may play a role in the promotion of the growth of the orange-spotted grouper.

  18. An expression analysis of 57 transcription factors derived from ESTs of developing seeds in Maize (Zea mays).

    Science.gov (United States)

    Wang, Guifeng; Wang, Hui; Zhu, Jia; Zhang, Jing; Zhang, Xiaowei; Wang, Fei; Tang, Yuanping; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2010-06-01

    Maize seeds are an important source of food, animal feed, and industrial raw materials. To understand global gene expression and regulation during maize seed development, a normalized cDNA library, covering most of the developmental stages of maize seeds, was constructed. Sequencing analysis of 10,848 randomly selected clones identified 6,630 unique ESTs. Among them, 57 putative transcription factors (TFs) were identified. The TFs belong to seven different super-families, specifically 17 Zinc-finger, 13 bZIP, 8 bHLH, 6 MADS, 7 MYB, 3 Homedomain, and 3 AP2/EREBP. The spatial and temporal expression of the TFs was analyzed by semi-quantitative RT-PCR with representative tissue types and seeds at different developmental stages, revealing their diverse expression patterns and expression levels. One-third (19) of the maize TFs was found their putative orthologs in Arabidopsis. Similar expression patterns were observed in both maize and Arabidopsis for the majority of orthologous pairs (15 out of 19), suggesting their conserved functions during seed development. In conclusion, the systematic analysis of maize seed TFs has provided valuable insight into transcriptional regulation during maize seed development.

  19. Subtractive transcriptome analysis of leaf and rhizome reveals differentially expressed transcripts in Panax sokpayensis.

    Science.gov (United States)

    Gurung, Bhusan; Bhardwaj, Pardeep K; Talukdar, Narayan C

    2016-11-01

    In the present study, suppression subtractive hybridization (SSH) strategy was used to identify rare and differentially expressed transcripts in leaf and rhizome tissues of Panax sokpayensis. Out of 1102 randomly picked clones, 513 and 374 high quality expressed sequenced tags (ESTs) were generated from leaf and rhizome subtractive libraries, respectively. Out of them, 64.92 % ESTs from leaf and 69.26 % ESTs from rhizome SSH libraries were assembled into different functional categories, while others were of unknown function. In particular, ESTs encoding galactinol synthase 2, ribosomal RNA processing Brix domain protein, and cell division cycle protein 20.1, which are involved in plant growth and development, were most abundant in the leaf SSH library. Other ESTs encoding protein KIAA0664 homologue, ubiquitin-activating enzyme e11, and major latex protein, which are involved in plant immunity and defense response, were most abundant in the rhizome SSH library. Subtractive ESTs also showed similarity with genes involved in ginsenoside biosynthetic pathway, namely farnesyl pyrophosphate synthase, squalene synthase, and dammarenediol synthase. Expression profiles of selected ESTs validated the quality of libraries and confirmed their differential expression in the leaf, stem, and rhizome tissues. In silico comparative analyses revealed that around 13.75 % of unigenes from the leaf SSH library were not represented in the available leaf transcriptome of Panax ginseng. Similarly, around 18.12, 23.75, 25, and 6.25 % of unigenes from the rhizome SSH library were not represented in available root/rhizome transcriptomes of P. ginseng, Panax notoginseng, Panax quinquefolius, and Panax vietnamensis, respectively, indicating a major fraction of novel ESTs. Therefore, these subtractive transcriptomes provide valuable resources for gene discovery in P. sokpayensis and would complement the available transcriptomes from other Panax species.

  20. Molecular cloning of feline resistin and the expression of resistin, leptin and adiponectin in the adipose tissue of normal and obese cats.

    Science.gov (United States)

    Takashima, Satoshi; Nishii, Naohito; Kato, Akiko; Matsubara, Tatsuya; Shibata, Sanae; Kitagawa, Hitoshi

    2016-01-01

    Resistin, one of the adipokines that has a cycteine-rich C-terminus, is considered to relate to the development of insulin resistance in rats. However, in cats, there is little knowledge regarding resistin. In this study, we cloned the feline resistin cDNA from adipose tissue by RT-PCR. The feline resistin clone contained an entire open reading frame encoding 107 amino acids that had 72.8%, 75.4%, 50.9% and 51.8% homology with bovine, human, mouse and rat homologues, respectively. In both subcutaneous and visceral adipose tissues, the transcription levels of feline resistin mRNA were significantly higher in obese cats than normal cats, and those of feline adiponectin mRNA were significantly lower in obese cats than normal cats. However, there was no difference in the expression of feline leptin between normal and obese cats. On the other hand, in both normal and obese cats, there were no significant differences in resistin, leptin and adiponectin mRNA levels between subcutaneous and visceral adipose tissues. In cats, the altered expression of resistin and adiponectin mRNA with obesity may contribute to the pathogenesis of insulin resistance and subsequent diabetes mellitus. In addition to feline adiponectin, the feline resistin cDNA clone obtained in this study will be useful for further investigation of the pathogenesis of obesity in cats.

  1. Gene cloning, expression, purification and characterization of lipoprotein- associated phospholipase A2 in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Fu-junZHANG; Yi-pingWANG

    2005-01-01

    AIM To express and purify Lipoprotein -associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors through the recombinant Lp-PLA2. METHODS The full-length gene of Lp-PLA2 was cloned from the differentiated THP-1 cells by RT-PCR and PCR. The Lp-PLA2 gene was subcloned into the Pichia expression vector pPIC9 and introduced a sequence encoding a C-terminal stretch of six histidine residues at the same time. The recombinant plasmid was transformed into Pichia pastoris GS115 by spheroplasting and the gene was then integrated into the GS115 genome. Lp- PLA2 was expressed in the yeast strain GS115 by inducing with 0.5% methanol.

  2. Cloning, Expression of Crocus sativus Phytoene Desaturase Gene and Preparation of Antiserum against It

    Institute of Scientific and Technical Information of China (English)

    Bai Jie; Miao Chen; Xu Ying; Tang Lin; Wang Zhi-tao; Chen Fang

    2004-01-01

    A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET -21a(+) and overexpressed in E. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma.

  3. Cloning and Expression of Recombinant Human Thymosin in Yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Cao Junxia(曹俊霞); Jin Liji; Duan Yanlong; An Lijia

    2003-01-01

    The gene of human thymosin alpha 1(hT(1)was synthesised according to favorite codons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24E that is N-acetylserine were replaced by hT(1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thymosin α1. And also,the Asn-Gly bond was designed to faciliate isolation of the target protein.The fusion gene was cloned into the expression vector, pPIC/9K. The constructs were transformed into HIS4 mutant strain GS115 by electroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.

  4. Molecular Cloning and Prokaryotic Expression of Non-Structural Protein NS1 Gene of Porcine Parvovirus

    Institute of Scientific and Technical Information of China (English)

    WU Dan; TONG Guang-zhi; QIU Hua-ji; XUE Qiang; ZHOU Yan-jun; LI Jing-peng

    2003-01-01

    Porcine parvovirus (PPV) is one of the major agents causing swine reproductive failure. NS1protein is a non-structural protein of PPV and can be used as a reagent for differentiation of vaccinated ani-mals and infected ones. In present study, a recombinant plasmid pET28a/NS1 was constructed by cloning thecoding sequence for NS1 of PPV into pET28a, a bacterial expression vector. The NS1 protein was expressed inE. coli BL21 (DE3) after induced by IPTG and the recombinant fusion protein was purified with affinity chro-matography. Expression amount of NS1 protein was improved by optimizing the inducing parameters. The re-combinant NS1 protein is reactive to PPV positive sera in Western blot and ELISA test and therefore can beapplicable in differential diagnosis of PPV infections.

  5. Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon shedaoensis Zhao

    Institute of Scientific and Technical Information of China (English)

    Qian JIN; Li-Xia YANG; Hao-Mang JIAO; Bin LU; Yu-Qun WU; Yuan-Cong ZHOU

    2004-01-01

    A protein with the activity of phospholipase A2 named asAPLA2 was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E. Coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the nativeenzyme.

  6. Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon shedaoensis Zhao

    Institute of Scientific and Technical Information of China (English)

    QianJIN; Li-XiaYANG; Hao-MangJIAO; BinLU; Yu-QunWU; Yuan-CongZHOU

    2004-01-01

    A protein with the activity of phospholipase A2 named asAPLA2 was pmified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1μmol/L ADP, and the IC50 was determined to be 6μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E.coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibc dy against the native enzyme.

  7. Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    Nan Wang

    2011-01-01

    Full Text Available Alcohol dehydrogenases (ADH are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD or nicotinamide adenine dinucleotide phosphate (NADP, as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+. The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3, and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH.

  8. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    Science.gov (United States)

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  9. Cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp.

    Directory of Open Access Journals (Sweden)

    Hamed Esmaeil Lashgarian

    2016-10-01

    Full Text Available Cholesterol oxidase (CHO is one of the valuable enzymes that play an important role in: measurement of serum cholesterol, food industry as a biocatalyst and agriculture as a biological larvicide. This enzyme was produced by several bacterial strains. Wild type enzyme produced by Rhodococcus sp. secret two forms of CHO enzyme: extra cellular and membrane bound type which its amount is low and unstable. The goal of the study was cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp. CHO gene was isolated from native bacteria and cloned into pET23a. In the next step, the construct was expressed in E.coli BL21 and induced by different concentration of IPTG ranges from 0.1 - 0.9 mM. This gene contains 1642 bp and encodes a protein consists of 533 amino acids. It has about 96 % homology with CHO gene isolated from Rhodococcus equi. The high expression was obtained in 0.5 mM concentration of IPTG after 4 hour induction. This recombinant enzyme had a molecular weight of 55 kDa, that secretion of intra cellular type is much more than extracellular form. The optimum pH and temperature conditions for the recombinant enzyme were 7.5 and 45°C, respectively. CHO enzyme obtained from Rhodococcus sp. is a cheap enzyme with medical and industrial applications that can be produced easily and purified in large scale with simple methods.

  10. The cloning and expression characterization of the centrosome protein genes family (centrin genes) in rat testis

    Institute of Scientific and Technical Information of China (English)

    SUN; Xiaodong(孙晓冬); GE; Yehua(葛晔华); MA; Jing(马静); YU; Zuoren(俞作仁); LI; Sai(李赛); WANG; Yongchao(王永潮); XUE; Shepu(薛社普); HAN; Daishu(韩代书)

    2002-01-01

    Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.

  11. A metagenomic alkaline protease from saline habitat: cloning, over-expression and functional attributes.

    Science.gov (United States)

    Purohit, Megha K; Singh, Satya P

    2013-02-01

    Metagenomics has opened new horizon to unlock the biotechnological potential for novel enzymes. An alkaline protease gene was obtained from the total environmental DNA extracted from a saline habitat. After cloning and sequencing, it was identified that the protease gene related to uncultivable bacteria (HM219181). The protease was over expressed at 6h of induction with optimum induction at 1mM IPTG and 27°C. The purified enzyme was characterized with respect to various factors; temperature, pH, NaCl and chemical denaturant. The sequence analysis indicated a hydrophobic tendency of the protein, while the predicted 3D structure indicated the enzyme as a serine protease.

  12. Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Gidekel Manuel

    2009-10-01

    Full Text Available Abstract Background Deschampsia antarctica shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the D. antarctica Cu/ZnSOD gene into Pichia pastoris and to characterize the heterologous protein. Findings The Copper/Zinc superoxide dismutase (Cu/ZnSOD gene, SOD gene, was isolated from a D. antarctica by cDNA library screening. This SOD gene was cloned in the expression vector pGAPZαA and successfully integrated into the genome of the yeast P. pastoris SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg. Conclusion D. antarctica Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40°C for 30 minutes and it is stable (80% of activity between -20°C and 20°C. The recombinant SOD described in this study can be used in various biotechnological applications.

  13. Cloning, Sequencing and Expression Analysis of the First Cellulase Gene Encoding Cellobiohydrolase 1 from a Cold-adaptive Penicillium chrysogenum FS010

    Institute of Scientific and Technical Information of China (English)

    Yunhua HOU; Tianhong WANG; Hao LONG; Huiyuan ZHU

    2007-01-01

    A cellobiohydrolase 1 gene (cbh1) was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). DNA sequencing shows that cbh1 has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbh1 promoter (1.3 kb) was also cloned and sequenced, which contains seven putative binding sites (5'-SYGGRG-3') for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbh1 transcription of P. chrysogenum was examined by Northern analysis,suggesting that the expression of cbh1 is regulated at transcriptional level. The cbh1 gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.

  14. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    Science.gov (United States)

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.

  15. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  16. The potato developer (D) locus encodes an R2R3 MYB transcription factor that regulates expression of multiple anthocyanin structural genes in tuber skin.

    Science.gov (United States)

    Jung, Chun Suk; Griffiths, Helen M; De Jong, Darlene M; Cheng, Shuping; Bodis, Mary; Kim, Tae Sung; De Jong, Walter S

    2009-12-01

    A dominant allele at the D locus (also known as I in diploid potato) is required for the synthesis of red and purple anthocyanin pigments in tuber skin. It has previously been reported that D maps to a region of chromosome 10 that harbors one or more homologs of Petunia an2, an R2R3 MYB transcription factor that coordinately regulates the expression of multiple anthocyanin biosynthetic genes in the floral limb. To test whether D acts similarly in tuber skin, RT-PCR was used to evaluate the expression of flavanone 3-hydroxylase (f3h), dihydroflavonol 4-reductase (dfr) and flavonoid 3',5'-hydroxylase (f3'5'h). All three genes were expressed in the periderm of red- and purple-skinned clones, while dfr and f3'5'h were not expressed, and f3h was only weakly expressed, in white-skinned clones. A potato cDNA clone with similarity to an2 was isolated from an expression library prepared from red tuber skin, and an assay developed to distinguish the two alleles of this gene in a diploid potato clone known to be heterozygous Dd. One allele was observed to cosegregate with pigmented skin in an F(1) population of 136 individuals. This allele was expressed in tuber skin of red- and purple-colored progeny, but not in white tubers, while other parental alleles were not expressed in white or colored tubers. The allele was placed under the control of a doubled 35S promoter and transformed into the light red-colored cultivar Désirée, the white-skinned cultivar Bintje, and two white diploid clones known to lack the functional allele of D. Transformants accumulated pigment in tuber skin, as well as in other tissues, including young foliage, flower petals, and tuber flesh.

  17. Population differences in transcript-regulator expression quantitative trait loci.

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    Pierre R Bushel

    Full Text Available Gene expression quantitative trait loci (eQTL are useful for identifying single nucleotide polymorphisms (SNPs associated with diseases. At times, a genetic variant may be associated with a master regulator involved in the manifestation of a disease. The downstream target genes of the master regulator are typically co-expressed and share biological function. Therefore, it is practical to screen for eQTLs by identifying SNPs associated with the targets of a transcript-regulator (TR. We used a multivariate regression with the gene expression of known targets of TRs and SNPs to identify TReQTLs in European (CEU and African (YRI HapMap populations. A nominal p-value of <1×10(-6 revealed 234 SNPs in CEU and 154 in YRI as TReQTLs. These represent 36 independent (tag SNPs in CEU and 39 in YRI affecting the downstream targets of 25 and 36 TRs respectively. At a false discovery rate (FDR = 45%, one cis-acting tag SNP (within 1 kb of a gene in each population was identified as a TReQTL. In CEU, the SNP (rs16858621 in Pcnxl2 was found to be associated with the genes regulated by CREM whereas in YRI, the SNP (rs16909324 was linked to the targets of miRNA hsa-miR-125a. To infer the pathways that regulate expression, we ranked TReQTLs by connectivity within the structure of biological process subtrees. One TReQTL SNP (rs3790904 in CEU maps to Lphn2 and is associated (nominal p-value = 8.1×10(-7 with the targets of the X-linked breast cancer suppressor Foxp3. The structure of the biological process subtree and a gene interaction network of the TReQTL revealed that tumor necrosis factor, NF-kappaB and variants in G-protein coupled receptors signaling may play a central role as communicators in Foxp3 functional regulation. The potential pleiotropic effect of the Foxp3 TReQTLs was gleaned from integrating mRNA-Seq data and SNP-set enrichment into the analysis.

  18. A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

    Science.gov (United States)

    Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

    2013-08-20

    Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior

  19. Transcription profiling reveals stage- and function-dependent expression patterns in the filarial nematode Brugia malayi

    Directory of Open Access Journals (Sweden)

    Li Ben-Wen

    2012-05-01

    Full Text Available Abstract Background Brugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring and disabiling tropical disease. Although a first draft genome sequence was released in 2007, very little is understood about transcription programs that govern developmental changes required for the parasite’s development and survival in its mammalian and insect hosts. Results We used a microarray with probes that represent some 85% of predicted genes to generate gene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% of transcripts with detectable expression signals were differentially expressed across lifecycle stages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentially expressed transcripts revealed five major transcription patterns that were associated with parasite lifecycle stages or gender. Examination of known stage-associated transcripts validated these data sets and suggested that newly identified stage or gender-associated transcripts may exercise biological functions in development and reproduction. The results also indicate that genes with similar transcription patterns were often involved in similar functions or cellular processes. For example, nuclear receptor family gene transcripts were upregulated in gene expression pattern four (female-enriched while protein kinase gene family transcripts were upregulated in expression pattern five (male-enriched. We also used pair-wise comparisons to identify transcriptional changes between life cycle stages and sexes. Conclusions Analysis of gene expression patterns of lifecycle in B. malayi has provided novel insights into the biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specific transcripts are likely to be critically important for key parasite functions such as establishment and maintenance of

  20. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

    Science.gov (United States)

    Goswami, Suneha; Kumar, Ranjeet R; Dubey, Kavita; Singh, Jyoti P; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C; Kala, Yugal K; Singh, Gyanendra P; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat.

  1. SSH analysis of endosperm transcripts and characterization of heat stress regulated expressed sequence tags in bread wheat

    Directory of Open Access Journals (Sweden)

    Suneha Goswami

    2016-08-01

    Full Text Available Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h wheat cv. HD2985 by suppression subtractive hybridization (SSH. We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger’s sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs. Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs. We observed eight different types of post-translational modifications (PTMs in the DEPs corresponds to the cloned ESTs—147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant, as compared to HD2329 (thermosusceptible during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat – a novel step towards the development of

  2. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

    Science.gov (Uni