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Sample records for cdna clones mammalian

  1. cDNA expression cloning in mammalian cells.

    Science.gov (United States)

    Hoffman, B J

    2001-05-01

    This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library. PMID:18428491

  2. Cloning vectors for expression of cDNA libraries in mammalian cells.

    OpenAIRE

    Murphy, A.J.; Efstratiadis, A

    1987-01-01

    We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient...

  3. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

    OpenAIRE

    Udo, Hiroshi

    2015-01-01

    One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as...

  4. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen;

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k...

  5. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination.

    Directory of Open Access Journals (Sweden)

    Hiroshi Udo

    Full Text Available One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter. Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15 resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75% and directional cloning (99% by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.

  6. Molecular cloning and characterization of pig, cow and sheep MAdCAM-1 cDNA and the demonstration of cross-reactive epitopes amongst mammalian homologues.

    Science.gov (United States)

    Tachedjian, M; Yu, M; Lew, A M; Rockman, S; Boyle, J S; Andrew, M E; Wang, L

    2006-05-01

    Full-length cDNA clones for the pig, cow and sheep mucosal addressin cellular adhesion molecule (MAdCAM)-1 homologues were isolated from Peyer's patches by a combination of reverse transcription (RT)-polymerase chain reaction and 5' and 3' RACE strategies. Degenerate primers based on conserved amino acid (aa) sequences within the N-terminal immunoglobulin (Ig)-like domains of the human and rodent MAdCAM-1 molecules were used for initial sequencing of the Ig-like domains. MAdCAM-1 transcripts of 1425 bp, 1525 bp and 1510 bp obtained for the pig, cow and sheep contained an open-reading frame for proteins of 390, 424 and 418 aa, respectively. The pig and ruminant MAdCAM-1 had two N-terminal Ig-like domains, a mucin-like region and a third Ig-like domain found in rodent but not human MAdCAM-1. Antibodies raised against bacterially expressed N-terminal Ig-like domains of pig, human and sheep MAdCAM-1 demonstrated the existence of cross-reactive epitopes, raising the possibility of producing monoclonal antibodies which can be used as multi-species MAdCAM-1-targeting reagent for the development of mucosal vaccines. PMID:16671951

  7. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  8. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A;

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  9. 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.

  10. 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.

  11. Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (Huso huso) and the disruptive effect of methylmercury on gene expression.

    Science.gov (United States)

    Gharaei, Ahmad; Mahboudi, Fereidoun; Esmaili-Sari, Abbas; Edalat, Rozita; Adeli, Ahmad; Keyvanshokooh, Saeed

    2010-09-01

    Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg(-1) [control]; 0.76 mg kg(-1) [low]; 7.8 mg kg(-1) [medium]; 16.22 mg kg(-1) [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga. PMID:19821139

  12. 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones Data detail Data name 5'-end sequence...s of budding yeast full-length cDNA clones Description of data contents cDNA sequence...e Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive ...

  13. First characterization of infectious cDNA clones of Olive mild mosaic virus

    OpenAIRE

    Cardoso, Joana M.S.; Félix, M. Rosário; Clara, M.Ivone; Oliveira, Solange

    2012-01-01

    Full-length cDNA clones of an Olive mild mosaic virus (OMMV) isolate were constructed in order to find infectious cDNA clones. The sequencing of three individual full-length clones revealed some differences between them. In vitro transcription of these clones was performed and the effect of spontaneous mutations in the biological behaviour of the in vitro transcripts was evaluated by symptomatology, RNA accumulation and virus replication in inoculated plants. In vitro synthesized RNA from one...

  14. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor

    Energy Technology Data Exchange (ETDEWEB)

    Dobner, P.R.; Barber, D.L.; Villa-Komaroff, L.; McKiernan, C.

    1987-05-01

    Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis using /sup 32/P-labeled nucleotides, has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional coding domain, resembling neuromedin N, occurs immediately after an Arg-Arg basic amino acid pair located in the central region of the precursor. Additional amino acid homologies suggest that tandem duplications have contributed to the structure of the gene. RNA blot analysis, using the cloned cDNA probe, has revealed several mRNA species ranging in size from 500 to 980 nucleotides in the canine enteric mucosa. In contrast a single RNA species of 1500 nucleotides was detected in bovine hypothalamus poly-(A)/sup +/ RNA. The ability of the canine probe to cross-hybridize with bovine mRNA suggest that this probe can be used to isolate neurotensin/neuromedin N genes from other mammalian species.

  15. Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus.

    Science.gov (United States)

    Ogawa, Yuko; Murayama, Nobuhiro; Fujita, Yoshiaki; Yanoshita, Ryohei

    2007-06-15

    The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile. PMID:17383704

  16. 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.

  17. Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

    Science.gov (United States)

    Gao, Jinliang; Luo, Jianxun; Fan, Ruiquan; Fingerle, Volker; Guan, Guiquan; Liu, Zhijie; Li, Youquan; Zhao, Haiping; Ma, Miling; Liu, Junlong; Liu, Aihong; Ren, Qiaoyun; Dang, Zhisheng; Sugimoto, Chihiro; Yin, Hong

    2008-03-01

    The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

  18. 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.

  19. 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.

  20. Construction of infectious cDNA clones for RNA viruses: Turnip crinkle virus.

    Science.gov (United States)

    Ryabov, Eugene V

    2008-01-01

    Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious cDNA clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription, PCR amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, and selection of infectious clones. PMID:18370276

  1. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    International Nuclear Information System (INIS)

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  2. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  3. Cloning and cDNA sequence of the regulator subunit of cAMP-dependent protein kinase from Dictyostelium discoideum

    International Nuclear Information System (INIS)

    cDNA clones encoding the regulatory subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from Dictyostelium discoideum were isolated by immunoscreening of a cDNA library constructed in the expression vector λgt11, using autoradiography. High-affinity cAMP binding activity was detected in extracts from bacteria lysogenized with these clones. Nucleotide sequence analysis of three overlapping clones allowed the determination of a 1195-base-pair cDNA sequence coding for the entire regulatory subunit and containing nontranslated 5' and 3' sequences. The open reading frame codes for a protein of 327 amino acids, with molecular weight 36,794. The regulatory subunit from Dictyostelium shares a high degree of homology with its mammalian counterparts, but is lacking the NH2-terminal domain required for the association of regulatory subunits into dimers in other eukaryotes. On the basis of the comparison of the regulatory subunits from Dictyostelium, yeast, and bovine tissues, a model for the evolution of these proteins is proposed

  4. Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

    International Nuclear Information System (INIS)

    Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

  5. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    Science.gov (United States)

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  6. Expression cloning of a cDNA encoding the bovine histamine H1 receptor.

    OpenAIRE

    Yamashita, M; Fukui, H; Sugama, K; Horio, Y; Ito, S.; Mizuguchi, H.; Wada, H

    1991-01-01

    A functional cDNA clone for the histamine H1 receptor was isolated from a cDNA library of bovine adrenal medulla by a combination of molecular cloning in an expression vector and electrophysiological assay in Xenopus oocytes. The H1 receptor cDNA encodes a protein of 491 amino acids (Mr 55,954) with seven putative transmembrane domains, illustrating the similarity to other receptors that couple with guanine nucleotide-binding regulatory proteins (G protein-coupled receptors). The sequence hom...

  7. Cloning and expression of cDNA for salmon growth hormone in Escherichia coli

    OpenAIRE

    Sekine, Susumu; Mizukami, Tamio; Nishi, Tatsunari; Kuwana, Yoshihisa; Saito, Akiko; Sato, Moriyuki; Itoh, Seiga; Kawauchi, Hiroshi

    1985-01-01

    cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putativ...

  8. Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

    Institute of Scientific and Technical Information of China (English)

    Xiaohui ZHANG; Shangzhong XU; Xue GAO; Hongyan REN; Jinbao CHEN

    2008-01-01

    The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3' RACE and 5' RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% sim-ilarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, car-diac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle.

  9. Genetic instability of Japanese encephalitis virus cDNA clones propagated in Escherichia coli.

    Science.gov (United States)

    Zheng, Xuchen; Tong, Wu; Liu, Fei; Liang, Chao; Gao, Fei; Li, Guoxin; Tong, Guangzhi; Zheng, Hao

    2016-04-01

    The genetic instability of Flavivirus cDNA clones in transformed bacteria is a common phenomenon. Herein, a cDNA fragment of the nucleotide (nt) 1-2913 of the genome of a flavivirus, Japanese encephalitis virus (JEV), was used to investigate factors that caused the instability of cDNA clones. Several cDNA fragments with different 5'- or 3'-termini of the 2913-nt cDNA were obtained by PCR amplification or restriction enzyme digestion and cloned into a pCR-Blunt II-TOPO vector. All the cDNA fragments were stably propagated at 25 °C. However, the 5'-untranslated region and half of the 3'-E gene could cause the instability of the 2913-nt cDNA at 37 °C. The 5'-terminus sequences of the 2913-nt fragment were subjected to testing of the prokaryotic promoter activity by luciferase assay and Western blot. The sequences of 54-120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. These findings revealed that the high prokaryotic promoter activity of the 54-120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5'-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria. PMID:26888374

  10. Systematic sequencing of cDNA clones using the transposon Tn5

    OpenAIRE

    Shevchenko, Yuriy; Bouffard, Gerard G.; Butterfield, Yaron S.N.; Blakesley, Robert W.; Hartley, James L.; Young, Alice C.; Marco A. Marra; Jones, Steven J M; Touchman, Jeffrey W.; Green, Eric D.

    2002-01-01

    In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inser...

  11. Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

    OpenAIRE

    Erma Sulistyaningsih; Sukarti Moeljopawiro; Jarot Subandono; Wayan T. Artama

    2015-01-01

    Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recom...

  12. The Arabian camel Camelus dromedarius heat shock protein 90α: cDNA cloning, characterization and expression.

    Science.gov (United States)

    Saeed, Hesham; Shalaby, Manal; Embaby, Amira; Ismael, Mohammad; Pathan, Akbar; Ataya, Farid; Alanazi, Mohammad; Bassiouny, Khalid

    2015-11-01

    Heat shock protein 90 (Hsp90) is a highly conserved ubiquitous molecular chaperone contributing to assisting folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In the present study, a heat shock protein 90α full length coding cDNA was isolated and cloned from the Arabian one-humped camel by reverse transcription polymerase chain reaction (RT-PCR). The full length cDNA sequence was submitted to NCBI GeneBank under the accession number KF612338. The sequence analysis of the Arabian camel Hsp90α cDNA showed 2202bp encoding a protein of 733 amino acids with estimated molecular mass of 84.827kDa and theoretical isoelectric point (pI) of 5.31. Blast search analysis revealed that the C. dromedarius Hsp90α shared high similarity with other known Hsp90α. Comparative analyses of camel Hsp90α protein sequence with other mammalian Hsp90s showed high identity (85-94%). Heterologous expression of camel Hsp90α cDNA in E. coli JM109 (DE3) gave a fusion protein band of 86.0kDa after induction with IPTG for 4h.

  13. CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

    Institute of Scientific and Technical Information of China (English)

    Liu Qingyong(刘庆勇); Ruan Xiyun(阮喜云); Liu Xiaogong(刘效恭); Ji Zongzheng(纪宗正); Dang Jiangong; Nan Xunyi(南勋义); Wang Quanying(王全颖); Yang Guangxiao(杨广笑)

    2003-01-01

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

  14. 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.

  15. Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    S. S. Hasson

    2010-01-01

    Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

  16. Serine protease variants encoded by Echis ocellatus venom gland cDNA: cloning and sequencing analysis.

    Science.gov (United States)

    Hasson, S S; Mothana, R A; Sallam, T A; Al-balushi, M S; Rahman, M T; Al-Jabri, A A

    2010-01-01

    Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent. PMID:20936075

  17. 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.

  18. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    OpenAIRE

    CLAUDIA TEREZIA SOCOL; A. VLAIC; VIORICA COSIER

    2013-01-01

    An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, cont...

  19. Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco.

    Science.gov (United States)

    Farmer, M J; Czernic, P; Michael, A; Negrel, J

    1999-08-01

    The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl

  20. Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA3 application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity with AAIR genes from other plant species. This cDNA was cloned into expression vector and recombinant E. coli DH5α cells with remarkable AAIR enzyme activity were obtained.

  1. Cloning and expression of cDNA for anti-müllerian hormone.

    OpenAIRE

    Picard, J Y; Benarous, R; Guerrier, D.; Josso, N; Kahn, A.

    1986-01-01

    Messenger RNA, prepared from fetal bovine testicular tissue, was used to construct a cDNA library in lambda gt11 phage. The library was screened with an antibody probe directed against bovine anti-Müllerian hormone and three positive clones were isolated. Cross-hybridizing cDNA inserts carried by clones 4 and 5 (1.2 and 0.08 kilobases long, respectively) code for a fragment of authentic anti-Müllerian hormone, as shown by the ability of the anti-epitope antibodies eluted from fusion protein 4...

  2. Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

    OpenAIRE

    Zarucki-Schulz, T; Kulomaa, M S; Headon, D R; N.L. Weigel; Baez, M; Edwards, D.P.; McGuire, W L; Schrader, W T; O'Malley, B W

    1984-01-01

    A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 10...

  3. Molecular cloning of cDNA encoding the small subunit of Drosophila transcription initiation factor TFIIF.

    OpenAIRE

    Gong, D W; Mortin, M A; Horikoshi, M; Nakatani, Y

    1995-01-01

    Transcription initiation factor TFIIF is a tetramer consisting of two large subunits (TFIIF alpha or RAP74) and two small subunits (TFIIF beta or RAP30). We report here the molecular cloning of a Drosophila cDNA encoding TFIIF beta. The cDNA clone contains an open-reading frame encoding a 277 amino acid polypeptide having a calculated molecular mass of 32,107 Da. Comparison of the deduced amino acid sequence with the corresponding sequences from vertebrates showed only 50% identity, with four...

  4. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2013-12-01

    Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

  5. Molecular cloning of human interferon cDNA.

    OpenAIRE

    Taniguchi, T.; Fujii-Kuriyama, Y; Muramatsu, M

    1980-01-01

    A hybrid plasmid, TpIF319, has been shown to contain the sequence for human fibroblast interferon mRNA [Taniguchi, T., Sakai, M., Fujii-Kuriyama, Y., Muramatsu, M., Kobayashi, S. & Sudo, T. (1979) Proc. Jpn. Acad. 55, Ser. B, 464-469]. This conclusion was confirmed by a hybridization-translation assay, using rabbit globin mRNA and its cDNA-containing plasmid as a control. Plasmid TpIF319 was used as probe and another recombinant plasmid, TpIF319-13, whose cDNA insert consists of about 800 bas...

  6. Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

    Directory of Open Access Journals (Sweden)

    Erma Sulistyaningsih

    2015-10-01

    Full Text Available Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA

  7. Creation and validation of a ligation-independent cloning (LIC retroviral vector for stable gene transduction in mammalian cells

    Directory of Open Access Journals (Sweden)

    Patel Asmita

    2012-01-01

    Full Text Available Abstract Background Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert. Results Utilizing ligation-independent cloning (LIC technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358. Conclusions Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.

  8. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine

    International Nuclear Information System (INIS)

    O6-Methylguanine-DNA methyltransferase a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O6-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-like cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada-) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGNT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A)+ RNA transcript

  9. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M;

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 p...

  10. CLONING AND CHARACTERIZATION OF CDNA ENCODING GIARDIA LAMBLIA d-GIARDIN

    Science.gov (United States)

    A cDNA coding for d-giardin was cloned from Giardia lamblia trophozoites in order to localize the protein and study its function in mediating surface attachment. Recombinant d-giardin antigen was produced in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatogr...

  11. Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

    Institute of Scientific and Technical Information of China (English)

    HUANG Jun-hua; LI Yong-feng; HE Fan; LI Dan; SUN Yuan; HAN Wen; QIU Hua-ji

    2013-01-01

    The reverse genetics for classical swine fever virus (CSFV) is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low-copy vector pOK12, producing pOKShimen-RzT . Direct transfection of pOKShimen-RzT into PK/T7 cells, a PK-15-derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

  12. The characterization of cDNA clones coding for wheat storage proteins.

    OpenAIRE

    Bartels, D.; Thompson, R D

    1983-01-01

    Poly(A)+ RNA isolated from the developing wheat endosperm var. Chinese Spring, has been used as template for the construction of a cDNA library. Within the library, clones have been identified by in vitro translation of hybrid-selected mRNA which encode alpha/beta gliadin related sequences and gamma-gliadin related sequences. The DNA sequence of one such clone has been determined and it shows homology with that of a clone encoding a barley storage protein, B-hordein. The sequence includes a t...

  13. A cDNA clone from Zea mays endosperm sucrose synthetase mRNA.

    OpenAIRE

    Geiser, M.; Döring, H P; Wöstemeyer, J; Behrens, U.; Tillmann, E; Starlinger, P.

    1980-01-01

    A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.

  14. Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

    Indian Academy of Sciences (India)

    T Venugopal; S Mathavan; T J Pandian

    2002-06-01

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.

  15. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5' UTS (untranslated sequences) is 253 bp, 3' UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.

  16. Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

    Institute of Scientific and Technical Information of China (English)

    SU Song-kun; ZHNEG Huo-qing; CHEN Sheng-lu; ZHONG Bo-xiong; Stefan Albert

    2005-01-01

    By screening the worker (Apis cerana cerana) heads cDNA library using a fragment of the mrjp3 gene ofApis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 (AccMRJP3) cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly (A) tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame (ORF) with 1 779 bp encoding 593 amino acids. The un-translated regions (UTR) of the 5' end and 3' end are 46 bp and 160 bp in length,respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

  17. Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

    Science.gov (United States)

    Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

    2011-06-01

    A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

  18. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  19. Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

    Directory of Open Access Journals (Sweden)

    Woodruff Teresa K

    2004-10-01

    Full Text Available Abstract Background Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA. Methods The subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR and 5' rapid amplification of cDNA ends (RACE RT-PCR from adult rhesus monkey testis RNA. Results Both the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0, which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1. Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained. Conclusion The data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.

  20. 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).

  1. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

  2. cDNA Cloning and Sequence Analysis of Rice Sbel and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHENXiu-hua; LIUQiao-quan; WuHsin-kan; WANGZong-yang; GuMing-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes SheI and Shed encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA libray, derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned SheI and Shed cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of She3 was the same as that of shed (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned She1 cDNA and the reported she1 (Genbank Accession No. D11082). The cloned SheI and Shed cDNAs make it possible to improve rice starch quality through genetic engineering.

  3. cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiu-hua; LIU Qiao-quan; WU Hsin-kan; WANG Zong-yang; GU Ming-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbe3 encoding SBE I and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbe3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned Sbe1 cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

  4. Random rapid amplification of cDNA ends (RRACE) allows for cloning of multiple novel human cDNA fragments containing (CAG)n repeats.

    Science.gov (United States)

    Carney, J P; McKnight, C; VanEpps, S; Kelley, M R

    1995-04-01

    We describe a new technique for isolating cDNA fragments in which (i) either a partial sequence of the cDNA is known or (ii) a repeat sequence is utilized. We have used this technique, termed random rapid amplification of cDNA ends (random RACE), to isolate a number of trinucleotide repeat (CAG)n-containing genes. Using the random RACE (RRACE) technique, we have isolated over a hundred (CAG)n-containing genes. The results of our initial analysis of ten clones indicate that three are identical to previously cloned (CAG)n-containing genes. Three of our clones matched with expressed sequence tags, one of which contained a CA repeat. The remaining four clones did not match with any sequence in GenBank. These results indicate that this approach provides a rapid and efficient method for isolating trinucleotide repeat-containing cDNA fragments. Finally, this technique may be used for purposes other than cloning repeat-containing cDNA fragments. If only a partial sequence of a gene is known, our system, described here, provides a rapid and efficient method for isolating a fragment of the gene of interest. PMID:7536696

  5. Molecular Cloning and Bacterial Expression of Germacrene A Synthase cDNA from Crepidiastrum sonchifolium

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Germacrene A synthase(GAS) catalyzes the biosynthesis of germacrene A, which is a key precursor for sesquiterpene lactones. Cloning of a novel full-length cDNA encoding GAS from the medicinal plant Crepidiastrum sonchifolium(designated CsGAS) is reported in this study. The cDNA is 1837 bp long and contains a 1680-bp open reading frame encoding a 559 amino-acid protein. The functional expression of the cDNA in Escherichia coli, as an N-terminal thioredoxin fusion protein, with the pET32a vector yielding a recombinant enzyme. Sequence analysis was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco, and the effect of possible involvement of a number of amino acids in sesquiterpene synthase on product specificity was also discussed.

  6. 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.

  7. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  8. Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus).

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    1999-08-01

    We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS-PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

  9. Identification of cDNA clones encoding HMG 2, a major protein of the mexican axolotl hydrocortisone-sensitive thymocytes.

    Science.gov (United States)

    de Guerra, A; Guillet, F; Charlemagne, J; Fellah, J S

    1995-01-01

    We have identified and analyzed cDNA clones encoding a major 26 kDa protein of the HMG1-2 family which is abundant in the cytoplasm and nucleus of axolotl hydrocortisone-sensitive thymocytes. The axolotl HMG2 protein is very similar to proteins belonging to the HMG1-2 family, from teleost fish to mammals. All the molecular features of the HMG1-2 proteins are conserved, including the high proportion of basic and aromatic residues, and the characteristic acidic C-terminus tail. The 3'-untranslated region of the HMG2 axolotl cDNA is also similar to the avian and mammalian HMG2 3'-UT sequences, suggesting that some selective events have acted at the DNA level to conserve this region, which could be important in the differential expression of the HMG1 and HMG2 genes. The axolotl HMG2 protein contains the two well conserved HMG boxes which are thought to be the DNA-binding domains of the molecule. Axolotl thymocytes and spleen cells contain almost identical amounts of HMG2 mRNAs but HMG2 polypeptide is undetectable in spleen cells using anti-26 kDa antibodies. The reason for the accumulation of HMG1-2 molecules in vertebrate hydrocortisone-sensitive thymocytes is discussed, as well as their possible role in apoptosis. PMID:8654668

  10. Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.

  11. CLONING AND CHARACTERIZATION OF HUMAN UBIQUITIN BINDING ENZYME 2 cDNA

    Institute of Scientific and Technical Information of China (English)

    李光涛; 吕鸿雁; 周严; 金鉴; 蒋科艺; 彭小忠; 袁建刚; 强伯勤

    2002-01-01

    Objective.To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern. Methods.According to the sequence of human EST,which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2),primers were synthesized to screen the human fetal brain cDNA library.The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple tissue Northern blot. Results.Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified.Both containing the ubiquitin conjugating domain,the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence.They belong to a highly conserved and widely expressed E2 enzyme family.Northern blot shows that they are expressed exclusively in adult human heart,placenta,and pancreas but no transcripts can be detected in brain,lung,liver,skeletal muscle or kidney.Conclusions.The gene encoding human ubiquitin binding enzyme is expressed under temporal control.As a key enzyme in the degradation of proteins,ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post translation.

  12. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  13. Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

    International Nuclear Information System (INIS)

    A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

  14. Cloning and sequencing of complete -crystallin cDNA from embryonic lens of Crocodylus palustris

    Indian Academy of Sciences (India)

    Raman Agrawal; Reena Chandrashekhar; Anurag Kumar Mishra; Jetty Ramadevi; Yogendra Sharma; Ramesh K Aggarwal

    2002-06-01

    -Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete -crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the -enolase gene from non-lenticular tissues. Quantitatively, the -crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile -crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete -crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′-and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme -enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of -crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

  15. Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.

    Science.gov (United States)

    Horii, T; Bzik, D J; Inselburg, J

    1988-07-01

    A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts. PMID:2456465

  16. Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone

    Science.gov (United States)

    Schwarz, Megan C.; Sourisseau, Marion; Espino, Michael M.; Gray, Essanna S.; Chambers, Matthew T.; Tortorella, Domenico

    2016-01-01

    ABSTRACT The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates. IMPORTANCE The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and

  17. Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone

    Science.gov (United States)

    Schwarz, Megan C.; Sourisseau, Marion; Espino, Michael M.; Gray, Essanna S.; Chambers, Matthew T.; Tortorella, Domenico

    2016-01-01

    ABSTRACT The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates. IMPORTANCE The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and

  18. Isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone.

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    2000-02-01

    We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS-PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5' and 3' ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71-74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47-49%) identity with salmonid and carp GHs.

  19. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.

    OpenAIRE

    Okazaki, K.; Okazaki, N.; Kume, K.; Jinno, S; Tanaka, K; Okayama, H

    1990-01-01

    We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The tran...

  20. Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.

    Science.gov (United States)

    Endo, Noriyuki; Kan-no, Nobuhiro; Nagahisa, Eizoh

    2007-06-01

    Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor. PMID:17350870

  1. Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

    OpenAIRE

    Lee, W S; Wells, R G; Sabbag, R V; Mohandas, T K; Hediger, M A

    1993-01-01

    We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the r...

  2. Expression cloning of cDNA encoding a seven-helix receptor from human placenta with affinity for opioid ligands

    OpenAIRE

    1992-01-01

    Here we report the expression cloning of cDNA encoding a putative opioid receptor from a human placenta cDNA library. Placental opioid receptors are of the kappa type. As the dynorphin opioid peptides are kappa-selective, a dynorphin ligand was used in an affinity-enrichment (panning) procedure to select transiently transfected COS-7 cells expressing kappa receptor binding sites. The cloned cDNA encodes a 440-residue protein of the seven-helix guanine nucleotide-binding protein (G-protein)-co...

  3. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

    2013-01-20

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

  4. Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-06-01

    Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the E(rns) genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation. PMID:27265471

  5. Molecular cloning of a cDNA related to vernalization(verc203) in winter wheat

    Institute of Scientific and Technical Information of China (English)

    种康; 谭克辉; 黄华梁; 梁厚果

    1995-01-01

    A cDNA clone related to the vernalization in winter wheat(verc203)was harvested from the en-riched cold-induced cDNA library of 10~4 pfu with differential screening.The insert of verc203 in λ gt10 vector wassubcloned into the sites between BamH Ⅰ and Hind Ⅲ in pUC19 plasmid after being amplified with PCR.the analysis of the Northern blotting with a probe of verc203 indicated that the verc203 has a negative signalfor the control and the devernalized mRNA and a positive signal for the vernalized winter wheat and non-vernalized spring wheat at about 2.6 kb.

  6. Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7-9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit of saccharomyces cerevisiae, schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTb. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that human CCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3-24 times of those in other tissues. The CCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327). The genomic structure of the 5′-end of CCTβ was also described in detail through comparative analysis.

  7. Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family.

    OpenAIRE

    Grove, G; Zarlengo, R P; Timmerman, K P; Li, N Q; Tam, M F; Tu, C P

    1988-01-01

    We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the ve...

  8. 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.

  9. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Murwantoko M

    2015-11-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory

  10. Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

    OpenAIRE

    Sureau, C; Taylor, J; Chao, M; Eichberg, J W; Lanford, R E

    1989-01-01

    A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of h...

  11. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor.

    OpenAIRE

    Dobner, P R; Barber, D L; Villa-Komaroff, L; McKiernan, C

    1987-01-01

    Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional c...

  12. Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences.

    OpenAIRE

    Feinberg, R. F.; Sun, L. H.; Ordahl, C P; Frankel, F R

    1983-01-01

    The expression of specific cellular genes in M1.19 rat hepatoma cells involves glucocorticoid regulation by mechanisms that are not well understood. To approach this problem we cloned cDNA prepared from dexamethasone-induced poly(A)-RNA and used a comparative colony hybridization method to identify recombinant clones containing hormone-regulated sequences. Two such cDNA clones, p1394 and p255, hybridize to a homogeneous RNA species of 900 nucleotides that is present in high abundance in 24-hr...

  13. Cloning and expression of a novel chicken sulfotransferase cDNA regulated by GH.

    Science.gov (United States)

    Cao, H; Agarwal, S K; Burnside, J

    1999-03-01

    We have used mRNA differential display to compare gene expression in normal and GH receptor-deficient dwarf chickens, and report here the characterization of one differentially expressed gene, which shows significant sequence identity to the sulfotransferase gene family. Partial cDNA clones were isolated from a chicken liver cDNA library and an additional sequence was obtained using 5' rapid amplification of cDNA ends. A complete cDNA probe hybridizes to three transcripts (2.4, 2.0 and 1.45 kb) on Northern blots of chicken liver RNA, which differ in the length of the 3' untranslated region. All three transcripts are expressed at higher levels in normal vs dwarf chickens, as expected for a GH-regulated gene. The expression of this sulfotransferase mRNA was also detected in skeletal muscle, but not other tissues. The administration of GH to chickens increased the hepatic expression within 1 h, suggesting this sulfotransferase could be directly regulated by GH. Sulfotransferase activity, using estradiol or corticosterone as substrate, is detected in cells transfected with an expression vector containing the full-length cDNA. The sequence of this sulfotransferase does not show significant similarity with any subfamily of the sulfotransferases and its endogenous substrate is presently unknown. However, we speculate that GH activation of sulfotransferase activity could play a role in reducing concentrations of growth-antagonistic steroid hormones in GH target tissues. These results demonstrate the usefulness of differential display in this model system to identify genes that play a role in mediating GH action. PMID:10076195

  14. The identification of specific cDNA clones from tall and dwarf rice plants

    International Nuclear Information System (INIS)

    Full text: The use of dwarfing genes in rice breeding has proceeded for several years without a clear understanding of the genetic, hormonal and physiological mechanisms involved. This issue was addressed by focussing on the isolation of specific clones from tall- and dwarf-derived cDNA libraries. The materials used include near-isogenic lines of the tall rice cultivar 'Shiokari', differing at the DGWG or 'Tanginbozu' dwarfing gene loci. Also used were tall and dwarf 'Ginbozu' rice, the latter having been induced by treatment with 5-azacytidine, a potent demethylating agent. Subtractive and differential hybridisation have, to date, identified several candidate tall- and dwarf-specific clones. Their further characterisation is currently underway. (author)

  15. Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

    Institute of Scientific and Technical Information of China (English)

    Wan-Song Ni; Zhi-Yong Lei; Xi Chen; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    Salt cress (Thellungiella halophila), a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.

  16. Molecular cloning and sequencing of a cDNA encoding partial putative molt-inhibiting hormone from Penaeus chinensis

    Science.gov (United States)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  17. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeue chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  18. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymer ase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A s pecific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 ba se pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  19. 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.

  20. cDNA cloning and expression of a collectin from red-spotted grouper ( Epinephelus akaara)

    Science.gov (United States)

    Zhang, Zhiwen; Ding, Shaoxiong; Wang, Ying; Mao, Yong; Su, Yongquan; Wang, Jun

    2009-09-01

    Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin (EALec1) from Epinephelus akaara using RACE. The complete EALec1 cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail. The EALec1 cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain (CRD), indicating that EALec1 is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALec1 in 12 different tissues using RT-PCR. EALec1 was expressed in all tissues, though at different levels. In addition, we inserted EALec1 into an expression vector (pET-28a) for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALec1 recombinant expression vector.

  1. Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

    Science.gov (United States)

    Villand, P; Olsen, O A; Kleczkowski, L A

    1993-12-01

    PCR amplification of cDNA prepared from poly(A)+ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis. PMID:8292792

  2. cDNA cloning and expression of a collectin from red-spotted grouper (Epinephelus akaara)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; DING Shaoxiong; WANG Ying; MAO Yong; SU Yongquan; WANG Jun

    2009-01-01

    Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin (EALec1) from Epinephelus akaara using RACE. The complete EALec1 cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail. The EALec1 cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain (CRD), indicating that EALec1 is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALec1 in 12 different tissues using RT-PCR. EALec1 was expressed in all tissues, though at different levels. In addition, we inserted EALec1 into an expression vector (pET-28a) for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALec1 recombinant expression vector.

  3. Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jingpu; WANG Weixian; ZHU Shaoxia

    2004-01-01

    Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. Β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.

  4. cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain.

    Science.gov (United States)

    Lang, W H

    1988-09-20

    A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified. PMID:3207675

  5. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    Energy Technology Data Exchange (ETDEWEB)

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. (Agouron Institute, La Jolla, CA (USA))

    1988-07-01

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg){sub 9} repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.

  6. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Wayan T. Artama

    2015-10-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory

  7. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides

    Energy Technology Data Exchange (ETDEWEB)

    Robakis, N.K.; Ramakrishna, N.; Wolfe, G.; Wisniewski, H.M.

    1987-06-01

    Deposits of amyloid fibers are found in large numbers in the walls of blood vessels and in neuritic plaques in the brains of patients with Alzheimer disease and adults with Down syndrome. The authors used the amino acid sequence of the amyloid peptide to synthesize oligonucleotide probes specific for the gene encoding this peptide. When a human brain cDNA library was screened with this probe, a clone was found with a 1.7-kilobase insert that contains a long open reading frame coding for 412 amino acid residues including the 28 amino acids of the amyloid peptide. RNA gel blots revealed that a 3.3-kilobase mRNA species was present in the brains of individuals with Alzheimer disease, with Down syndrome, or with not apparent neurological disorders. Southern blots showed that homologous genes are present in the genomic DNA of humans, rabbits, sheep, hamsters, and mice, suggesting that this gene has been conserved through mammalian evolution. Localization of the corresponding genomic sequences on human chromosome 21 suggest a genetic relationship between Alzheimer disease and Down syndrome, and it may explain the early appearance of large numbers of neuritic plaques in adult Down syndrome patients.

  8. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality ...scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality scores De...from the budding yeast full-length cDNA library by the vector-capping method, the sequence quality score gen...s accession only. Sequence 5'-end sequence data of budding yeast full-length cDNA clones. FASTA format. Quality Phred's quality... Update History of This Database Site Policy | Contact Us 5'-end sequences of budding yeast full-length cDNA clones and quality

  9. An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

    Science.gov (United States)

    Shan, Chao; Xie, Xuping; Muruato, Antonio E; Rossi, Shannan L; Roundy, Christopher M; Azar, Sasha R; Yang, Yujiao; Tesh, Robert B; Bourne, Nigel; Barrett, Alan D; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-06-01

    The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies. PMID:27198478

  10. 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).

  11. MOLECULAR-CLONING AND CHARACTERIZATION OF A CDNA FOR THE BETA-SUBUNIT OF HUMAN ALCOHOL-DEHYDROGENASE

    OpenAIRE

    Duester, G; Hatfield, G.; Buhler, R; Hempel, J; Jornvall, H; Smith, M.

    1984-01-01

    Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing c...

  12. Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80S ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this rice S4 gene is from a multigene family.

  13. Recombinant selection by microinjection: a simple cDNA cloning procedure for production of exclusively sense RNA transcripts

    OpenAIRE

    Digweed, M; Günthert, U

    1989-01-01

    A new strategy for cDNA cloning is presented, designed particularly for identification of recombinants by functional analysis, after microinjection into somatic cells. First-strand synthesis is primed by the oligodeoxyribonucleotide: (formula; see text) After second-strand synthesis and blunting, double-stranded cDNA is formed, which carries restriction sites for NotI and ApaI downstream from the coding sequence. The cDNA is ligated into a plasmid, between two promoters for phage T7 and T3 RN...

  14. Recovery of Virulent and RNase-Negative Attenuated Type 2 Bovine Viral Diarrhea Viruses from Infectious cDNA Clones

    OpenAIRE

    Meyer, Christiane; von Freyburg, Martina; Elbers, Knut; Meyers, Gregor

    2002-01-01

    Cloned cDNA derived from the genome of the virulent type 2 bovine viral diarrhea virus (BVDV) strain NY'93/C was sequenced and served for establishment of the infectious cDNA clone pKANE40A. Virus recovered from pKANE40A exhibited growth characteristics similar to those of wild-type BVDV NY'93/C and proved to be clinically indistinguishable from the wild-type virus in animal experiments. A virus mutant in which the RNase residing in the viral glycoprotein Erns was inactivated, revealed an att...

  15. In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system

    OpenAIRE

    Bürkle, L.; Meyer, S.; Dortay, H.; Lehrach, H; Heyl, A.

    2005-01-01

    In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between differen...

  16. Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

    Institute of Scientific and Technical Information of China (English)

    Guo-Qing DONG; Xiao-Ling YUAN; Ya-Jun SHAN; Zhen-Hu ZHAO; Jia-Pei CHEN; Yu-Wen CONG

    2004-01-01

    The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eiseniafoetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The eDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

  17. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  18. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    Science.gov (United States)

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-01-01

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia. PMID:25366775

  19. cDNA cloning and expression of earthworm fibrinolytic enzyme component A

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Earthworm fibrinolytic enzyme component A (EFEa), a protein with dual fibrinolytic activity, is one of the major therapeutically important earthworm fibrinolytic enzyme components. The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida) by the RT-PCR technique. The deduced amino acid sequence of the EFE component A shows high homology with some members of serine proteases trypsin family, and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family. The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E. coli. The resulting expression plasmids, pQE-efea and pMAL-efea, were used to transform the E. coli strain M15. Recombinant protein bands corresponding with calculated molecular weights were induced. The induced His6-EFEa fusion protein with pQE- efea was accumulated into inclusion body, while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinolytic activities.

  20. Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

    Science.gov (United States)

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Sun, Hongjuan; Chen, Zhong; Yang, Aifu; Gao, Shan; Wang, Bai; Jiang, Bei; Guan, Xiaoyan

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. PMID:24355405

  1. Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

    International Nuclear Information System (INIS)

    In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

  2. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

    Directory of Open Access Journals (Sweden)

    De Paepe Anne

    2004-02-01

    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  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. cDNA cloning and sequence analysis of NIb gene of soybean mosaic virus

    Institute of Scientific and Technical Information of China (English)

    刘俊君; 彭学贤; 莽克强

    1995-01-01

    cDNA of soybean mosaic virus (Beijing isolate, SMV-BJ) has been synthesized, using viralgenomic RNA as template and random hexanucleotides as primers. Based on the sequences of SMV-BJ coat protein (CP) gene as well as SMV- and WMV-II-related regions, oligonucleotides were made as primers for polymerase chain reaction (PCR). NIb gene of SMV-BJ was amplified by PCR, and cloned into pBluescript SK. The complete sequence was determined. The comparison of NIb genes between SMV-BJ and WMV-II . (USA) shows that similarities for nucleotide sequence reach 80.3%, and the deduced amino acid sequence. 91 3%. In consideration of the high identities in between the CP gene and the 3’-non-coding region between them, WMV-II might be considered as a watermelon strain of SMV Besides, some unexpected sequences were found in the 3’-region of 2 NIb gene clones. Following modification and splicing, a binary vector of NIb gene has been constructed for its expression in higher plant for the purpose of studying the possible repl

  5. cDNA cloning and characterization of a gibberellin-responsive gene in hypocotyls of Cucumis sativus L.

    Science.gov (United States)

    Chono, M; Yamauchi, T; Yamaguchi, S; Yamane, H; Murofushi, N

    1996-07-01

    A cDNA clone corresponding to a gibberellin-responsive gene (CRG16) was isolated from cucumber hypocotyls. CRG16 was deduced to encode an extremely hydrophobic protein of 65 amino acids. The deduced sequence exhibited no significant homology to other proteins. Levels of CRG16 mRNA reflected the gibberellin-induced elongation of cucumber hypocotyls.

  6. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  7. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system

    International Nuclear Information System (INIS)

    CD28 (Tp44) is a human T-cell-specific homodimer surface protein that may participate in T-cell activation. The authors have isolated a cDNA clone encoding CD28 by a simple and highly efficient cloning strategy based on transient expression in COS cells. Central to this strategy is the use of an efficient method to prepare large plasmid cDNA libraries. The libraries are introduced into COS cells, where transient expression of surface antigen allows the isolation of cDNAs by way of monoclonal antibody binding. The CD28 cDNA encodes a highly glycosylated membrane protein with homology to the immunoglobulin superfamily and directs the production of a homodimer in transfected COS cells

  8. 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. PMID:24381103

  9. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  10. Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

    OpenAIRE

    Wolf, D; Harris, N; Goldfinger, N; Rotter, V

    1985-01-01

    Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-...

  11. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corre-sponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  12. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    李权; 闻宏; 敖世洲

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corresponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  13. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

    OpenAIRE

    Yanagi, Masayuki; Purcell, Robert H.; Emerson, Suzanne U.; Bukh, Jens

    1997-01-01

    We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA tra...

  14. Cloning a Full-length cDNA Encoding UDP-glucose Pyrophosphorylase from Amorpha fruticosa by PCR-based Methods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGP), a key enzyme producing UDP-glucose in the synthesis of sucrose and cell ulose, is cloned by using this method. We design 5' RACE primers based on UGP A1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5' and 3' end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.

  15. cDNA cloning and characterization of two trehalases from Spodoptera litura (Lepidoptera; Noctuidade).

    Science.gov (United States)

    Zou, Q; Wei, P; Xu, Q; Zheng, H Z; Tang, B; Wang, S G

    2013-01-01

    The oriental leafworm moth, Spodoptera litura, is a major agricultural pest in southeast Asia and nearby Pacific regions. Two distinct trehalases have been identified in insects: soluble trehalase (Treh1) and membrane-bound trehalase (Treh2), although there is currently no information on these genes in S. litura. To characterize the distribution and function of treh, cDNAs of Treh proteins were cloned from S. litura. SpoliTreh1 cDNA has an open reading frame of 1758 nucleotides, which encodes a protein of 585 amino acids, with a predicted mass of approximately 67.07 kDa and an isoelectric point of 4.86. SpoliTreh2 cDNA has an open reading frame of 2325 nucleotides, encoding a protein of 645 amino acids, a mass of approximately 73.62 kDa, and an isoelectric point of 5.90. Northern blotting analysis revealed that SpoliTreh1 transcripts are in the midgut, fat body, tracheae, and epidermis, but not in the brain and Malpighian tubules of S. litura larvae, whereas SpoliTreh2 transcripts were found in all 6 tissues. SpoliTreh1 transcripts were highly expressed in the fat body of the pre-pupal stage, and SpoliTreh2 transcripts were highly expressed in the fat body of 3-day-old larvae of the 6th instar and during the 1st 6 days of the pupal stage, except the 2nd day. Both SpoliTreh1 and SpoliTreh2 were highly expressed in third-instar larvae. PMID:23613237

  16. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

    OpenAIRE

    Nolden, T.; Pfaff, F; Nemitz, S.; Freuling, C. M.; Höper, D.; Müller, T; Stefan Finke

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T re...

  17. Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding

    Energy Technology Data Exchange (ETDEWEB)

    Harada, N.; Castle, B.E.; Gorman, D.M.; Itoh, A.; Schreurs, J.; Barrett, R.L.; Howard, M.; Miyajima, A. (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA (USA))

    1990-02-01

    Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, the authors have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a K{sub d} = 165 pM. Crosslinking of {sup 125}I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the {beta} chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.

  18. A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase.

    Science.gov (United States)

    Shah; Cha

    2000-05-29

    The differential display method was used to isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the mesocarp of two oil palms, Elaeis oleifera and Elaeis guineensis, Tenera. DNA-free total RNA from mesocarp and kernel of E. guineensis, Tenera and E. oleifera (15 WAA) were used to obtain differential gene expression patterns between these tissues from the two species. In this report, we describe the isolation and characterization of a specific cDNA clone, MO1 (434 bp) which was shown to be mesocarp-specific as well as species-specific for E. oleifera Sequencing of this fragment showed homology to the enzyme sesquiterpene synthase. Its longer cDNA clone, pMO1 (1072 bp), isolated from a 15-week E. oleifera mesocarp cDNA library confirmed that it encodes for sesquiterpene synthase. The complete sequence of 1976 bp was obtained using 5'RACE method. Northern hybridization showed that MO1 and pMO1 mRNA transcripts are highly expressed only in the mesocarp of E. oleifera from 5 to 20 WAA. No expression was detected in the kernel (12-17 WAA) and vegetative tissues of both species nor in the mesocarp of E. guineensis. This is the first communication to document on the isolation and characterisation of a mesocarp-and species-specific cDNA clone from oil palm. PMID:10729614

  19. A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

    Science.gov (United States)

    Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

    2014-03-01

    Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.

  20. A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

    Science.gov (United States)

    Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

    2014-03-01

    Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants. PMID:24388933

  1. Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines.

    Science.gov (United States)

    Johnson, K N; Ball, L A

    2001-12-01

    Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly. PMID:11711613

  2. Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

    OpenAIRE

    Domdey, H; Wiebauer, K; Kazmaier, M; Müller, V.; Odink, K.; Fey, G

    1982-01-01

    Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the...

  3. Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

    OpenAIRE

    Mohammad Hadi Sekhavati; Mojtaba Tahmoorespur; Kamran Ghaedi; Kianoush Dormiani, Pharm; Mohammad Reza Nassiri; Yahya Khazaie; Mahboubeh Foruzanfar; Morteza Hosseini; Mohammad Hossein Nasr Esfahani

    2013-01-01

    Objective: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro fu...

  4. Human CSF-1: molecular cloning and expression of 4-kb cDNA encoding the human urinary protein

    Energy Technology Data Exchange (ETDEWEB)

    Wong, G.G.; Temple, P.A.; Leary, A.C.; Witek-Giannotti, J.S.; Yang, Y.; Ciarletta, A.B.; Chung, M.; Murtha, P.; Kriz, R.; Kaufman, R.J.; Ferenz, C.R.

    1987-03-20

    A 4-kilobase complementary DNA (cDNA) encoding human macrophage-specific colony-stimulating factor (CSF-1) was isolated. When introduced into mammalian cells, this cDNA directs the expression of CSF-1 that is structurally and functionally indistinguishable from the natural human urinary CSF-1. Direct structural analysis of both the recombinant CSF-1 and the purified human urinary protein revealed that these species contain a sequence of at least 40 amino acids at their carboxyl termini which are not found in the coding region of a 1.6-kilobase CSF-1 cDNA that was previously described. These results demonstrate that the human CSF-1 gene can be expressed to yield at least two different messenger RNA species that encode distinct but related forms of CSF-1.

  5. 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.

  6. Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

    Institute of Scientific and Technical Information of China (English)

    Haibo Wang; Duoduo Yuan; Rong Xu; Cheng-Wu Chi

    2011-01-01

    Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum, In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structurefunction studies.

  7. Cloning and Expression of the cDNA of a Murine Soluble Fas

    Institute of Scientific and Technical Information of China (English)

    胡中波; 邹萍; 李爱香; 肖娟; 仲照东; 刘凌波

    2002-01-01

    Summary: In order to regulate the apoptosis induced by Fas-FasL system, a soluble isoform of mouse Fas was cloned from thymocytes of immature mice with the primers designed according to the full-length Fas cDNA sequence in the GeneBank. It was directionally inserted into the intermedium vector pUC19. DNA sequencing proved that it was consistent with the expected sequence.Then it was subcloned into the eukaryotic expression vector pCA13, which was used to construct the recombinant vector pCA13-FasC. By lipofectamine (LF2000)-mediated transfection, pCA13FasC was transfected into the 293 cells. RT-PCR and Western blot indicated that the murine soluble Fas C protein was expressed in the 293 cells. Apoptosis inducing test showed that the expression of this murine Fas C could block the Fas-induced apoptosis, which confirmed the biological activity of the recombinant Fas C.

  8. Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

    International Nuclear Information System (INIS)

    The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH2-terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

  9. Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

    OpenAIRE

    Moore, R. E.; Davies, M S; O'Connell, K M; Harding, E I; Wiegand, R C; Tiemeier, D C

    1986-01-01

    The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The...

  10. Primary structure of pregnancy zone protein. Molecular cloning of a full-length PZP cDNA clone by the polymerase chain reaction.

    Science.gov (United States)

    Devriendt, K; Van den Berghe, H; Cassiman, J J; Marynen, P

    1991-01-17

    A full-length cDNA clone of the human pregnancy zone protein (PZP) was cloned from the hepatocellular carcinoma cell line Hep3B. Based on the exon sequences of the PZP gene (Devriendt et al. (1989) Gene 81, 325-334; Marynen et al., unpublished data), primer pairs were designed to amplify six overlapping fragments of the PZP cDNA. The obtained cDNA is 4609 bp long and contains an open reading frame coding for 1482 amino acids, including a signal peptide of 25 amino acid residues. Comparison with the published partial PZP amino acid sequence (Sottrup-Jensen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7353-7357) and the PZP genomic sequences confirmed the identity as a PZP cDNA. 71% of the corresponding amino acid residues in PZP and human alpha 2-macroglobulin (alpha 2M) are identical and all cysteine residues are conserved. A typical internal thiol ester site and a bait domain were identified. A Pro/Thr polymorphism was identified at amino acid position 1180, and an A/G nucleotide polymorphism at bp 4097.

  11. cDNA cloning and characterization of a mannose-binding lectin from Zingiber officinale Roscoe (ginger) rhizomes

    Indian Academy of Sciences (India)

    Zhonghai Chen; Guoyin Kai; Xiaojun Liu; Juan Lin; Xiaofen Sun; Kexuan Tang

    2005-03-01

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed that zoa expressed in all the tested tissues of Z. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed in Escherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae.

  12. Construction of infectious cDNA clones of PRRSV: Separation of coding regions for nonstructural and structural proteins

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing "porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high fre- quency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. We developed an infectious cDNA clone of an attenuated strain of Type II PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, en- coding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vac- cine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5′ terminus of genome. To dis- cern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon transfection of the in vitro transcripts of both the original and Mlu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently, PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA, was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains, and a platform for further develop- ment of

  13. Construction of infectious cDNA clones of PRRSV:Separation of coding regions for nonstructural and structural proteins

    Institute of Scientific and Technical Information of China (English)

    YUAN ShiShan; WEI ZuZhang

    2008-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing"porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great Interest. We developed an infectious cDNA clone of an attenuated strain of Type Ⅱ PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, encoding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vaccine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5' terminus of genome. To discern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon trensfection of the in vitro transcripts of both the original and MIu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently,PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA,was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering crose-protection to various genetically diversified PRRSV strains, and a platform for further development of PRRSV as a gene

  14. Epigenetic reprogramming in mammalian species after SCNT-based cloning.

    Science.gov (United States)

    Niemann, Heiner

    2016-07-01

    The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. PMID:27160443

  15. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    International Nuclear Information System (INIS)

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A)+ RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A)+ RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A)+ RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

  16. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, E.K.; Horton, R.; Bloem, L.; Bach, R.; Williams, K.R.; Guha, A.; Kraus, J.; Lin, T.C.; Nemerson, Y.; Konigsberg, W.H.

    1987-08-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).

  17. Cloning, expression, and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Zou, Li-Ping; Li, Han-Xia; Ouyang, Bo; Zhang, Jun-Hong; Ye, Zhi-Biao

    2006-08-01

    GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1,086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii. PMID:16939010

  18. Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii.

    Science.gov (United States)

    Satou, Motoyasu; Kaiya, Hiroyuki; Nishi, Yoshihiro; Shinohara, Akio; Kawada, Shin-Ichiro; Miyazato, Mikiya; Kangawa, Kenji; Sugimoto, Hiroyuki

    2016-06-01

    Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1-27], [1-19], [1-16] and [1-15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5'-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals. PMID:27102942

  19. 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.

  20. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M. (Charing Cross Sunley Research Centre, Hammersmith, London (England))

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF{alpha} with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNF{alpha} with an affinity of 2.5 {times} 10{sup {minus}9} M. This binding can be competitively inhibited with unlabeled TNF{alpha} or lymphotoxin (TNF{beta}).

  1. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  2. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Science.gov (United States)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  3. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H;

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...

  4. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  5. Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA.

    Science.gov (United States)

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1988-09-01

    We report the isolation of cDNA clones for a Plasmodium falciparum gene that encodes the complete amino acid sequence of a previously identified exported blood stage antigen. The Mr of this antigen protein had been determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis, by different workers, to be 113,000, 126,000, and 140,000. We show, by cDNA nucleotide sequence analysis, that this antigen gene encodes a 989 amino acid protein (111 kDa) that contains a potential signal peptide, but not a membrane anchor domain. In the FCR3 strain the serine content of the protein was 11%, of which 57% of the serine residues were localized within a 201 amino acid sequence that included 35 consecutive serine residues. The protein also contained three possible N-linked glycosylation sites and numerous possible O-linked glycosylation sites. The mRNA was abundant during late trophozoite-schizont parasite stages. We propose to identity this antigen, which had been called p126, by the acronym SERA, serine-repeat antigen, based on its complete structure. The usefulness of the cloned cDNA as a source of a possible malaria vaccine is considered in view of the previously demonstrated ability of the antigen to induce parasite-inhibitory antibodies and a protective immune response in Saimiri monkeys. PMID:2847041

  6. 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.

  7. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    Science.gov (United States)

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector. PMID:26478540

  8. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  9. Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

    OpenAIRE

    Devos, René; Plaetinck, Geert; Cheroutre, Hilde; Simons, Guus; Degrave, Wim,; Tavernier, Jan; Remaut, Erik; Fiers, Walter

    1983-01-01

    A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal seq...

  10. Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite.

    OpenAIRE

    Chaudhuri, A; Polyakova, J; Zbrzezna, V; K Williams; Gulati, S.; Pogo, A O

    1993-01-01

    cDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplified DNA fragment encoding an internal peptide sequence of glycoprotein D (gpD) protein. The open reading frame of the 1267-bp cDNA clone indicated that gpD protein was composed of 338 amino acids, predicting a M(r) of 35,733, which was the same as a deglycosylated gpD protein. Portions of the predicted amino acid sequence, matched with six CNBr/pepsin peptides ...

  11. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA.

    OpenAIRE

    Ruggli, N; Tratschin, J D; Mittelholzer, C.; Hofmann, M A

    1996-01-01

    The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of...

  12. 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...... 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...

  13. cDNA cloning and analysis of betaine aldehyde dehydrogenase, a salt inducible enzyme in sugar beet

    Energy Technology Data Exchange (ETDEWEB)

    McCue, K.F.; Hanson, A.D. (Michigan State Univ., East Lansing (USA))

    1990-05-01

    Betaine accumulates and serves as a compatible osmolyte in some plants subjected to drought or salinity stress. The last enzyme in the betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). The activity of BADH increases in response to increasing salinity levels. This increase in activity corresponds to an increase in protein detectable by immunoblotting, and to an increase in the translatable BADH mRNA. BADH was cloned from a cDNA library constructed in {lambda}gt10 using poly(A){sup +} RNA from sugar beets salinized to 500 mM NaCl. cDNAs were size selected (>1kb) before ligation into the vector, and the library was screened with a spinach BADH cDNA probe. Three nearly full length clones obtained were confirmed as BADH by their nucleotide and deduced amino acid homology to spinach BADH. Clones averaged 1.8 kb and contained open reading frames of 500 amino acids at 80% identity with spinach BADH. RNA gel blot analysis of poly(A){sup +} RNA indicated that salinization to 500 mM NaCl resulted in a 5-fold increase of BADH mRNA level.

  14. Molecular Cloning of Myostatin Partial cDNA of Beijing Duck and Its Expression in Breast Muscle

    Institute of Scientific and Technical Information of China (English)

    WANG Yong-sheng; HOU Shui-sheng; HUANG Wei; KANG Jun-mei

    2006-01-01

    In this experiment, 500 bp cDNA of myostatin gene was cloned from a Beijing duck's breast. The duck myostatin gene was found to have 98, 96, 95, 88, and 87% sequence similarity at the cDNA level with domestic goose, chicken, domestic pigeon, human, and pig, respectively. The predicted amino acid sequence has an overall similarity with a comparable region of turkey 99%, domestic goose 98%, and chicken 99%. Conserved domains of deduced amino acids showed that it belonged to the TGF-beta family. Myostatin expression in breast muscle was higher at 28, 35, and 42 days than at 7, 14, and 21 days. The pattern of myostatin expression was closely parallel to the trend of breast muscle growth, suggesting that myostatin might play an important role in breast muscle development. It was possible to postulate that myostatin may be a major determinant of muscle mass in breast muscle, as shown in other species.

  15. Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

    Science.gov (United States)

    Yu, J; Cary, J W; Bhatnagar, D; Cleveland, T E; Keller, N P; Chu, F S

    1993-11-01

    Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8285664

  16. Isolation and Cloning of cDNA of Gene Encoding for Metallothionein Type 2 from Soybean [Glycine max (L.) (Merrill)] cv. Slamet

    OpenAIRE

    UTUT WIDYASTUTI; YASSIER ANWAR; SUHARSONO

    2009-01-01

    Metallothionein has an important role in the detoxification of metal ions. It has a low molecular weight and contains cysteine-rich residue. The objective of this research is to isolate and clone the cDNA of gene encoding for metallothionein from soybean [Glycine max (L.) (Merrill)] cv Slamet (GmMt2). We had successfully isolated total RNA by reverse transcription and synthesized total cDNA from total RNA as template. cDNA of GmMt2 had been isolated from total cDNA by PCR. It was successfully...

  17. Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom.

    Science.gov (United States)

    Clement, Herlinda; Olvera, Alejandro; Rodríguez, Mabel; Zamudio, Fernando; Palomares, Laura A; Possani, Lourival D; Odell, George V; Alagón, Alejandro; Sánchez-López, Rosana

    2012-12-01

    Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as "spreading factors" that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell. PMID:22982117

  18. Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder;

    1994-01-01

    -134]. The protein contains a nuclear targeting signal (KKKGK), and fractionation of transformed human amnion cells (AMA) in karyoplasts and cytoplasts confirmed that it is predominantly localized in the nucleus. Database searching indicated that IEF SSP 9502 is a putative human homologue of the Saccharomyces......We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

  19. Cloning and sequencing of Octopus dofleini hemocyanin cDNA: derived sequences of functional units Ode and Odf.

    OpenAIRE

    Lang, W H; van Holde, K E

    1991-01-01

    A number of additional cDNA clones coding for portions of the very large polypeptide chain of Octopus dofleini hemocyanin were isolated and sequenced. These data reveal two very similar coding sequences, which we have denoted "A-type" and "G-type." We have obtained complete A-type sequences coding for functional units Ode and Odf; consequently a total of three such unit sequences are now known from a single subunit of one molluscan hemocyanin. This presents the opportunity to make sequence co...

  20. Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

    Institute of Scientific and Technical Information of China (English)

    马凤蓉; 朱立平; 汪燚; 赵方萄; 史耕先; 李波; 李国燕; 张淑珍; 王讯

    2000-01-01

    A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

  1. Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS.

    Science.gov (United States)

    Wang, Y; Cao, Z; Newkirk, R F; Ivy, M T; Townsel, J G

    2001-05-01

    It is well documented that the sodium dependent, hemicholinium-3 sensitive, high affinity choline co-transporter is rate limiting in the biosynthesis of acetylcholine and is essential to cholinergic transmission. Until recently this transporter had eluded cloning. Okuda et al. (2000. Nature Neurosci. 3, 120-125) recently reported the successful cloning of the choline co-transporter in Caenorhabditis elegans (CHO-1) and rat (CHT1). We report herein the cloning of the choline co-transporter in the horseshoe crab, Limulus polyphemus. Through the use of a series of degenerate primers selected from consensus sequences of CHO-1 and CHT1, we generated two probes that were used to search a Limulus cDNA library produced from central nervous system (CNS) tissue. The full length nucleotide sequence of the Limulus homolog consists of 3368 bp which includes an open reading frame (ORF) that predicts a protein of 579 amino acids and two non-translation regions (NTR), one at the 3' end and the other at the 5' end. The amino acid sequence has 46% identity with rat CHT1 and 50% identity with both CHO-1 in C. elegans and the recently cloned human co-transporter (hCHT; Apparsundaram et al., 2000. Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga, 2000. FEBS Lett. 484, 92-97). Hydropathy plot analysis predicts the Limulus choline co-transporter (LChCoT) to have thirteen transmembrane domains (TMD), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analyses using cDNA probes designed from LChCoT cDNA sequences revealed its distribution specifically in central nervous system structures. On the other hand it was not found in non-nervous tissues. The successful cloning of LChCoT, which was shown to be a member of the sodium-dependent glucose transporter family (SLGT), should prove useful in the determination of its physiological regulation, including its intracellular trafficking. PMID:11368908

  2. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    Science.gov (United States)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  3. Molecular cloning, sequence analysis and expression in Escherichia coli of Camelus dromedarius glucose-6-phosphate dehydrogenase cDNA.

    Science.gov (United States)

    Saeed, Hesham Mahmoud; Alanazi, Mohammad Saud; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Khan, Zahid

    2012-06-01

    This study determined the full length sequence of glucose-6-phosphate dehydrogenase cDNA (G6PD) from the Arabian camel Camelus dromedarius using reverse transcription polymerase chain reaction. The C. dromedarius G6PD has an open reading frame of 1545 bp, and the cDNA encodes a protein of 515 amino acid residues with a molecular weight of 59.0 KDa. The amino acid sequence showed the highest identity with Equus caballus (92%) and Homo sapiens (92%). The G6PD cDNA was cloned and expressed into Escherichia coli as a fusion protein and was purified in a single chromatographic step using nickel affinity gel column. The purity and the molecular weight of the enzyme were checked on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity of G6PD was determined to be 289.6 EU/mg protein with a fold purification of 95.45 and yield of 56.8%. PMID:22538316

  4. 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.

  5. Cloning and Analysis of Full-Length cDNA of PumNPR1 Gene from Pyrus ussuriensis Maxim

    Institute of Scientific and Technical Information of China (English)

    CHE Daidi; FAN Jinping; WANG Jingang; XU Ping; YANG Tao; LIU Shenkui

    2008-01-01

    The purpose of this study is to find a new gene resource for the researches of molecular breeding of Rosaceae plants disease-resistance. Pyrus ussuriensis Maxim is used as a starting material to clone the full-length cDNA of NPR1(nonexpressor of pathogenesis- related genes 1) which is a key regulator in SA (salicylic acid)-mediated systemic acquired resistance (SAR) by homologous cloning and RACE techniques. The length of the cDNA sequence was 1 767 bp, the ORF was 1 761 bp, it coded 586 amino acids, pI=5.58, the relative molecular weight was 65.009 ku, contained 19 kinds of amino acids, and had full BTB/POZ and ANK domains. Compared the homology of NPR1 gene in GenBank database, the homology with Pyrus pyrifolia, Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Oryza sativa, Helianthus annuus were 98%, 62%, 68%, 65%, 57%, 63%. The homology of functional area were 99%, 78%, 82%, 79%, 74%, 77%. This NPR1 gene was considered as homologic gene of Pyrus ussuriensis Maxim and named PumNPR1.

  6. Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene by Homologous Cloning

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5' end of RNA transcript (SMART) approach. This library had a plaque titer of 1 × 109pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average eDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences.Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profies and find the major genes of prolificacy of Small Tail Han sheep.

  7. Molecular cloning of a gene that is necessary for G1 progression in mammalian cells

    International Nuclear Information System (INIS)

    The authors have cloned a human cDNA that complements the mutation of ts11, a temperature-sensitive (ts) mutant of the BHK hamster cell line that at the nonpermissive temperature is blocked in progression through the G1 phase of the cell growth cycle. After transfecting human chromosomal DNA into ts11 cells and selecting for cells that had acquired a non-ts phenotype, the authors screened a geonomic library constructed in the EMBL3 λ vector from a secondary non-ts transformant and isolated a recombinant phage containing human DNA sequences that were uniformly present in primary and secondary non-ts transformants. Genomic probes that recognized an mRNA of about 2 kilobases in human cells were used to isolate from a cDNA expression library two cDNA plasmids that could efficiently transform ts11 cells to a non-ts phenotype. Sequencing of one of these cDNAs revealed a single open reading frame, which could encode a 540 amino acid protein. The ts11 gene has at least two other homologs in human DNA and thus it appears to be part of a small gene/pseudogene family. Experiments with serum-synchronized cells indicate that the expression of the ts11 gene, which is necessary for G1 progression, is itself cell-cycle regulated, being induced in approximately mid-G1

  8. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

    Science.gov (United States)

    van Tegelen, Léon J.P.; Moreno, Paolo R.H.; Croes, Anton F.; Verpoorte, Robert; Wullems, George J.

    1999-01-01

    Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus. PMID:9952467

  9. Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

    Science.gov (United States)

    Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

  10. cDNA Cloning, Expression and Characterization of an Allergenic 60s Ribosomal Protein of Almond (Prunus dulcis

    Directory of Open Access Journals (Sweden)

    Abolhassani Mohsen

    2009-06-01

    Full Text Available Tree nuts, including almond (prunus dulcis are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  11. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  12. RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

    Science.gov (United States)

    Ferriol, I; Turina, M; Zamora-Macorra, E J; Falk, B W

    2016-05-01

    Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA. PMID:26756828

  13. cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.

    OpenAIRE

    Myerowitz, R; Proia, R L

    1984-01-01

    We have isolated a cDNA clone containing sequences complementary to mRNA encoding the alpha-chain of the lysosomal enzyme beta-hexosaminidase. RNA from a human lung fibroblast strain, IMR90, was enriched for beta-hexosaminidase messenger by polysome immunoselection with antiserum against beta-hexosaminidase A. This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure. After transformation of Escherichia coli, 385 ampicillin-resistant colonies...

  14. 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. PMID:24462869

  15. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  16. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    Energy Technology Data Exchange (ETDEWEB)

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  17. [Construction of cDNA infectious clones of EV71 highly-pathogenic and cell-culture-adapted strains].

    Science.gov (United States)

    Zhang, Yong-xin; Li, Xiao-yu; Huang, Yu-ming; Zhou, Yong-dong; Bi, Sheng-li; Cen, Shan

    2014-11-01

    The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.

  18. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-06-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  19. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Institute of Scientific and Technical Information of China (English)

    QI Fei; GUO Huarong; WANG Jian

    2008-01-01

    Reversible protein phosphorylation,catalyzed by protein kinases and phosphatases,is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes.Protein phosphatase 1(PP1) is the first and well-characterized member of the protein serine/threoninephosphatase family.In the present study.a full-length cDNA encoding the beta isolorm of the catalytic subunit of protein phosphatase 1(PP1cb).was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus,designated SmPP1cb,by the rapid amplification of cDNA ends (RACE) technique.The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame(ORF),flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region.The ORF encodes a putative 327 amino acid protein.and the N-terminal section of this protein iS highly acidic,Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp.a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B(PP2B).And its calculated molecular mass is 37 193 Da and pI 5.8.Sequence analysis indicated that,SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates.and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG,which is different from mammalian in two positions A-6 and G-3,indicating the possibility of different initiation of translation in turbot,and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals.especially zebrafish.The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  20. cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; YING Kang; XIE Yi

    2005-01-01

    Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

  1. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Kai-tai

    2001-01-01

    [1]Tom S, Andrew PR. Human Molecular Genetics [M]. John Wiley & Sons, Inc. United States of America 1996; 335.[2]Zhao Yong-liang, Jin Cui-zhen, Wu De-chang et al. Neoplastic transformation and cytogenetic changes of rat tracheal epithelial cells induced by a-particles irradiation [J]. Chin Med Sci J 1997; 12:202.[3]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[4]Frederick A, Roger B. Current Protocols in Molecular Biology [M]. John Wiley & Sons, Inc. United States of America 1998; 2.1.1.[5]Roux KH. Optimization and troubleshooting in PCR [J]. PCR Methods Appl 1995; 4:5158.[6]Sambrook, J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd Ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989; 54.[7]Zhang Y, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs [J]. Methods Mol Biol 1997; 69:61.[8]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[9]Iqbal S, Robinson J, Deere D, et al. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water [J]. Lett Appl Microbiol 1997; 24:498.[10]Schunck B, Kraft W, Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces [J]. J Virol Methods 1995; 55:427.

  2. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    International Nuclear Information System (INIS)

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma λgt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from λgt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents

  3. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  4. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

    DEFF Research Database (Denmark)

    Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten;

    1985-01-01

    '-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human...

  5. 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

    -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...

  6. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  7. 虹鳟 Ndufb2基因全长 cDNA 序列的克隆与分析%Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss

    Institute of Scientific and Technical Information of China (English)

    王家庆; 边佳; 李代宗; 马爽; 王亮; 那广宁

    2013-01-01

    Summary Rainbow trout belongs to Salmonidae aerobic fish,and it is necessary for high dissolved oxygen content of living water environment.If the dissolved oxygen content of living water is less than 5 mg/L,it will cause the increase of respiratory rate,which is the so-called“aquaculture floating head”phenomenon.Because the fish lives in hypoxia environment and the 90% oxygen consumption is in the mitochondria,the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal.At the mitochondrial inner membrane,electrons from NADH and succinate pass through the electron transport chain to oxygen,which is reduced to water.Complex I is one of the main sites at which premature electron leakage to oxygen occurs,thus being one of the main sites of production of harmful superoxide.The first isolation of mitochondrial complex I since 1 961,its composition and structure have had a primary understanding,but the specific mechanism of its participation in respiration,especially the function of each subunit is not clear.The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH:ubiquinone oxidoreductase(complex I). Mammalian complex I is composed of 45 different subunits.This protein has NADH dehydrogenase activity and oxidoreductase activity.It plays an important role in transferring electrons from NADH to the respiratory chain. Reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE)methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss .The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program.A pair of gene specific primers were designed to amplify the full-length cDNA sequence.ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences,and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed.Protein phosphorylation sites and

  8. Development of a full-genome cDNA clone of Citrus leaf blotch virus and infection of citrus plants.

    Science.gov (United States)

    Vives, María Carmen; Martín, Susana; Ambrós, Silvia; Renovell, Agueda; Navarro, Luis; Pina, Jose Antonio; Moreno, Pedro; Guerri, José

    2008-11-01

    Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a ~9-kb single-stranded, positive-sense genomic RNA encapsidated by a 41-kDa coat protein. CLBV isolates are associated with symptom production in citrus including leaf blotching of Dweet tangor and stem pitting in Etrog citron (Dweet mottle disease), and some isolates are associated with bud union crease on trifoliate rootstocks, but Koch's postulates for this virus were not fulfilled. A full-genome cDNA of CLBV isolate SRA-153, which induces bud union crease, was placed under the T7 promoter (clone T7-CLBV), or between the 35S promoter and the Nos-t terminator, with or without a ribozyme sequence downstream of the CLBV sequence (clones 35SRbz-CLBV and 35S-CLBV). RNA transcripts from T7-CLBV failed to infect Etrog citron and Nicotiana occidentalis and N. benthamiana plants, whereas agro-inoculation with binary vectors carrying 35SRbz-CLBV or 35S-CLBV, and the p19 silencing suppressor, caused systemic infection and production of normal CLBV virions. Virus accumulation was similar in citron plants directly agro-infiltrated, or mechanically inoculated with wild-type or 35SRbz-CLBV-derived virions from Nicotiana, and the three sources incited the symptoms characteristic of Dweet mottle disease, but not bud union crease. Our results show that (1) virions derived from an infectious clone show the same replication, movement and pathogenicity characteristics as the wild-type CLBV; (2) CLBV is the causal agent of Dweet mottle disease but not of the bud union crease syndrome; and (3) for the first time an RNA virus could be successfully agro-inoculated on citrus plants. This infectious clone may become a useful viral vector for citrus genomic studies.

  9. Cloning of a short HLA-DQ beta locus-specific cDNA probe: typing for DQw specificities.

    Science.gov (United States)

    Sood, S K; McCusker, C T; Singal, D P

    1989-01-01

    A short HLA-DQ beta locus-specific (141 bp) probe was cloned from the full-length pII-beta-1 cDNA. Pst 1-digested genomic DNA from homozygous typing cell lines (HTC) was hybridized with this short DQ beta locus-specific, pDQ beta 141, probe. Restriction fragment length polymorphism (RFLP) patterns generated with this DQ beta locus-specific probe were compared with those obtained with the full-length (627 bp) DQ beta, pII-beta-1, probe. The results demonstrate that the RFLP patterns with the pDQ beta 141 probe were very simple, and no crossreacting DR beta and DX beta bands were observed. DQw1, 2, 3 and 4 specificities could each be identified by a single RFLP. PMID:2467193

  10. Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Plants synthesize the osmoprotectant glycine betaine (GB) via choline→betaine aldehyde→glycine betaine[1]. Two enzymes are involved in the pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spinacia oleracea L. And Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. Tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42℃), as determined by immunoblot analysis, but did not respond to cold stress (4℃), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents.

  11. cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda.

    Science.gov (United States)

    Duan, Yafei; Liu, Ping; Li, Jitao; Li, Jian; Gao, Baoquan; Chen, Ping

    2013-12-01

    Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5' untranslated region (UTR) of 14 bp, a 3' UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda. PMID:24141991

  12. cDNA cloning and recombinant expression of the gen-eral odorant binding protein Ⅱ from Spodoptera litura

    Institute of Scientific and Technical Information of China (English)

    JIN FengLiang; DONG XiaoLin; XU XiaoXia; REN ShunXiang

    2009-01-01

    A cDNA encoding the general odorant binding protein Ⅱ (GOBP Ⅱ) was isolated from the antennae of Spodoptera fitura (SiGOBP Ⅱ, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SIGOBP Ⅱ was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pl of 5.72. SIGOPB Ⅱ shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SIGOPB Ⅱ shared significant identity with the GOBP Ⅱ from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SIGOBP Ⅱ was specifically expressed in the antennae, cDNA encoding SIGOBP Ⅱ was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coil BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPⅡ i.e, 32 kD, which has a 6xHis tag at the N-terminus. The recombinant SIGOBP U was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1 : 12800, while Western blot analysis showed that the recombinant SIGOBP Ⅱ was recognized as anti-SiGOBP Ⅱ an-tiserum.

  13. Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

    Science.gov (United States)

    Liu, Nan-Nan; Liu, Zeng-Shan; Lu, Shi-Ying; Hu, Pan; Li, Yan-Song; Feng, Xiao-Li; Zhang, Shou-Yin; Wang, Nan; Meng, Qing-Feng; Yang, Yong-Jie; Tang, Feng; Xu, Yun-Ming; Zhang, Wen-Hui; Guo, Xing; Chen, Xiao-Feng; Zhou, Yu; Ren, Hong-Lin

    2015-04-15

    Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis. PMID:25712755

  14. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E;

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  15. Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128.

    Science.gov (United States)

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2004-02-27

    A novel xyloglucan-specific endo-beta-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-beta-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (-2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained. PMID:14987996

  16. cDNA cloning, characterization, and pharmacologic evaluation of anticancer activity of a lectin gene in Pinellia integrifolia.

    Science.gov (United States)

    Liu, L L; Yang, Z J; Peng, Z S

    2016-01-01

    Plant lectins are proteins that possess at least one non-catalytic domain, which could reversibly bind to specific monosaccharides or oligosaccharides. The important roles played by plant lectins in immune regulation, signaling pathways, and plant defense could be attributed to their specific binding activities with carbohydrates. In this study, a Pinellia integrifolia lectin gene, designated pia, was cloned using rapid amplification of cDNA ends. The open reading frame (ORF) of pia was constructed into the pET-28a vector, and a 33-kDa recombinant protein was induced in Escherichia coli BL21. The hemagglutination and anticancer properties of the purified recombinant protein were assayed in vitro. The results indicated that the full-length cDNA of pia was 1210 bp long, containing an 807-bp ORF encoding a 268-amino acid peptide. The putative P. integrifolia lectin protein (PIA) contained three mannose-binding sites. The agglutinating activity exhibited by PIA was inhibited by D-mannose. PIA was also shown to exert an anti-proliferative activity against nasopharyngeal carcinoma, human cervical carcinoma, and human breast cancer cell lines in vitro. These results could be applied to determine the function of PIA in the future. PMID:27525949

  17. Cloning, Expression, and In Vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Mohammad Hadi Sekhavati

    2013-01-01

    Full Text Available Objective: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein.Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3. Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment.Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE of the purifi ed phiC31 integrase confirmed the size of protein (70 kDa. Finally, the functionality of purified phiC31 integrase was verified.Conclusion: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.

  18. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16+ natural killer cells and CD3+, CD16- T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  19. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; You-Yong Lu

    2002-01-01

    AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.

  20. Differential gene expression and characterization of tissue-specific cDNA clones in oil palm using mRNA differential display.

    Science.gov (United States)

    San, Cha Thye; Shah, Farida Habib

    2005-12-01

    The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein. PMID:16328884

  1. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas

  2. Detection and characterization of a mouse α-spectrin cDNA clone by its expression in Escherichia coli

    International Nuclear Information System (INIS)

    A cloned segment of mouse α-spectrin mRNA has been identified by radioimmunoassay and immunoautoradiography. Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an 125I-labeled antiserum to erythrocyte membrane ghost proteins. Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized. Transfer of the 750-base-pair insert to an expression vector containing the P/sub L/ promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin. The spectrin-like peptides made in E. coli elicited antibody that reacted only with the α-spectrin subunit of erythrocyte membranes. This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias

  3. Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of HumanLactase-Phlorizin Hydrolase

    Institute of Scientific and Technical Information of China (English)

    WEI HE; ZHEN-YU JI; CHENG-YU HUANG

    2006-01-01

    Objective To study the mechanism of lactose intolerance (LI) by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse (♂).Gene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase (LPH) in human, rat, and rabbit. A coding sequence (CDS) fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinformatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosy1 hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA (GenBank accession No: AY751548) provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

  4. Isolation and Cloning of cDNA of Gene Encoding for Metallothionein Type 2 from Soybean [Glycine max (L. (Merrill] cv. Slamet

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUTI

    2009-07-01

    Full Text Available Metallothionein has an important role in the detoxification of metal ions. It has a low molecular weight and contains cysteine-rich residue. The objective of this research is to isolate and clone the cDNA of gene encoding for metallothionein from soybean [Glycine max (L. (Merrill] cv Slamet (GmMt2. We had successfully isolated total RNA by reverse transcription and synthesized total cDNA from total RNA as template. cDNA of GmMt2 had been isolated from total cDNA by PCR. It was successfully inserted into pGEM-T Easy plasmid, and the recombinant plasmids were introduced into Escherichia coli strain DH5α. Sequence analysis by using T7 and SP6 primers showed that the length of PCR-isolated fragment is 257 bp containing 246 bp completed sequence of Mt2 cDNA encoding for 81 amino acids. Enzyme restriction analysis showed that GmMt2 does not contain any restriction sites found in the multi cloning sites of pGEM-T easy. Nucleotide and amino acid alignment analysis using BLAST program showed that GmMt2 is similar with completed cDNA of AtMt2A from Arabidopsis thaliana (L. Heynh. Amino acid sequence analysis showed that the motifs of Cys sequence of GmMT2 are Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys.

  5. cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67.7-kDa carboxyl-terminal fragment

    International Nuclear Information System (INIS)

    cDNA clones encoding human topoisomerase I were isolated from an expression vector library (λgt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus

  6. Virulence in pigs of vPader10 rescued from an infectious cDNA clone of the CSFV strain Paderborn

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Nielsen, Jens; Uttenthal, Åse;

    The BAC clone, pBeloPader10, contains a complete cDNA of the CSFV strain Paderborn. Virus, named vPader10, was rescued from this construct by electroporation of RNA transcripts into porcine PK15 cells. To further study the characteristics of vPader10, we evaluated the virulence of this virus...

  7. Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

    Science.gov (United States)

    Bhattacharya, A; Shepard, A R; Moser, D R; Roberts, L R; Holland, L J

    1991-02-01

    Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture. PMID:2050271

  8. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    Science.gov (United States)

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-01

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. PMID:25499296

  9. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    International Nuclear Information System (INIS)

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

  10. Cloning and characterization of cDNA encoding an elicitor of Phytophthora colocasiae.

    Science.gov (United States)

    Mishra, Ajay Kumar; Sharma, Kamal; Misra, Raj Shekhar

    2010-02-28

    The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. A cDNA encoding elicitor, the major secreted extracellular glycoprotein of Phytophthora colocasiae, a pathogen of taro (Colocasia esculenta) plants, was isolated, sequenced and characterized. The expression of the corresponding elicitor gene during the disease cycle of P. colocasiae was analyzed. Elicitor was shown to be expressed in mycelium grown in culture media, whereas it was not expressed in sporangiospores and zoospores. In planta, during infection of taro, particularly during the biotrophic stage, expression of elicitor was down-regulated compared to in vitro. The highest levels of expression of elicitor were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The elicitation of the suspension-cultured taro cells was effective in the induction of the enzyme activity of l-phenylalanine-ammonia lyase, peroxidase and lipoxygenase as well as the expression of defense-related endochitinase gene. All these biological activities were exerted within a low concentration range. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in taro plant against Phytophthora leaf blight. PMID:19230634

  11. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  12. [Construction of an infectious cDNA clone derived from foot-and-mouth disease virus O/QYYS/s/06].

    Science.gov (United States)

    Lu, Shousheng; Zhao, Qizu; Liu, Xiangtao; Sun, Yanwei; Ren, Tao; Zhang, Guihong; Qi, Wenbao; Zha, Yunfeng; Kong, Lingchen; Zhang, Han; Fan, Huiying; Liao, Ming

    2009-07-01

    After sequencing, we amplified and cloned foot-and-mouth disease virus (FMDV) O/QYYS/s/06 whole genome by three fragments. These three fragments were cloned into vector P43 one by one to construct recombinant plasmid P43C, which carried the full-length cDNA of FMDV O/QYYS/s/06. Then, plasmid P43C and plasmid T7 expressing T7 RNA polymerase were co-transfected into BHK-21 cells. After 48 h, we harvested the culture broth from transfected BHK-21 cells and inoculated into 2-3 day-old sucking mice. After four generation passage, the virus harvested from sucking mice was confirmed to be type O FMDV by the indirect hemagglutination test, sucking mice's neutralization test and sequencing. The results showed that we have successfully constructed the full-length cDNA clone of FMDV O/QYYS/s/06 strain. PMID:19835137

  13. Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

    Science.gov (United States)

    Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M

    2012-12-01

    Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives. PMID:22971818

  14. Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda.

    Science.gov (United States)

    Ding, J L; Navas, M A; Ho, B

    1995-03-01

    Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification. PMID:7538401

  15. cDNA cloning and recombinant expression of the general odorant binding protein Ⅱ from Spodoptera litura

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A cDNA encoding the general odorant binding protein Ⅱ(GOBP Ⅱ) was isolated from the antennae of Spodoptera litura(SlGOBP Ⅱ,GenBank Accession No.EU086371) by homologous cloning and rapid amplification of cDNA ends(RACE).Sequencing and structural analyses revealed that the open reading frame(ORF) of SlGOBP Ⅱ was 489 bp,encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72.SlGOPB Ⅱ shared typical structural features of odorant binding proteins with other insects,including the six conservative cysteine residues.The deduced amino acid sequence of SlGOPB Ⅱ shared significant identity with the GOBP Ⅱ from S.frugiperda and S.exigua.RT-PCR and Northern blot analyses showed that SlGOBP Ⅱ was specifically expressed in the antennae.cDNA encoding SlGOBP Ⅱ was constructed into the pET-32a vector and the recombinant protein was highly expressed in Escherichia coli BL21(DE3) after induction with IPTG.SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPⅡ i.e,32 kD,which has a 6×His tag at the N-terminus.The recombinant SlGOBP Ⅱ was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits.ELISA showed that the titer of antiserum was 1:12800,while Western blot analysis showed that the recombinant SlGOBP Ⅱ was recognized as anti-SlGOBP Ⅱ antiserum.

  16. 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.

  17. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries

    Directory of Open Access Journals (Sweden)

    Nan-Nan Liu

    2016-07-01

    Full Text Available Lysophospholipase I (LYPLA1 is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1 was cloned using primers and rapid amplification of cDNA ends (RACE technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR of 24 bp, a 3′-UTR of 1740 bp with a poly (A tail, and an open reading frame (ORF of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.

  18. Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries.

    Science.gov (United States)

    Liu, Nan-Nan; Liu, Zeng-Shan; Hu, Pan; Zhang, Ying; Lu, Shi-Ying; Li, Yan-Song; Yang, Yong-Jie; Zhang, Dong-Song; Zhou, Yu; Ren, Hong-Lin

    2016-01-01

    Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. PMID:27483239

  19. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  20. cDNA cloning of conotoxins with framework XII from several Conus species

    Institute of Scientific and Technical Information of China (English)

    Zhuguo Liu; Zheng Yu; Na Liu; Chongjia Zhao; Jie Hu; Qiuyun Dai

    2010-01-01

    In our efforts for cloning novel I2-superfamily conotoxins using the signal peptide sequence,we identified a novel eonotoxin Lt12.4 from Conus iitteratus.This gene has a fra mework Ⅻ(-C-C-C-C-CC-C-C-),which is distinct from the cysteine pattern I2-superfamily conotoxin(-C-C-CC CC-C-C-).Subsequently,we found the signal peptide sequence of Lt12.4 by 5'-RACE.Using this new sequence,we identified another five novel conotoxins with this cysteine pattern from four Conus species(Conus eburneus,Conus imperialis,Conus marmoreus,and C.litteratus).These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII,and may contain Gla residues.Furthermore,they have the highly conserved signal peptide and hypervariable mature peptide sequences,and widely exist in Conus species.Therefore,it could be defined as a new superfamily of E-conotoxins.

  1. Structure of a cannabinoid receptor and functional expression of the cloned cDNA.

    Science.gov (United States)

    Matsuda, L A; Lolait, S J; Brownstein, M J; Young, A C; Bonner, T I

    1990-08-01

    Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana. PMID:2165569

  2. Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm.

    Science.gov (United States)

    Harn, C; Knight, M; Ramakrishnan, A; Guan, H; Keeling, P L; Wasserman, B P

    1998-07-01

    Two starch synthase clones, zSSIIa and zSSIIb, were isolated from a cDNA library constructed from W64A maize endosperm. zSSIIa and zSSIIb are 3124 and 2480 bp in length, and contain open reading frames of 732 and 698 amino acid residues, respectively. The deduced amino acid sequences of the two clones share 58.1% sequence identity. Amino acid sequence identity between the zSSIIa and zSSIIb clones and the starch synthase II clones of potato and pea ranges between 45 to 51%. The predicted amino acid sequence from each SSII cDNA contains the KXGGL consensus motif at the putative ADP-Glc binding site. Both clones also contain putative transit peptides followed by the VRAA(E)A motif, the consensus cleavage site located at the C-terminus of chloroplast transit peptides. The identity of the zSSIIa and zSSIIb clones as starch synthases was confirmed by expression of enzyme activity in Escherichia coli. Genomic DNA blot analysis revealed two copies of zSSIIa and a single copy of zSSIIb. zSSIIa was expressed predominantly in the endosperm, while transcripts for zSSIIb were detected mainly in the leaf at low abundance. These findings establish that the zSSIIa and zSSIIb genes are characteristically distinct from genes encoding granule-bound starch synthase I (Waxy protein) and starch synthase I. PMID:9687068

  3. Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase.

    OpenAIRE

    Lee, D C; Carmichael, D F; Krebs, E G; McKnight, G S

    1983-01-01

    A cDNA clone for the type I regulatory subunit (RI) of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from bovine testis by a differential screening method. mRNA coding for RI was enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RI and protein A-Sepharose. Poly(A)+ RNA from these polysomes was utilized to construct a cDNA library in pBR322, and this library was screened for hybridization to 32P-la...

  4. Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2).

    OpenAIRE

    Binkert, C; Landwehr, J; Mary, J L; J. Schwander; Heinrich, G

    1989-01-01

    Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325....

  5. 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.

  6. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Science.gov (United States)

    Li, Xing-Xia; Yu, Wen-Chao; Cai, Zhong-Qiang; He, Cheng; Wei, Na

    2016-01-01

    The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization. PMID:27703977

  7. Cloning and sequencing of a rice ( Oryza sativa L.) RAPB cDNA using yeast one-hybrid system

    Institute of Scientific and Technical Information of China (English)

    姚泉洪; 邢彦彦; 王宗阳; 张景六; 彭日荷; 洪孟民

    1999-01-01

    Cis-acting elements containing CCAAT core sequence are located in 5’ upstream regions of numerous eukaryotic genes. CCAAT-binding factors interact with these cis-acting elements as heteromeric complex and therefore control the gene transcription. CCAAT binding factors contain at least three subunits and each subunit alone cannot bind to CCAAT box. The cloning of a rice cDNA called RAPB which homologizes to yeast HAP2 (one of the subunits in CCAAT-binding factors) using yeast one-hybrid system and functional complementation approaches is reported. The analytic results indicate that the deduced amino acid sequence in the C terminal of RAPB also contains the functional domain of 60 amino acids highly homologous with yeast HAP2, whereas the deduced amino acids in N terminal region differs significantly, and no Gln-rich region is found in the RAPB protein as in HAP2. The Southern blotting analysis demonstrates that only one copy of RAPB gene exists in rice genome.

  8. Identification, cDNA Cloning, and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla.

    Science.gov (United States)

    Rather, Mohd Ashraf; Bhat, Irfan Ahmad; Sharma, Rupam

    2016-01-01

    Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp. PMID:26980432

  9. Characterization of an abaecin-like antimicrobial peptide identified from a Pteromalus puparum cDNA clone.

    Science.gov (United States)

    Shen, Xiaojing; Ye, Gongyin; Cheng, Xiongying; Yu, Chunyan; Altosaar, Illimar; Hu, Cui

    2010-09-01

    Abaecin is a major antimicrobial peptide, initially identified from the honeybee. In our effort to discover new antimicrobial peptides from the endoparasitoid wasp Pteromalus puparum, we identified an antibacterial cDNA clone that codes a fragment with high amino acid sequence similarity to abaecin. The proline-rich peptide (YVPPVQKPHPNGPKFPTFP, named PP30) was chemically synthesized and characterized in this study. Antimicrobial assays indicated that the cationic peptide was active against both Gram-negative and positive bacteria, but not active against fungi tested. No hemolytic activity was observed against human erythrocytes after 1h incubation at concentration of 125 microM or below. The antibacterial activity of PP30 against Escherichia coli was attenuated in the presence of increasing concentrations of NaCl. Transmission electron microscopic (TEM) examination of PP30-treated E. coli cells showed morphological changes in the cells and extensive damage to the cell membranes. The circular dichroism (CD) spectroscopy studies indicated that PP30 formed random coil structures in phosphate buffer (pH 7.4), 50% TFE and 25 mM SDS solution. Expression analysis of the gene coding for the peptide indicated that its expression was upregulated upon bacterial infection, indicating that the gene may play a role in preventing potential infection by microorganisms during parasitization in Pieris rapae. PMID:20466006

  10. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas

    Directory of Open Access Journals (Sweden)

    Xing-Xia Li

    2016-01-01

    Full Text Available The shell of the pearl oyster (Pinctada fucata mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

  11. Molecular cloning and characterization of nitrate reductase gene cDNA from non-heading Chinese cabbage

    Institute of Scientific and Technical Information of China (English)

    SUN Feifei; HOU Xilin; LI Ying; CUI Xiumin

    2007-01-01

    Four non-heading Chinese cabbage (Brassica campestris ssp.chinensis Makino) cultivars,Suzhouqing,Xuekeqing,Huangxinwu,Aijiaohuang,were planted to investigate the activity of nitrate reductase (NRA) in leaves.After being induced by KNO3 at 50 mmol/L for different hours,the NRAs of the four cultivars were determined in vivo.The results showed that the NRAs changing trends of these four cultivars were similar.The highest NRAs in leaves reached their maximum at the 4th,4th,6th,and the 6th hour of induction,respectively.According to these results,the level of NR mRNA in plants could be enhanced by nitrate inducement.Then,the total RNA was isolated from the leaves of Suzhouqing that was induced by KNO3 at 50 mmol/L for four hours,and two fragments ofNR cDNA were obtained through RT-PCR using specific primers.The products of PCR were cloned and sequenced.They are 1125 and 438 base pairs,which were named nr1125 and nr438,encoding 374 and 135 amino acids,respectively.Finally,nr1125 was accepted and released by GenBank (accession number DQ001901).

  12. Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

    Science.gov (United States)

    Dabrowska, M; Jagielska, E; Cieśla, J; Płucienniczak, A; Kwiatowski, J; Wranicz, M; Boireau, P; Rode, W

    2004-02-01

    The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle. PMID:15030008

  13. Cloning and expressional analyses of a cinnamoyl CoA reductase cDNA from rice seedlings

    Institute of Scientific and Technical Information of China (English)

    BAI Yong; GONG Wei; LIU Tianyun; ZHU Yuxian

    2003-01-01

    Cinnamoyl CoA reductase (CCR: EC 1.2.1.44), the entry-point enzyme of the lignin specific biosynthetic pathway, catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes. Multiple sequence alignment showed that the deduced polypeptide shared 70% similarity and 30% sequence identity at the amino acid level with defined CCR genes from other plant species and they all contain the common signature sequences thought to be the catalytic site as well as the putative NADP binding domain. Using a conserved OsCCR cDNA fragment as the probe for library screening, we isolated the genomic DNA that covered the whole coding region of OsCCR with total length of 3045 bp including 4 introns and 5 exons. The open reading frame for our OsCCR gene contains 337 amino acids. Northern blot indicated that OsCCR was expressed in different organs with the highest level found in stems. In situ hybridization results showed that OsCCR mRNA was localized mainly along the vascular bundles in stems and leaves, and also in lateral roots that was differentiating from the tillering node. We conclude that the vascular-localized expression of OsCCR gene may suggest its possible involvement in lignin biosynthesis. Cloning and characterization of OsCCR will help to clarify how lignifications in plants are regulated and will provide a physical basis for creating genetically engineered rice plants with optimal lignin contents.

  14. Cloning and sequencing of Octopus dofleini hemocyanin cDNA: derived sequences of functional units Ode and Odf.

    Science.gov (United States)

    Lang, W H; van Holde, K E

    1991-01-01

    A number of additional cDNA clones coding for portions of the very large polypeptide chain of Octopus dofleini hemocyanin were isolated and sequenced. These data reveal two very similar coding sequences, which we have denoted "A-type" and "G-type." We have obtained complete A-type sequences coding for functional units Ode and Odf; consequently a total of three such unit sequences are now known from a single subunit of one molluscan hemocyanin. This presents the opportunity to make sequence comparisons within one hemocyanin subunit. Domains within one subunit show on the average 42% identity in amino acid residues; corresponding functional units from hemocyanins of different species show degrees of identity of 53-75%. Therefore, molluscan hemocyanins already existed before the individual molluscan classes diverged in the early Cambrian. Sequence comparisons of molluscan hemocyanins with arthropodan hemocyanins and tyrosinases allow us to identify the ligands of the "Copper B" site with high probability. Possible ligands for the "Copper A" site are proposed, based on sequence comparisons between molluscan hemocyanins and tyrosinases. Besides two histidine side chains, a methionine side chain might be involved in binding of Copper A, a result not in conflict with spectroscopic studies. PMID:1898774

  15. Some properties and cDNA cloning of proteinaceous toxins from two species of lionfish (Pterois antennata and Pterois volitans).

    Science.gov (United States)

    Kiriake, Aya; Shiomi, Kazuo

    2011-11-01

    Lionfish, members of the genera Pterois, Parapterois and Dendrochirus, are well known to be venomous, having venomous glandular tissues in dorsal, pelvic and anal spines. The lionfish toxins have been shown to cross-react with the stonefish toxins by neutralization tests using the commercial stonefish antivenom, although their chemical properties including structures have been little characterized. In this study, an antiserum against neoverrucotoxin, the stonefish Synanceia verrucosa toxin, was first raised in a guinea pig and used in immunoblotting and inhibition immunoblotting to confirm that two species of Pterois lionfish (P. antennata and P. volitans) contain a 75kDa protein (corresponding to the toxin subunit) cross-reacting with neoverrucotoxin. Then, the amino acid sequences of the P. antennata and P. volitans toxins were successfully determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Notably, either α-subunits (699 amino acid residues) or β-subunits (698 amino acid residues) of the P. antennata and P. volitans toxins share as high as 99% sequence identity with each other. Furthermore, both α- and β-subunits of the lionfish toxins exhibit high sequence identity (70-80% identity) with each other and also with the β-subunits of the stonefish toxins. As reported for the stonefish toxins, the lionfish toxins also contain a B30.2/SPRY domain (comprising nearly 200 amino acid residues) in the C-terminal region of each subunit. PMID:21878347

  16. Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings.

    Science.gov (United States)

    Mato, M; Ozeki, Y; Itoh, Y; Higeta, D; Yoshitama, K; Teramoto, S; Aida, R; Ishikura, N; Shibata, M

    1998-11-01

    Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.

  17. Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

    International Nuclear Information System (INIS)

    In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.)

  18. Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein.

    Science.gov (United States)

    Pogue-Geile, K; Geiser, J R; Shu, M; Miller, C; Wool, I G; Meisler, A I; Pipas, J M

    1991-08-01

    We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

  19. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA of OH/CHA/99

    Institute of Scientific and Technical Information of China (English)

    LIU Guangqing; LIU Zaixin; XIE Qingge; CHEN Yingli; BAO Huifang; LIU Xiangtao

    2004-01-01

    The China foot-and-mouth virus (FMDV) isolate OH/CHA/99 was isolated from swine, which was unable to infect bovine thyroid cells in vitro or to cause typical disease in bovines following intradermal inoculation in the tongue. To enhance antigenicity, replication, maturation and pathogenicity studies of OH/CHA/99, an infectious full- length cDNA clone, designated pBlFMDV, was prepared. The in vitro and in vivo biological properties of the virus derived from pBlFMDV were studied by analyzing antigenicity, plaque morphology and virulence in pigs. The results showed that the virus derived from pBlFMDV had the same biological properties as the parent strain OH/CHA/99; the full- length infectious cDNA clone, pBlFMDV, will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of FMDV.

  20. Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1.

    Science.gov (United States)

    Ferriol, Inmaculada; Ambrós, Silvia; da Silva, Dorivaldo M; Falk, Bryce W; Rubio, Luis

    2016-06-01

    Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, has a genome composed of two single-stranded positive-sense RNAs of ∼5.8 (RNA1) and 3.4kb (RNA2). Full-length cDNA clones of both genomic RNAs (pBenR1 and pBenR2) from BBWV-1 isolate Ben were constructed under the control of the T7 promoter. In vitro derived capped transcripts were infectious in Nicotiana benthamiana, Chenopodium quinoa and Vicia faba plants. The biological activity of viral transcripts was not affected by extra bases at the 5'-terminus introduced during in vitro transcription. Virions derived from the infectious cDNA clones displayed similar viral infectivity and accumulation, as well as symptom induction as the wild-type BBWV-1 isolate. PMID:26951858

  1. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    Energy Technology Data Exchange (ETDEWEB)

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. (Wistar Inst. of Anatomy and Biology, Philadelphia, PA (USA))

    1990-09-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

  2. Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2002-10-01

    Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

  3. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains

    Energy Technology Data Exchange (ETDEWEB)

    Wun, T.C.; Kretzmer, K.K.; Girard, T.J.; Miletich, J.P.; Broze, G.J. Jr.

    1988-05-05

    Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor X/sub a/ directly, and in a X/sub a/-dependent fashion also inhibits the VII/sub a/-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI antiserum was used to screen human placental and fetal liver lambdagt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind /sup 125/I-factor X/sub a/. Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (lambdaP9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that lambdaP9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH/sub 2/ terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH/sub 2/ terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA which hybridize with LACI cDNA.

  4. Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties

    DEFF Research Database (Denmark)

    Dan, Ippeita; Ong, Shao-En; Watanabe, Norinobu M;

    2002-01-01

    We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK do...... apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells. Udgivelsesdato: 2002-Feb-22...

  5. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    OpenAIRE

    Alamar Santiago; Arribas Raquel; Forment Javier; Alonso-Cantabrana Hugo; Marques M Carmen; Conejero Vicente; Perez-Amador Miguel A

    2009-01-01

    Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information an...

  6. Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Y. Cui

    2008-05-01

    Full Text Available Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1 allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+, expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG, 267-277 (NYHAVNIVGYG and 284-303 (YWIVRNSWDTTWGDSGYGYF. Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%, an extended strand (17.13%, a ß turn (5.61%, and a random coil (43.30%. A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

  7. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

    OpenAIRE

    Hohman, V S; Schluter, S F; Marchalonis, J J

    1992-01-01

    A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constru...

  8. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

    Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

  9. Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony-stimulating factor activities: relationship to interleukin 5

    International Nuclear Information System (INIS)

    Conditioned medium from the Con A-treated mouse helper T-cell clone Ly1+2-/9 contains activities that enhance the production of IgA by mouse B cells and induce human cord blood cells to form eosinophil colonies. We have isolated a cDNA sequence that expresses IgA-enhancing factor and eosinophil colony-stimulating factor activities from a cDNA library prepared from activated Ly1+2-/9 cells. Based on homology with the mouse cDNA sequence, a human cDNA sequence coding for an interleukin with IgA-enhancing factor and eosinophil colony-stimulating factor activities was isolated from a cDNA library prepared from a human T-cell clone stimulated with anti-T3 antibody and phorbol 12-myristate 13-acetate. DNA sequence analyses revealed that mouse and human cDNA clones encode proteins of 133 and 134 amino acids, respectively, that are identical to cDNA clones encoding the T-cell replacing factor I and B-cell growth factor II activities. These results establish that a single cDNA clone encodes a protein that acts as a growth and differentiation factor for both B cell and eosinophils

  10. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    Energy Technology Data Exchange (ETDEWEB)

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  11. Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.).

    Science.gov (United States)

    Zeh, Michaela; Leggewie, Georg; Hoefgen, Rainer; Hesse, Holger

    2002-02-01

    A potato cDNA clone, StMS1, that encodes a methionine synthase was isolated. This protein was identified on the basis of both structural and functional evidence. The predicted sequence of the protein encoded by StMS1 shows a high degree of similarity to methionine synthases from other organisms and the expression of StMS1 in bacterial mutant strains restored the mutant's ability to synthesize methionine. Genomic organization and expression analyses suggest that StMS1 is a low-copy gene and is differentially expressed in potato organs. StMS1 expression was found in all tissues, but at elevated levels in flowers, basal levels in sink and source leaves, roots and stolons, and low levels in stems and tubers. RNA expression data were confirmed by western blot analysis except that the protein content in leaves was less than expected from the RNA data. Western blot analysis of subcellular fractions revealed that the protein is located in the cytosol. However, the changing pattern of gene expression during the day/night period implied a light-dependent control of MS transcription normally seen for enzymes localized in plastids. The expression of MS was shown to be light-inducible with its highest expression at midday. These RNA data were not confirmed at the protein level since protein content levels remained constant over the whole day. Feeding experiments of detached leaves revealed that sucrose or sucrose-derived products are responsible for StMS1 induction. This induction can be blocked by treatment with DCMU during the light period. Western analysis revealed that the amount of StMS1 is not affected by either treatment. This experiment confirmed the presence of a day/night rhythm. Methionine synthase expression is regulated by photoassimilates but this seems not to detectably alter protein levels. PMID:11855727

  12. cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

    Institute of Scientific and Technical Information of China (English)

    HUANG; Shengbing; SONG; Wei; LIN; Qishui

    2005-01-01

    A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-Qop. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

  13. Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus.

    Science.gov (United States)

    Kuhn, R J; Niesters, H G; Hong, Z; Strauss, J H

    1991-06-01

    We have constructed a full-length cDNA clone of the virulent T48 strain of Ross River virus, a member of the alphavirus genus. Infectious RNA can be transcribed from this clone using SP6 or T7 RNA polymerase. The rescued virus has properties indistinguishable from those of the T48 strain of Ross River virus. We have used this clone, together with a full-length cDNA clone of Sindbis virus, to construct chimeric plasmids in which the 5' and the 3' nontranslated regions of the Sindbis and Ross River genomes were exchanged. The nontranslated regions of the two viral genomes differ in both size and sequence although they maintain specific conserved sequence elements. Virus was recovered from all four chimeras. Chimeras containing heterologous 3' nontranslated regions had replicative efficiencies equal to those of the parents. In contrast, the chimeras containing heterologous 5' nontranslated regions were defective in RNA synthesis and virus production, and the severity of the defect was dependent upon the host. Replication of a virus containing a heterologous 5' nontranslated region may be inefficient due to the formation of defective protein-RNA complexes, whereas, the presumptive complexes formed between host or virus proteins and the 3' nontranslated region to promote RNA synthesis appear to function normally in the chimeras.

  14. Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family.

    Science.gov (United States)

    Tucker, M L; Durbin, M L; Clegg, M T; Lewis, L N

    1987-05-01

    A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

  15. 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.

  16. cDNA cloning,sequence analysis,and recombinant expression of akitonin beta,a C-type lectin-like protein from Agkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    Xiang-dong ZHA; Jing LIU; Kang-sen XU

    2004-01-01

    AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structurefunction relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template.The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBADTOPO as vector and transformed into E. coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin β expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.

  17. The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1: mRNA cloning, structure analysis and comparison with mammalian homologues

    Directory of Open Access Journals (Sweden)

    Thomas Anne VT

    2005-10-01

    Full Text Available Abstract Background Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2, the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX -producing bacteria. Results The porcine-LFA-1 CD11a (alpha subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. Conclusion Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.

  18. Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina

    International Nuclear Information System (INIS)

    Type A γ-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA ρ1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family

  19. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Directory of Open Access Journals (Sweden)

    Carolyn A. Owen

    2016-06-01

    Full Text Available Cucurbit yellow stunting disorder virus (CYSDV, a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

  20. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Science.gov (United States)

    Owen, Carolyn A.; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A.; Eliasco, Eleonora; Aranda, Miguel A.; Coutts, Robert H. A.; Livieratos, Ioannis C.

    2016-01-01

    Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. PMID:27314380

  1. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts.

    Science.gov (United States)

    Owen, Carolyn A; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A; Eliasco, Eleonora; Aranda, Miguel A; Coutts, Robert H A; Livieratos, Ioannis C

    2016-01-01

    Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5'- and 3'-UTRs plus the 3'-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. PMID:27314380

  2. Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Daucus carota L.,and their partial amino acid sequences were determined.The homological analysis showed that the sequence from the 64 ku protein was highly homological to b -glucosidase,and that from the 55 ku protein had no significant homologue in GenBank.Using conservative sequence of animal IF proteins as primer,we cloned a cDNA fragment from Daucus carota L.Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves.Homological analysis revealed that it had moderate homology to a variety of a -helical proteins.Our results might shed more light on molecular characterization of IF existence in higher plant.

  3. Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

    Institute of Scientific and Technical Information of China (English)

    赵大中; 陈丹英; 杨橙; 翟中和

    2000-01-01

    Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Caucus carota L, and their partial amino acid sequences were determined. The homological analysis showed that the sequence from the 64 ku protein was highly homological to p-glucosidase, and that from the 55 ku protein had no significant homologue in GenBank. Using conservative sequence of animal IF proteins as primer, we cloned a cDNA fragment from Daucus carota L. Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves. Homological analysis revealed that it had moderate homology to a variety of a-helical proteins. Our results might shed more light on molecular characterization of IF existence in higher plant.

  4. Purification, characterization and partial cDNA cloning of high-temperature stress-induced protein from French bean (Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Nagesh Babu, R.

    2013-02-01

    Full Text Available In order to identify the components of high temperature response in French bean, three heat shock proteins induced under high temperature were purified to homogeneity by Carboxy methyl cellulose and sephadex G-100 chromatography followed by preparative SDS-PAGE. Two of these, Hsp1 and Hsp3 were further characterized by immuno-detection with polyclonal antibodies. Hsp3 exhibited ATPase and chaperone activity with malate dehydrogenase and citrate synthase. Partial cDNA for Hsp3 synthesized using the primer derived from amino-terminal sequence was cloned and expressed in Escherichia coli. The recombinant protein possesses ATPase activity, and showed thermal protection at 50°C in Escherichia coli. The translated partial cDNA showed homology with stress induced proteins including ATPases from higher plants. These results supported the fact that French bean response to high temperature stress involves Hsps as one of the principal components.

  5. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Xu, W.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States); Kozak, C.A. [National Institute of Health, Bethesda, MD (United States)

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  6. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

    OpenAIRE

    Crock, John; Wildung, Mark; Croteau, Rodney

    1997-01-01

    (E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a...

  7. Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity

    DEFF Research Database (Denmark)

    Vorum, H; Liu, X; Madsen, Peder;

    1998-01-01

    By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid N...... and pancreas and at very low levels in brain and liver. Calumenin belongs to a family of multiple EF-hand proteins that include the ER localized proteins reticulocalbin and ERC-55 and the Golgi localized Cab45. Since its Ca2+ binding may be important for the function of the protein we have used microdialysis...

  8. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T;

    2000-01-01

    . donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16...... out of 25 patients with visceral leishmaniasis and four out of 18 patients with cutaneous leishmaniasis contained IgG antibodies which reacted with the purified LePa fusion protein as evaluated in an ELISA. The LePa DNA sequence was inserted into an eukaryotic expression vector and Balb/c mice were...

  9. In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone

    OpenAIRE

    YANAGI, MASAYUKI; St. Claire, Marisa; Emerson, Suzanne U.; Purcell, Robert H.; Bukh, Jens

    1999-01-01

    Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a ...

  10. Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis

    NARCIS (Netherlands)

    Berezikov, Eugene; van Tetering, Geert; Verheul, Mark; van de Belt, Jose; van Laake, Linda; Vos, Joost; Verloop, Robert; van de Wetering, Marc; Guryev, Victor; Takada, Shuji; van Zonneveld, Anton Jan; Mano, Hiroyuki; Plasterk, Ronald; Cuppen, Edwin

    2006-01-01

    MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA cand

  11. Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

    Directory of Open Access Journals (Sweden)

    Mi Sa Vo Phan

    2014-06-01

    Full Text Available Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV from Glycine soja (wild soybean, named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean and Pisum sativum (pea as well as N. benthamiana, but not the other legume species.

  12. Cloned yeast and mammalian transcription factor TFIID gene products support basal but not activated metallothionein gene transcription

    International Nuclear Information System (INIS)

    Transcription factor IID (TFIID), the TATA binding factor, is thought to play a key role in the regulation of eukaryotic transcriptional initiation. The authors studied the role of TFIID in the transcription of the yeast metallothionein gene, which is regulated by the copper-dependent activator protein ACE1. Both basal and induced transcription of the metallothionein gene require TFIID and a functional TATA binding site. Crude human and mouse TFIID fractions, prepared from mammalian cells, respond to stimulation by ACE1, In contrast, human and yeast TFIID proteins expressed from the cloned genes do not respond to ACE1, except in the presence of what germ or yeast total cell extracts. These results indicate that the cloned TFIID gene products lack a component(s) or modifications(s) that is required for regulated as compared to basal transription

  13. Cloning and Bioimformatic Analysis of Full Length cDNA of LHY Gene from Cicer arietinum L.%鹰嘴豆 LHY 基因 cDNA 克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    韩慧; 王希东; 姚正培; 夏木斯亚·卡坎

    2014-01-01

    从鹰嘴豆中克隆生物节律钟 LHY 基因的 cDNA 全长序列,进行序列信息学分析.通过同源克隆策略,利用 RT-PCR 技术获得核心片段,结合5′-RACE 和3′-RACE 技术,克隆得到鹰嘴豆生物节律钟基因 LHY 的 cD-NA 全长序列,其核苷酸序列长度为3061 bp,包括2220 bp 的完整开放阅读框(ORF),编码739个氨基酸.验证后命名为 CarLHY 基因,获得基因登录号为 KJ558378.生物信息学研究表明 CarLHY 基因 cDNA 序列与其他植物 LHY 基因具有较高的相似性;预测 CarLHY 蛋白不具有跨膜结构;为转录因子,定位于细胞核中;不具备信号肽.对 CarLHY 蛋白功能结构域预测表明,蛋白质核心结构存在符合转录因子与 DNA 结合的常见功能域 HTH.蛋白系统进化树显示,与大豆分子进化距离最近,其次是黑杨、拟南芥.%The sequence analysis was carried out to investigate the full-length cDNA sequence of biothythm clock gene LHY cloned from Cicer arietinum Linn with 5′-RACE and 3′-RACE.RT-PCR tech-nology was used to obtain core fragment with the homologous cloning strategy.The cDNA sequence was 3061 bp,and the open reading frame (ORF)was 2 220 bp,encoded 739 amino acids.It was named CarLHY after checked.The registration number was KJ558378 in Genebank datebase.The research of biological in-formation science indicated that cDNA sequence of CarLHY gene and other plant LHY gene have higher similarity.It was predicted that CarLHY protein have no transmembrane structure.It was speculated that CarLHY protein have no transmembrane spiral;It was a transcriptional factors and located in the nucleus. It has no signal peptide.CarLHY protein functional domains forecast indicated that there were transcription factor and HTH domain binded to DNA in protein core structure.The phylogenetic tree showed that CarL-HY and Glycine LHY are closest in molecular evolution distance,followed by Populus LHY and Arabii-dopsis thalana LHY gene.

  14. A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

    Directory of Open Access Journals (Sweden)

    Moritz Eißmann

    Full Text Available Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

  15. Can mammalian cloning combined with embryonic stem cell technologies be used to treat human diseases?

    OpenAIRE

    Hadjantonakis, Anna-Katerina; Papaioannou, Virginia E.

    2002-01-01

    Cloning is commonly perceived as a means of generating genetically identical individuals, but it can also be used to obtain genetically matched embryo-derived stem cells, which could potentially be used in the treatment of patients. A recent report offers the first 'proof of principle' of such cloning for therapeutic purposes, referred to as nuclear transplantation to produce stem cells for autologous transplantation.

  16. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  17. Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

    Institute of Scientific and Technical Information of China (English)

    JIAO Chuanzhen; WANG Zaizhao; LI Fuhua; ZHANG Chengsong; XIANG Jianhai

    2004-01-01

    The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5′- and 3′-untranslated region(5′- and 3′-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1-547 bp, but they did not exist in the region of 548-2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.

  18. Characterization of variation induced by low-energy N+ and cloning of differentially expressed cDNA of a mutant in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using Arabidopsis thaliana as experimental materials, the variations induced by low-energy N+ have been investigated. Germination rate of the treated seeds is lower than that of the control, and it decreases with the intensification of the radiation. The phenotypic variations have been observed in M2 plants irradiated with higher doses, such as chlorisis, semilethality, plant morphology, and changes of blooming habit and fertility. In random amplified polymorphic DNA (RAPD) analysis on M2 seedlings, some differences including band deletions or additions are found in treated plants compared to the control and the differences are associated with the radiation doses. One of the M1 plants from the seeds irradiated with the dose of 80×1015 N+/cm2 is a dwarf variant. Its stable M6 generation, mutant T80II, is used to construct subtractive cDNA library and to clone differentially expressed cDNA. A 721 bp cDNA fragment is partly homologous with GRF7 gene.

  19. Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

    Science.gov (United States)

    Sakurai, Tetsuya; Plata, Germán; Rodríguez-Zapata, Fausto; Seki, Motoaki; Salcedo, Andrés; Toyoda, Atsushi; Ishiwata, Atsushi; Tohme, Joe; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Ishitani, Manabu

    2007-01-01

    Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs). Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. PMID:18096061

  20. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  1. Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2007-12-01

    Full Text Available Abstract Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs. Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.

  2. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Miyamura, Tatsuo; Saito, Izumu (National Institute of Health, Tokyo (Japan)); Katayama, Tohru (Tokyo National Chest Hospital (Japan)); Kikuchi, Shu; Tateda, Akira (Sendai National Hospital (Japan)); Houghton, M.; Choo, Quilim; Kuo, G. (Chiron Corporation, Emeryville, CA (USA))

    1990-02-01

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented.

  3. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

    International Nuclear Information System (INIS)

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented

  4. Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

    Science.gov (United States)

    Stankovic, B.; Vian, A.; Henry-Vian, C.; Davies, E.

    2000-01-01

    Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca (flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

  5. [Rescue of bovine Asia 1 serotype foot-and-mouth disease virus from a full-length cDNA clone].

    Science.gov (United States)

    Li, Shuang; Zhang, Runxiang; Song, Ge; Gao, Mingchun; Liu, Xiangtao; Wang, Junwei

    2009-11-01

    After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5' fragment was 1.8 kb in length including 15Cs, and the 3' fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD. PMID:20222458

  6. Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

    Institute of Scientific and Technical Information of China (English)

    Xiangru FENG; Yilong CHEN; Xiao ZHAO; Wendong WANG; Junhui ZHANG; Zhenguo YANG SUN; Shengmei JIA; Qiang LU

    2012-01-01

    Abstract [Objective] This study aimed to obtain IL-IO (interleukin 10) full-length cD- NA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. []~lethod] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-IO full-length cDNA was cloned from 0.8x104 pfu of recombinant phages, and the sequence analysis and homology com- parison were carried out. [Result] Sequence analysis indicated that the IL-IO full- length cDNA of common carp was 1 117 bp long, containing a.55 bp 5'-UTR, a 522 bp 3"-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3"-untranslated region. The deduced protein sequence shared typical sequence features of the IL-IO family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-IO gene from GenBank. [Conclusion] This study laid foun- dation for further study of the expression manner, functional characteristic and regu- lation mechanism of IL-IO in vivo and the interaction mechanism in the inflammatory reaction and immune response.

  7. IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O6-METHYLGUANINE-DNA-METHYLTRANSFERASE CDNA INTO HUMAN UMBILICAL CORD BLOOD CD34+ CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT-PCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8′ 105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT-cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

  8. Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

    OpenAIRE

    Yu, J.; Cary, J W; D. Bhatnagar; Cleveland, T E; Keller, N. P.; Chu, F S

    1993-01-01

    Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type...

  9. Stabilization of a Full-Length Infectious cDNA Clone of Transmissible Gastroenteritis Coronavirus by Insertion of an Intron

    OpenAIRE

    González, José M; Pénzes, Zoltan; Almazán, Fernando; Calvo, Enrique; Enjuanes, Luis

    2002-01-01

    The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different po...

  10. Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis

    OpenAIRE

    Jennewein, Stefan; Wildung, Mark R.; Chau, MyDoanh; Walker, Kevin; Croteau, Rodney

    2004-01-01

    Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus...

  11. Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

    OpenAIRE

    Ebina, Y; Edery, M; Ellis, L; Standring, D; Beaudoin, J; Roth, R A; Rutter, W J

    1985-01-01

    We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growt...

  12. cDNA Fragment Cloning of L-Galactono-1,4-Lactone Dehydrogenase and It's Expression in Different Organs of R.roxburghii Tratt

    Institute of Scientific and Technical Information of China (English)

    AN Hua-ming; CHEN Li-geng; FAN Wei-guo

    2004-01-01

    A 855 bp cDNA encoding L-galactono-1,4-lactone dehydrogenase (GalLDH) fragment was cloned from fruit of R. roxburghii Tratt by the method of RT-PCR, on the basis of the homologous genes of Arabidopsis thaliana, cauliflower, sweet potato, strawberry, etc. in GenBank. Sequence analysis showed 79-92% identity in nucleotide sequence and 75-87%identity in amino acid sequence to that of strawberry and Arabidopsis thaliana, etc.Northern blot showed that the expression of GalLDH was significantly different in different organs. The transcription level of GalLDH in fruit was significantly higher than that in leaf, stem and root respectively. Furthermore, this expression mode was highly correlated with AsA levels.

  13. Cloning of cDNA and expression analysis of a DnaJ-like gene under heavy metal stress in bean

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A clone of PvSR6 encoding a new member of the DnaJ-like protein family was isolated from a mer curic-chloride-treated bean ( Phaseolus vulgaris L. )cDNA library by differential screening using cDNAs derived from treated and untreated plants. The predicted protein contains the highly conserved J domain only, which is present in all DnaJ-like proteins and is considered to play a critical role in DnaJ protein-protein interactions. PvSR6 gene is constitu tively expressed in roots but weakly expressed in stems and leaf tissue. Northern blot analysis revealed the transcripts of PvSR6 were at low levels in unstressed bean leaves, but the genes expression was strongly stimulated by heavy metals. These suggest that the PvSR6 might play an important role in resistance to the damage caused by heavy metals.

  14. Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone

    DEFF Research Database (Denmark)

    Pallesen, Lone Tjener; Andersen, Mikkel Holmen; Nielsen, Rune L.;

    2001-01-01

    acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced...... amino acid sequence demonstrated structural features characteristic for mucins, including an extracellular tandem repeat region with 11 partially conserved repeats (20 amino acids each), a membrane-proximal SEA module, a transmembrane domain, and a cytoplasmic C-terminal region. Monosaccharide...... composition determinations suggested significant structural differences between O-linked glycans of MUC1 originating from either bovine or human milk. Interspecies differences of the consensus repeat sequence in MUC1 and the physiological functions are discussed....

  15. cDNA Cloning of a Vacuolar H+-Pyrophosphatase and Its Expression in Hordeum brevisubulatum (Trin.) Link in Response to Salt Stress

    Institute of Scientific and Technical Information of China (English)

    L(U) Shi-you; JING Yu-xiang; PANG Xiao-bin; ZHAO Hua-yan; MA Lan-qing; LI Yan-fang

    2005-01-01

    A cDNA clone encoding a vacuolar H+-pyrophosphatase (V-H+-PPase) was isolated from Hordeum brevisubulatum (Trin.) Link by using RACE method. Sequence analysis revealed that HbVP1 contained 2 319 nucleotides of open reading frame (ORF) and 420 nucleotides of 3'-untranslated region. Its encoding protein consisted of 773 amino acid residues,which includes 14 transmembrane helices. The predicated molecular mass is 80.4 kDa with pI of 4.90. The V-H+-PPases in higher plants shared low identity (40-55%) with those of protozoa, marine alga and archaebacteria. HbVP1 transcripts accumulated abundantly in roots, shoots and seeds, and it was also strongly induced by salt treatment.

  16. Sequencing and rescuing a highly virulent classical swine fever virus: Chinese strain cF114 from a full-length cDNA clone

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The complete nucleotide sequence of classical swine fever virus (CSFV) strain cF114 (F114 strain propa- gated on PK-15 cells) was cloned by RT-PCR. The analyses of nucleotide and amino acids identity between cF114 and F114, Brescia, Alfort or C strain were 99.41%, 96.80%, 86.03%, 95.70% and 99.28%, 98.54%, 93.33%, 97.41% re- spectively. The cDNA fragments with correct sequence were ligated into a full-length cDNA and inserted into pMC18 plasmid (pMC12297). A full-length infectious viral RNA was synthesized by runoff transcription and transfected to PK15 cells. Viruses were recovered from transfected cells which wese titrated on PK-15 cells by endpoint dilution and indirect immunofluorescence with a CSFV-specific monoclonal antibody. The antigenicity and replication kinetics of the plasmid-derived virus (vM12297) were similar to the parental virus in vitro. The E01 or E2 gene was replaced with the genes from strain C and the pM/CE01 and pM/CE2 with chimeric full-length cDNA of cF114 were generated. The infectious viruses were obtained from pM/CE01 and pM/CE2. Both of the chimeric viruses can infect PK-15, SK- 6 and primary testicle cell of swine. The chimeric viruses can grow to a titer of 8×105 F-PFU/mL. These results are very important for understanding the genes related to the CSFV propagation and pathogenesis.

  17. Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon.

    Science.gov (United States)

    Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Joonyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung

    2002-02-01

    We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development. PMID:11867694

  18. A cDNA Cloning of a Novel Alpha-Class Tyrosinase of Pinctada fucata: Its Expression Analysis and Characterization of the Expressed Protein

    Directory of Open Access Journals (Sweden)

    Ryousuke Takgi

    2014-01-01

    Full Text Available Tyrosinase plays an important role in the formation of the shell matrix and melanin synthesis in mollusks shells. A cDNA clone encoding a 47 kDa protein was isolated from the pearl oyster Pinctada fucata. The cDNA was 1,957 base pairs long and encodes a 417 residue protein that has extensive sequence identity with tyrosinase (polyphenol oxidase: EC 1.14.18.1. This tyrosinase-like protein, termed PfTy, contains an N-terminal signal sequence and the two copper-binding domain signatures (CuA and CuB, suggesting that PfTy belongs to the α-subclass of type-3 copper proteins. Enzyme activity of PfTy was examined by a spectrophotometric method using the translation product derived from an S30 T7 high-yield protein expression system. Tyrosinase activity was seen in this recombinant product. RT-PCR analysis showed that PfTy mRNA was expressed in the mantle pallial, but not in the mantle edge. Therefore, PfTy may participate in insoluble shell matrix formation of the nacreous layer. PfTy expression was also observed in the foot, liver, and adductor muscle, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes. This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata.

  19. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    Science.gov (United States)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( Panimals.

  20. Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

    Science.gov (United States)

    Charlesworth, A; Georgieva, T; Gospodov, I; Law, J H; Dunkov, B C; Ralcheva, N; Barillas-Mury, C; Ralchev, K; Kafatos, F C

    1997-07-15

    Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed. PMID:9266686

  1. Cloning of the Full-length cDNA of the Wheat Involved in Salt Stress: Root Hair Defective 3 Gene (RHD3)

    Institute of Scientific and Technical Information of China (English)

    Lei SHAN; Shuang-Yi ZHAO; Guang-Min XIA

    2005-01-01

    The full-length cDNA of the wheat (Triticum aestivum L.) root hair defective 3 gene (RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5′ rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3 gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701-718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned,including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3 gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed.

  2. 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.

  3. Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta Var. esculenta).

    Science.gov (United States)

    Lin, Da-Gin; Jeang, Chii-Ling

    2005-10-01

    Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340 bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 +/- 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms.

  4. Full length cDNA cloning and expression analysis of annexinA2 gene from deer antler tissue

    Institute of Scientific and Technical Information of China (English)

    Li Hao; Xianghong Xiao; Heping Li

    2014-01-01

    ANXA2(AnnexinA2), a calcium-dependent phospholipid bind-ing protein, is involved in various Ca2+-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer (Cervus nippon hortulorum);the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the open-reading frame (ORF) encoding 339 amino acids; its relative mo-lecular weight was 38.3 kDa; and isoelectric point was 6.72. Sequence analysis indicates that the protein includes four conserved tan-dem-duplication ANX domains. The gene-accession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re-veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza-tion.

  5. Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection

    OpenAIRE

    Mehdy, Mona C.; Lamb, Christopher J.

    1987-01-01

    The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene encoding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A λgt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 105 recombinants with an antiserum to purified bean CHI. ...

  6. Murine branched chain alpha-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development.

    Science.gov (United States)

    Doering, C B; Coursey, C; Spangler, W; Danner, D J

    1998-06-01

    These studies were designed to demonstrate the structural and functional similarity of murine branched chain alpha-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain alpha-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain alpha-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system. PMID:9611264

  7. Cloning of lea cDNA fragment of carrot (Daucus carota L.) and analysis of its expression features

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Addition of concentrated sucrose to MS culture arrests the development of carrot somatic embryo at the stage of cotyledon embryo and, with the sucrose concentration restored to normal level, the embryo thus arrested is reactivated into post-embryonic development. Using the method of RT-PCR, the cDNA fragment of a new member of the Dc3 family of lea has been obtained from carrot somatic embryo under regulated state. As revealed by Northern blotting, strong expression has been observed in carrot somatic embryo under regulated state but the expression was much reduced 12 h after deregulation, and nearly disappeared 24 h after. Based on this finding as well as results of related studies, it is surmised that changing the sucrose concentration in culture enabled carrot somatic embryo under suspension culture to undergo a specific course of development which is comparable to the dormancy-germination process of seeds.

  8. Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

    International Nuclear Information System (INIS)

    The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero

  9. Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, R.J. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Mestrado em Farmácia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mantovani, M.S.; Silva, A.F. da [Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR (Brazil); Pesarini, J.R. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta”, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Mauro, M.O. [Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica, Núcleo de Hospital Universitário, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Programa de Doutorado em Biotecnologia e Biodiversidade - Rede Pró Centro-Oeste, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS (Brazil); Ribeiro, L.R. [Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto de Biociências de Rio Claro, Universidade Estadual Paulista, Rio Claro, SP (Brazil); Programa de Pós-Graduação em Patologia, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP (Brazil)

    2014-03-28

    The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

  10. Cu,Zn superoxide dismutase: cloning and analysis of the Taenia solium gene and Taenia crassiceps cDNA.

    Science.gov (United States)

    Parra-Unda, Ricardo; Vaca-Paniagua, Felipe; Jiménez, Lucia; Landa, Abraham

    2012-01-01

    Cytosolic Cu,Zn superoxide dismutase (Cu,Zn-SOD) catalyzes the dismutation of superoxide (O(2)(-)) to oxygen and hydrogen peroxide (H(2)O(2)) and plays an important role in the establishment and survival of helminthes in their hosts. In this work, we describe the Taenia solium Cu,Zn-SOD gene (TsCu,Zn-SOD) and a Taenia crassiceps (TcCu,Zn-SOD) cDNA. TsCu,Zn-SOD gene that spans 2.841 kb, and has three exons and two introns; the splicing junctions follow the GT-AG rule. Analysis in silico of the gene revealed that the 5'-flanking region has three putative TATA and CCAAT boxes, and transcription factor binding sites for NF1 and AP1. The transcription start site was a C, located at 22 nucleotides upstream of the translation start codon (ATG). Southern blot analysis showed that TcCu,Zn-SOD and TsCu,Zn-SOD genes are encoded by a single copy. The deduced amino acid sequences of TsCu,Zn-SOD gene and TcCu,Zn-SOD cDNA reveal 98.47% of identity, and the characteristic motives, including the catalytic site and β-barrel structure of the Cu,Zn-SOD. Proteomic and immunohistochemical analysis indicated that Cu,Zn-SOD does not have isoforms, is distributed throughout the bladder wall and is concentrated in the tegument of T. solium and T. crassiceps cysticerci. Expression analysis revealed that TcCu,Zn-SOD mRNA and protein expression levels do not change in cysticerci, even upon exposure to O(2)(-) (0-3.8 nmol/min) and H(2)O(2) (0-2mM), suggesting that this gene is constitutively expressed in these parasites.

  11. Complete genome sequence and construction of infectious full-length cDNA clones of tobacco ringspot Nepovirus, a viral pathogen causing bud blight in soybean.

    Science.gov (United States)

    Zhao, Fumei; Hwang, Un Sun; Lim, Seungmo; Yoo, Ran Hee; Igori, Davaajargal; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun

    2015-08-01

    Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions. PMID:26159876

  12. Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Crock, John E. (Moscow, ID)

    1999-01-01

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  13. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Science.gov (United States)

    Croteau, Rodney Bruce; Crock, John E.

    2005-01-25

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  14. Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase from mung beans.

    Science.gov (United States)

    Leiter, H; Mucha, J; Staudacher, E; Grimm, R; Glössl, J; Altmann, F

    1999-07-30

    Substitution of the asparagine-linked GlcNAc by alpha1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase (core alpha1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6. 8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core alpha1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial alpha1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4GlcNAcbeta1-Asn, GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4(Fucalpha1-6)GlcNAcbeta1-Asn, and GlcNAcbeta1-2Manalpha1-3[Manalpha1-3(Manalpha1-6 )Manalpha1-6]Manbeta1 -4GlcNAcbeta1-4GlcNAcbeta1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core alpha1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass

  15. Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli

    Science.gov (United States)

    Küpper, Hans; Keller, Walter; Kurz, Christina; Forss, Sonja; Schaller, Heinz

    1981-02-01

    Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage λ PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

  16. B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

    in turn react with authentic B-G proteins. None of the clones represent a complete message, some--if not all--bear introns, and none of them match with any sequences presently stored in the data banks. The following new information did, however, emerge. At least two homologous transcripts are present...... could explain the bewildering variation in size of B-G proteins within and between haplotypes. Southern blots of genomic chicken DNA gave complex patterns for most probes, with many bands in common using different probes, but few bands in common between haplotypes. The sequences detected are all present...

  17. Purification, cDNA cloning and functional expression of NADPH-cytochrome P450 reductase from Centaurium erythraea cell cultures.

    Science.gov (United States)

    Schwarz, H; Liu, B; Peters, S; Barillas, W; Beerhues, L

    2009-05-01

    Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury (Centaurium erythraea) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N-glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation. PMID:19470102

  18. Cloning and expression of canine clotting factor Ⅸ cDNA in vitro mediated by retroviral vector

    Institute of Scientific and Technical Information of China (English)

    高啸波; 邱信芳; 卢大儒; 薛京伦

    1999-01-01

    Oligonucleotide of cFIX eDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cDNA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX eDNA was obtained which covered the entire cFIX coding region. Furthermore, GlNaCcIX (driven by hCMV promoter) and GlNaMBcIX (driven by MCK enhancer and β-aetin promoter) were constructed using the retroviral vector backbone of GlNa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/10~6 cell/24 h (GlNaCcIX) and 211 ng/10~6 cell/24 h (GlNaMBcIX) respectively. Those data offered a promising result for further animal study.

  19. Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus).

    Science.gov (United States)

    Zhou, Long-Zhen; Ruan, Mi-Mi; Cai, Qiu-Feng; Liu, Guang-Ming; Sun, Le-Chang; Su, Wen-Jin; Cao, Min-Jie

    2012-03-01

    A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40°C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45°C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M(-1). Apparent K(m) was 1.02 μM and k(cat) was 148 S(-1) for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5'-RACE and 3'-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.

  20. cDNA Cloning, Sequence Analysis of the Porcine LIM and Cysteine-rich Domain 1 Gene

    Institute of Scientific and Technical Information of China (English)

    Jun WANG; Chang-Yan DENG; Yuan-Zhu XIONG; Bo ZUO; Lei XING; Feng-E LI; Ming-Gang LEI; Rong ZHENG; Si-Wen JIANG

    2005-01-01

    LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase Ⅱ phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.

  1. Cloning and Sequence Analysis of PGIP gene and cDNA from Prunus persica%桃PGIP基因及cDNA的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    谌悦; 张军科; 熊帅

    2009-01-01

    通过PCR扩增了桃的PGIP基因及cDNA序列,并进行了序列之间的比对及分析.结果表明,桃的PGIP基因全长1 092 bp,而其cDNA序列只有1 045 bp,PGIP基因中含有一段长147 bp的内含子,且内含子符合GT-AG规律.这与其他核果类植物的PGIP基因构成基本相同.其基因序列与GenBank中已登录的3个桃以及其他核果的序列比对,其同源性达92%~99%;与苹果、梨和大桉的同源性分别为85%、84%和84%.%PGIP gene and cDNA were cloned from Prunus persica and the sequences were compared and analyzed. The result showed that the length of PGIP gene was 1 092 bp, while the length of cDNA was only 1 045 bp. The PGIP gene contained 147 bp length intron, and according to GT-AG law, which basically the same with PGIP gene of other stone fruit plants. its gene sequence and logged 3 peaches in GenBank and other stone fruit were compared the homology can reached to 92%-99% . The homology rates of PGIPs ,compared peach with Malus, Pyrus and Eucalyptus , were 85%, 84% and 84% respectively.

  2. Cloning, expression and protective immunity evalua- tion of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokary- otic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (posi- tive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also sug- gested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.

  3. Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    YU JunLong; WANG ShiPing; LI WenKai; DAI Gan; XU ShaoRui; HE Zhuo; PENG XianChu; ZHOU SongHua; LIU XueQin

    2007-01-01

    1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coll.SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.

  4. cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes).

    Science.gov (United States)

    Xia, Xiaohua; Zhao, Jie; Du, Qiyan; Chang, Zhongjie

    2010-08-01

    The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads. PMID:20861569

  5. cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes).

    Science.gov (United States)

    Xia, Xiaohua; Zhao, Jie; Du, Qiyan; Chang, Zhongjie

    2010-08-01

    The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.

  6. cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes)

    Indian Academy of Sciences (India)

    Xiaohua Xia; Jie Zhao; Qiyan Du; Zhongjie Chang

    2010-08-01

    The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.

  7. cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua%葱蝇ADH基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    陈春露; 陈斌; 司风玲; 何正波

    2012-01-01

    [ Objective ] The aim was to clone the ADH gene of Delia antiqun, and carry out a sequence analysis. [ Method ] The cDNA sequence of ADH gene was cloned with the method of RACE, and then studied with homology analysis, comparison of amino acid sequence and phylogenetic analysis. [Result] The full length of cDNA obtained was 1 088 bp, among which there were 771 bp of ORF, encoding a protein of 256 amino acids with a calculated molecular weight of 30.80 kKa and a theoretical isolectric point of 8.22. The deduced amino acid sequence had the highest identity with that of Glossina morsitans based on homological analysis,and a phylogenic tree was inferred with homological ADH sequences from other insects. [ Conclusion ] The study provides a basis for the further research of ADH gene.%[目的]对葱蝇(Delia antiqua)ADH基因进行克隆,并对其进行序列分析.[方法]通过RACE的方法克隆葱蝇ADH基因的cDNA序列,同时对该序列进行同源性分析、氨基酸序列比对和系统发育分析.[结果]试验获得的cDNA全长1 088 bp,其中ORF 771 bp,编码256个氨基酸,推测其相对分子质量为30.80 kDa,等电点为8.22;通过该基因推导的氨基酸序列与其他物种的ADH进行相似性比较和系统发育分析,发现葱蝇与刺舌蝇(Glossina morsuans)氨基酸序列的同源性最高.[结论]该研究为ADH基因的进一步研究提供了基础.

  8. cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua%葱蝇ADH基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    陈春露; 陈斌; 司风玲; 何正波

    2012-01-01

    【目的】对葱蝇(De如antiqua)ADH基因进行克隆,并对其进行序列分析。【方法】通过RACE的方法克隆葱蝇ADH基因的cDNA序列,同时对该序列进行同源性分析、氨基酸序列比对和系统发育分析。[结果]试验获得的cDNA全长1088bp,其中ORF771bp,编码256个氨基酸,推测其相对分子质量为30.80kDa,等电点为8.22;通过该基因推导的氨基酸序列与其他物种的ADH进行相似性比较和系统发育分析,发现葱蝇与刺舌蝇(Glossina morsitans morsitoas)氨基酸序列的同源性最高。【结论】该研究为ADH基因的进一步研究提供了基础。%[Objective] This study aims to conduct cloning and sequence analysis of ADH gene in D. Antiqua. [Method] Full-length cDNA of ADH gene in D. antiqua was cloned by using RACE technology (GenBank access number: JQ666006). Analysis of the homology, characteristics and functional domains of ADH sequence and the phy- Iogenetic relationship to other dipteran ADH were conducted. [Result] The full length of ADH cDNA is 1 088 bp containing a 771 bp of ORF, encoding 256 amino acids, with a calculated relative molecular weight of 30.80 kDa and a theoretical isoelectric point of 8.22. The deduced amino acid sequence shares the highest homology with Glossina morsitans morsitans based on homological analysis and phylogenetic analysis. [Conclusion] This study provides basis for further research of ADH gene.

  9. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    Science.gov (United States)

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  10. cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

    Energy Technology Data Exchange (ETDEWEB)

    Kuefer, M.U.; Valentine, V.; Behm, F.G. [St. Jude Children`s Research Hospital, Memphis, TN (United States)] [and others

    1996-07-15

    A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.

  11. 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.

  12. Cloning and sequence of cDNA encoding 1-aminocyclo- propane-1-carboxylate oxidase in Vanda flowers

    Directory of Open Access Journals (Sweden)

    Pattana Srifah Huehne

    2013-08-01

    Full Text Available The 1-aminocyclopropane-1-carboxylate oxidase (ACO gene in the final step of ethylene biosynthesis was isolated from ethylene-sensitive Vanda Miss Joaquim flowers. This consists of 1,242 base pairs (bp encoding for 326 amino acid residues. To investigate the specific divergence in orchid ACO sequences, the deduced Vanda ACO was aligned with five other orchid ACOs. The results reveal that the ACO sequences within Doritaenopsis, Phalaenopsis and Vanda show highly conserved and almost 95% identical homology, while the ACOs isolated from Cymbidium, Dendrobium and Cattleya are 8788% identical to Vanda ACO. In addition, the 2-oxoglutarate- Fe(II_oxygenase (Oxy domain of orchid ACOs consists of a higher degree of amino acid conservation than that of the non-haem dioxygenase (DIOX_N domain. The overall homology regions of Vanda ACO are commonly folded into 12 α-helices and 12 β-sheets similar to the three dimensional template-structure of Petunia ACO. This Vanda ACO cloned gene is highly expressed in flower tissue compared with root and leaf tissues. In particular, there is an abundance of ACO transcript accumulation in the column followed by the lip and the perianth of Vanda Miss Joaquim flowers at the fully-open stage.

  13. Revisiting the identification and cDNA cloning of T cell-replacing factor/interleukin-5

    Directory of Open Access Journals (Sweden)

    Kiyoshi eTakatsu

    2014-12-01

    Full Text Available This is a perspective based on the paper Cloning of complementary DNA encoding T cell replacing factor and identity with B cell growth factor II, by Kinashi T, Harada N, Severinson E, Tanabe T, Sideras P, Konishi M, Azuma C, Tominaga A, Bergstedt-Lindqvist S, Takahashi M, Matsuda F, Yaoita Y, Takatsu K, and Honjo, T. Nature (1986 32(6092: 70-3. We have been interested in understanding the molecular basis of T-B cell cooperation for antibody formation. Although many investigators had described a number of different soluble factors that appeared to have biological relevance to T-B cell interactions, molecular basis of such active substances remained unknown for a long period of time. In this perspective, I will briefly summarize the history of the initial discovery of T cell-replacing factor/B cell growth factor II that appeared to be involved in B-cell growth and differentiation, and outline the discovery and characterization of interleukin-5. Studies of interleukin-5 have provided strong evidence that a single cytokine exerts a variety of activities on diverse target cells.

  14. Cloning, sequence analysis and radiation hybrid mapping of a mammalian KRT2p gene.

    Science.gov (United States)

    Miller, A B; Lowe, J K; Ostrander, E A; Galibert, F; Murphy, K E

    2001-09-01

    We report here on the cloning, characterization and radiation hybrid mapping of the canine basic keratin gene KRT2p. The gene spans 8.3 kb, consists of nine exons and eight introns, and is characterized by the typical features of both basic keratins and keratins in general, including glycine-rich head and tail domains, which flank an alpha-helical rod domain of approximately 310 amino acids. Comparisons of sequence and structure reveal that canine KRT2p is strikingly similar to human KRT2p. Alignment of the predicted amino acid sequences for human and dog reveals greater than 80% identity. In the rod domain, the amino acid identity exceeds 90%. We note, however, that canine KRT2p encodes a protein 21 residues longer than human K2p due to the insertion of a glycine repeat motif, GG(G)X, in the head and tail domains of the canine gene. This is the first report of the nearly complete genome sequence for KRT2p of any organism. Radiation hybrid mapping of canine KRT2p to chromosome 27 of the dog is also reported. PMID:11793249

  15. Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone.

    Science.gov (United States)

    Nagashima, Shigeo; Kobayashi, Tominari; Tanaka, Toshinori; Tanggis; Jirintai, Suljid; Takahashi, Masaharu; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-09-01

    To characterize the genomic mutations of hepatitis E virus (HEV) during consecutive passages associated with adaptation to growth in cell culture, a cloned genotype 3 HEV [pJE03-1760F/wt, starting virus (SV)] was passaged 10 times in A549 cells, and the entire genomic sequence of the passage 10 (P10) progeny was determined. Compared to SV, P10 virus possessed two non-synonymous (T2808C and A5054G) and four synonymous mutations (C1213T, T2557C, C3118T and C4435T) in the ORF1. Full-length infectious cDNA clones with a single, double (T2808C and A5054G), or all six mutations, identical to P10, were constructed, and their replication capacity was compared. Four (C1213T, T2557C, T2808C and A5054G) of the six viruses with a single mutation grew more efficiently than SV. The P10 virus propagated more rapidly and grew more efficiently than SV and T2808C+A5054G and reached a higher viral load (95.1- and 8.5-fold, respectively) at 20days post-inoculation. An immunofluorescence analysis revealed that a high percentage (>80%) of cells inoculated with the P10 virus expressed ORF2 proteins, while relatively low percentages (nearly 30% or 5%) inoculated with T2808C+A5054G or SV, respectively, expressed ORF2 proteins. We found that not only non-synonymous but also synonymous HEV mutations are independently associated with increased virus production. PMID:27485920

  16. cDNA cloning and functional characterization of ETHYLENE INSENSITIVE 3 orthologs from Oncidium Gower Ramsey involved in flower cutting and pollinia cap dislodgement.

    Science.gov (United States)

    Chen, Shin-Yu; Tsai, Hsing-Chun; Raghu, Rajasekaran; Do, Yi-Yin; Huang, Pung-Ling

    2011-10-01

    The cDNAs encoding ETHYLENE INSENSITIVE3 (EIN3) transcription factor, OgEIL1 and OgEIL2 of Oncidium were cloned, sequenced and characterized. The deduced amino acid sequences of OgEIL1 and OgEIL2 of identified cDNA clones contain all structural features found in the Arabidopsis EIN3, such as an amino terminal acidic domain, a proline-rich region, and five basic conserved domains. Complementation test for OgEIL1 in Arabidopsis ein3 mutant indicate that function of OgEIL1 is the same as Arabidopsis EIN3. RNA gel blot analysis indicated that OgEIL1 and OgEIL2 expressed differentially in the roots, stem, leaves and flower buds of Oncidium. OgEIL1 and OgEIL2 mRNA levels in fully opened flowers increased as time progressed after cutting and reached a maximum in the fifth day and decreased on seventh day, which is consistent with the hypothesis that flowers initiated to wilt when ethylene raised abruptly. In de-capped flowers, OgEIL2 mRNA showed a decrease, while OgEIL1 mRNA exhibited an increase. Exogenous application of ethylene increased the mRNA levels of OgEIL1 and OgEIL2 in flower buds and flowers after cutting compared prior to ethylene treatment, however, in pollinia de-capped flowers, both OgEIL1 and OgEIL2 mRNA levels responded to a decline to exogenous ethylene immediately after treatment. Collectively, it is suggested that the main functions of OgEIL1 and OgEIL2 are to modulate the senescence of Oncidium flowers.

  17. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone

    International Nuclear Information System (INIS)

    We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions

  18. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

    DEFF Research Database (Denmark)

    Gottwein, Judith; Scheel, Troels; Callendret, Benoit;

    2010-01-01

    Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic...... resulted in robust infection with peak HCV RNA titers of approximately 5.5 log(10) international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with...... animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes....

  19. cDNA cloning and transient expression of the Epstein-Barr virus-determined nuclear antigen EBNA3B in human cells and identification of novel transcripts from its coding region.

    OpenAIRE

    Kerdiles, B; Walls, D; Triki, H.; Perricaudet, M; Joab, I

    1990-01-01

    Recombinant plasmids containing sequences from the BamHI-E rightward reading frames 2a and 2b (BERF2a and 2b) of the Epstein-Barr virus (EBV) genome were isolated from a library of cDNA clones which had been previously made from the EBV B95-8 lymphoblastoid cell line (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:7103-7114, 1986). The characterization of these clones in combination with RNase mapping experiments led to the identification of one leftward and several rightwa...

  20. 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

  1. cDNA cloning, purification, properties, and function of a beta-1,3-glucan recognition protein from a pyralid moth, Plodia interpunctella.

    Science.gov (United States)

    Fabrick, J A; Baker, J E; Kanost, M R

    2003-06-01

    Microorganisms possess distinctive biochemical or molecular patterns on their cell surfaces, such as those formed by the lipopolysaccharides, lipoteichoic acids, and/or peptidoglycans of bacteria and the beta-1,3-glucans of fungi. Pattern recognition proteins that bind to these surface moieties have been implicated in the activation of the innate immune response in insects and other invertebrates. We report the purification and cloning of a cDNA for a 53-kDa beta-1,3-glucan recognition protein (betaGRP) from the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). BetaGRP cDNA contains an open reading frame that encodes 488 amino acids, of which the first 17 residues comprise the secretion signal peptide. The calculated molecular mass of the 471-residue mature protein is 53,311 Da. The protein consists of a carboxyl-terminal domain that is similar to other recognition proteins from invertebrates, beta-1,3-glucanases from bacteria, and a beta-1,3-glucanase from the sea urchin, Strongylocentrotus purpuratus. The amino-terminus of betaGRP shares sequence similarity with other invertebrate recognition molecules and the beta-1,3-glucanase from S. purpuratus. Affinity purification of a 53-kDa protein and subsequent sequencing of a peptide produced by tryptic cleavage confirmed the presence of the betaGRP in P. interpunctella larval hemolymph. RT-PCR analysis indicates that betaGRP is constitutively expressed in all life-stages, with no detectable induction following exposure of wandering larvae to microbial elicitors. Northern blot analysis indicates that the 1.8-kb betaGRP transcript is transcribed within the fat body. Recombinant betaGRP retains beta-1,3-glucan-binding activity, binds to lipopolysaccharide and lipoteichoic acid in vitro, causes aggregation of microorganisms, and activates the prophenoloxidase cascade in the presence of soluble beta-1,3-glucan. These data support the hypothesis that the 53-kDa betaGRP functions to recognize

  2. cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.

    Institute of Scientific and Technical Information of China (English)

    HU Zhen-di; FENG Xia; LIN Qing-sheng; CHEN Huan-yu; LI Zhen-yu; YIN Fei; LIANG Pei; GAO Xi-wu

    2016-01-01

    Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a ful-length cDNA of carboxylesterasepxCCE016b was cloned and exogenously expressed inEscherichia coliat the ifrst time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of thepxCCE016b was signiifcantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore,pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfuly with prokaryotic cel. Western blot analysis showed that it was coded by pxCCE016b. Al the above ifndings provide important information for further functional study, although we are uncertainty

  3. Rescue of infective virus from a genome-length cDNA clone of the FMDV serotype O (IND-R2/75) vaccine strain and its characterization.

    Science.gov (United States)

    Rajasekhar, R; Hosamani, Madhusudan; Basagoudanavar, Suresh H; Sreenivasa, B P; Tamil Selvan, R P; Saravanan, Paramasivam; Venkataramanan, Ramamurthy

    2013-08-01

    We report here the construction and characterization of an infectious cDNA clone of the Indian vaccine strain of foot and mouth disease virus (FMDV) serotype O, IND-R2/75. Viral genome was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in five fragments and subsequently assembled sequentially in a plasmid vector to generate a complete cDNA clone, flanked by the T7 RNA polymerase promoter and poly (A) tail at 5' and 3' ends, respectively. Transfection of BHK-21 cells with the RNA transcribed from this genome-length cDNA construct allowed the recovery of infectious recombinant FMDV particles as evidenced by cytopathic effect in BHK-21 cells. Characterization of the recombinant virus revealed its similarity to the parental strain. Recombinant virus could be distinguished from the parental virus based on the presence of a unique marker sequence in the former, which was incorporated in the cDNA using a silent mutation. The virus showed no significant amino acid changes in the capsid-coding region when serially passaged up to ten times in BHK-21 cells, while retaining the marker sequence. PMID:23465777

  4. Whitefly (Bemisia tabaci genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous cDNA libraries

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

    Full Text Available Abstract Background The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae. Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. Results To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV and Tomato mottle virus (ToMoV. In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production

  5. Methods to determine the transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cDNA amplification.

    Directory of Open Access Journals (Sweden)

    Julius Mulindwa

    2014-04-01

    Full Text Available Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq. We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

  6. Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.

  7. cDNA cloning, genomic structure, and chromosome mapping of the human epithelial membrane protein CL-20 gene (EMP1), a member of the PMP22 family.

    Science.gov (United States)

    Chen, Y; Medvedev, A; Ruzanov, P; Marvin, K W; Jetten, A M

    1997-04-01

    CL-20 is a novel gene encoding a protein that is structurally related to but distinct from the peripheral myelin protein PMP22. Like PMP22, CL-20 is likely to play important roles in the regulation of cell proliferation, differentiation, and cell death. In this study, we describe the cloning and sequencing of a cDNA encoding the human homologue of CL-20 and characterize the genomic structure of this gene. The hCL-20 gene (HGMW-approved symbol EMP1) encodes a protein of 157 amino acids that exhibits 76% identity to the rabbit CL-20 and to the rat EMP-1, which have been described recently, and 39% identity to human PMP22. CL-20 contains four hydrophobic domains, suggesting that it is an integral membrane protein. In particular the second hydrophobic domain encoded within the fourth exon is highly conserved among CL-20, EMP-1, and PMP22, suggesting a functional role for this region. CL-20 mRNA is abundant in squamous-differentiated bronchial epithelial cells; however, low levels of CL-20 mRNA can be detected in several human tissues by Northern analysis. Retinoic acid, which inhibits squamous differentiation, represses CL-20 expression in normal human bronchial epithelial cells. The genomic structure of the hCL-20 gene was analyzed using a P1 vector containing this gene. The hCL-20 gene contains five exons about 0.2, 0.12, 0.1, 0.14, and 2.2 kb and four introns about 15, 1.9, 0.1, and 0.7 kb. We have mapped the hCL-20 gene to chromosome 12p12 by fluorescence in situ hybridization. PMID:9126480

  8. Engineering infectious foot-and-mouth disease virus in vivo from a full-lensth genomic cDNA clone of the A/AKT/58 strain

    Institute of Scientific and Technical Information of China (English)

    BAI XingWen; LI PingHua; CAO YiMei; LI Dong; LU ZengJun; GUO JianHong; SUN DeHui; ZHENG HaiXue; SUN Pu; LIU XiangTao; LUO JianXun; LIU ZaiXin

    2009-01-01

    Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10-7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10-6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran-scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.

  9. 家蝇防御素基因的cDNA克隆及序列分析%Cloning and sequence analysis of the full-length cDNA encoding defensin, an antimicrobial peptide from the housefly (Musca domestica)

    Institute of Scientific and Technical Information of China (English)

    王来城; 王金星; 王来元; 赵小凡

    2003-01-01

    Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.

  10. 醛糖还原酶cDNA的克隆和表达%Cloning and Expression cDNA of Aldose Reductase

    Institute of Scientific and Technical Information of China (English)

    项军强; 蒋红亮; 张虹; 丁明; 赵辅昆

    2011-01-01

    Aldose reductase(AR), the rate-limiting enzyme of the polyol pathway of sugar metabolism, has been implicated in the pathogenesis of diabetic complications, in particular D-glucose reduction to sorbitol. Aldose reductase cDNA is amplified by reverse transcription-PCR (RT-PCR) from the isolated SMMC7721 total RNA. The PCR product is cloned and inserted into E. coli expression vector pET22b(+) to create a recombinant plasmid with 6× His Tag, which is named pET22b(+)-AR. The AR-(His)6 fusion protein is expressed in E. coli BL21(DE3) by IPTG. And AR-(His)6 is purified by Ni-NTA affinity chromotagraphy. Finally the purified AR-(His)6 activity enzyme is measured by AR assay with ultraviolet spectrophotometry. The results of this study may represent a basis for development of potential drug in preventing diabetic complications.%醛糖还原酶能促使葡萄糖转化成山梨醇,是多元醇代谢通路的限速酶,与糖尿病并发症的发生和发展有密切关系.用RT-PCR扩增醛糖还原酶基因,将扩增产物克隆到大肠杆菌表达载体pET22b(+),构建重组表达载体pET22b(+)-AR.经PCR、双酶切和序列测定鉴定后,转化E.coli BL21(DE3),经IPTG诱导表达,SDS-PAGE和Western blotting对重组蛋白进行分析和鉴定后,利用Ni-NTA琼脂糖亲和层析纯化获得重组蛋白AR-(His)6.紫外分光光度法对AR-(His)6进行酶活检测,其比活力为0.45 U/mg,为下一步筛选具有抑制醛糖还原酶活性的化合物及开发有临床应用价值的醛糖还原酶抑制剂提供参考.

  11. 梅花鹿卵泡刺激素α-亚基cDNA的分子克隆与序列分析%Nucleotide sequence of cloned cDNA for α-subunit of sika follicle stimulating hormone

    Institute of Scientific and Technical Information of China (English)

    关洪斌; 李庆章; 张莉

    2002-01-01

    从新屠宰的母梅花鹿脑垂体中提取总RNA,反转录获得cDNA,以此cDNA为模板用PCR法扩增目的片段,获得长为380 bp的梅花鹿卵泡刺激素α--亚基cDNA片段,将它克隆至pMD-18-T-Verctor.随机挑选3个阳性重组子进行测序,并将测序结果与绵羊、牛、猪等多种哺乳动物该基因的核苷酸序列及相应氨基酸序列进行比较.结果表明,梅花鹿卵泡刺激素α--亚基基因编码的氨基酸序列与绵羊、水牛的该基因同源性最高,达97%,只有4个氨基酸不同;与牛的该基因同源性达96%.与人的该基因氨基酸序列同源性较低,为75%.其编码的核苷酸序列与绵羊、水牛、牛的同源性最高,达96%,只有14~16个碱基不同;与人的该基因核苷酸同源性最低,为84%.总的来说,哺乳动物的卵泡刺激素α-亚基具有很高的同源性.%Total RNA was prepared from pituitary gland of new butchered sika.cDNA was synthesized by RT-PCRreaction and this cDNA was used as model in PCR amplification for α-subunit of sika follicle stimulating hormone.The PCR product was 380bp in 1.2% agarose gel electro-phoresis which just was the target fragment of predictedFSHα-subunit. It was cloned it to pMD-18-T vector. 3 positive recombinant was selected at random to analyze itssequence by DNA analysis apparatus. Its amino acid sequence was compared with some other mammalian. The resultshows that it has the highest homology with sheep and buffalo,which it reaches 97%. There are only 4 amino acidsdifference among sika ,sheep and buffalo. It has lower homology in amino acid with human, its homology is 75%. Ithas the highest homology among sika ,sheep, buffalo and bovine in nucleotide sequence, which it reaches 96%.There are 14-16 nucleotides difference among them. It has lower homology in nucleotide sequence with human, it isonly 84%. It was found that the nucleotide sequence of the o-subunit in these mammalian species are highly con-servative. According to our

  12. Introduction by molecular cloning of artifactual inverted sequences at the 5' terminus of the sense strand of bovine parathyroid hormone cDNA.

    OpenAIRE

    Weaver, C A; Gordon, D. F.; Kemper, B

    1981-01-01

    To study the structure and function of the gene for parathyroid hormone, we obtained recombinant plasmids containing bovine parathyroid hormone cDNA. The nucleotide sequence at the 5' terminus (relative to the sense strand) of the cDNA insert in a recombinant plasmid, pPTHi4, was different from that previously reported for the bovine parathyroid hormone cDNA insert of another recombinant plasmid, pPTHm1 [Kronenberg, H. M., McDevitt, B. F., Majzoub, J. A., Nathans, J., Sharp, P. A., Potts, J. ...

  13. Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

    Science.gov (United States)

    Nishi, Tatsuya; Onozato, Hiroyuki; Ohashi, Seiichi; Fukai, Katsuhiko; Yamada, Manabu; Morioka, Kazuki; Kanno, Toru

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus. PMID:27234555

  14. Cloning and Sequence Analysis of Tocopherol Cyclase cDNA from Wheat (T. Aestivum L.)%小麦生育酚环化酶基因全长cDNA的克隆与分析

    Institute of Scientific and Technical Information of China (English)

    邹礼平; 高和平

    2009-01-01

    生育酚环化酶(TC)是维生素E生物合成途径中的%Tocopherol cyclase (TC) is a key enzyme for plant vitamin E biosynthesis. The cDNA of wheat TC was cloned by RACE-PCR with primers designed based on a EST (BQ161011) sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1404 bp, which encoding 467 amino acid residues. Homology analysis of the deduced amino acid showed 91%, 88%, 71%, 68% and 67% identity with TC from rice, maize, sesame, potato and Arabidopsis thaliana, respectively. The result of phylogenetic analysis indicated that various TC sequences were clustered into three groups.

  15. 阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析%Molecular Cloning and Characterization of a Rac1 Homologue cDNA from Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    傅玉才; 章家新; 郑晓虹; 刘红

    2004-01-01

    Objective To clone and characterize a Racl homologue from Trichomonas vaginalis for studying cell cycle of the organism. Methods A cDNA library derived from T. vaginalis mRNA was constructed into λ TriplEx2 phage vector. An expression sequence tag program was launched. Sequences of cDNA clones were analyzed using NCBI BLAST algorithms, and ClustalW and Treeview programs. Results A cDNA clone with a length of 714 base pairs was isolated. The sequence analysis showed that the cDNA clone has an open reading frame with 600 bp. The deduced amino acid sequence from the open reading frame contains 200 residuals and is most homologous to Rac1 subfamily of Rho GTPases with > 60% identity. The conserved sequence elements of Rho GTPases, such as GTP-binding sites, GTPase-activating protein (GAP) interaction motifs, GTP-dissociation inhibitors (GDI) interaction motifs, guanine nucleotide exchange factor (GEF) interaction elements, etc, were detected in the amino acid sequence. The phylogenetic analysis showed that the cDNA clone is grouped in the Rac subfamily and is more closely related to Rac1 proteins of protozoa. Conclusion The cDNA clone isolated belongs to Rac subfamily of Rho GTPases and is probably a Rac1 protein of T. vaginalis.%目的获得阴道毛滴虫Rac1蛋白的cDNA克隆,研究其在细胞周期中的调解作用.方法提取阴道毛滴虫总RNA,构建cDNA表达文库,随机分离cDNA克隆并测序.用在线生物分析软件NCBI BLAST、ClustalW以及Treeview等程序进行序列分析.结果获得一株有714 bp的cDNA克隆.序列分析表明,该克隆开放阅读框具600 bp,推测肽链具200个氨基酸.该肽链与Rho家族中Rac1鸟苷三磷酸(GTP)酶同源性最高(>60%),并具多种Rho GTP酶的保守基序,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等.进化树分析显示该克隆属于Rac亚家族GTP酶,与原虫Rac1蛋白最接近.结论该克隆

  16. Cloning and Characterization of a Novel cDNA Encoding Late Embryogenesis-Abundant Protein 5 Like (LEA-5) Gene from Cara Cara Navel Orange Fruit(Citrus sinensis Osbeck)

    Institute of Scientific and Technical Information of China (English)

    TAO Neng-guo; YE Jun-li; XU Juan; DENG Xiu-xin

    2006-01-01

    LEA5 gene was postulated related with both stress and hormone responses. In an attempt to find genes exclusively expressed during fruit ripening of Cara Cara navel orange, a novel cDNA clone encoding late embryogenesis-abundant protein 5 like gene (CitLEA5-1) was obtained. It was 582 bp in length, containing 97 deduced amino acids. Compared with the stress-induced LEA5 from leaves of Citrus sinensis, CitLEA5-1 had a shorter 3' untranslated region (UTR). Semiquantitative RT-PCR analysis revealed that CitLEA5-1 was transcriptional regulated during fruit ripening of Cara Cara navel orange.

  17. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    Science.gov (United States)

    Tsetsarkin, Konstantin A.; Kenney, Heather; Chen, Rubing; Liu, Guangping; Manukyan, Hasmik; Whitehead, Stephen S.; Laassri, Majid

    2016-01-01

    ABSTRACT An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics. PMID:27555311

  18. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Konstantin A. Tsetsarkin

    2016-08-01

    Full Text Available An arthropod-borne virus, Zika virus (ZIKV, has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics.

  19. Construction of an infectious cDNA clone of foot-and-mouth disease virus type O1BFS 1860 and its use in the preparation of candidate vaccine

    Indian Academy of Sciences (India)

    M Hema; D Chandran; S B Nagendrakumar; M Madhanmohan; V A Srinivasan

    2009-03-01

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA, RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines.

  20. Construction of an infectious cDNA clone of foot-and-mouth disease virus type O 1 BFS 1860 and its use in the preparation of candidate vaccine.

    Science.gov (United States)

    Hema, M; Chandran, D; Nagendrakumar, S B; Madhanmohan, M; Srinivasan, V A

    2009-03-01

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable,full-length cDNA clone of FMDV type O 1 BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEM R- - 7Zf(-). An 8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription.Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA,RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines. PMID:19430118

  1. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class

    International Nuclear Information System (INIS)

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

  2. Cloning and Sequence Analysis of the Full-length cDNA of a Novel yp05 Gene Associated With Citrinin Production in Monascus aurantiacus

    Institute of Scientific and Technical Information of China (English)

    YON-GHUA XIONG; YANG XU; WEI-HUA LAI; YAN-PIN LI; HUA WEI

    2007-01-01

    Objective To obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

  3. 人UCA1基因新剪接变异体全长cDNA序列的克隆%Cloning of the full-length cDNA sequence of a novel human UCA1 spliced variant

    Institute of Scientific and Technical Information of China (English)

    王宇; 陈葳; 李旭

    2012-01-01

    Objective To clone the full-length cDNA sequence of novel UCA1 spliced isoforms for understanding the exact mechanism of this type of alternative splicing. Methods The full-length cDNA was amplified from BLZ-211 cells by using the in silicon sequence elongation technique, 5'-RACE and 3'-RACE techniques. Products of RT-PCR were sequenced and further assembled. Results The new UCA1 spliced isoform sequence was 2 202 bp. Conclusion A combination of the in silicon sequence elongation, 5'-RACE and 3'-RACE techniques is an effective way to obtain the full-length cDNA, which will guide further research on the mechanism of this type of alternative splicing.%目的 克隆新的UCA1剪接变异体全长cDNA序列,为研究其可变剪接机制奠定基础.方法 用电子克隆技术和cDNA序列末端快速扩增技术(rapid amplification of cDNA ends,RACE)扩增细胞系BLZ-211 cDNA并进行产物测序和序列拼接.结果 新克隆的UCA1剪接变异体全长cDNA序列为2 202 bp.结论 综合采用电子克隆技术与RACE技术是获得全长cDNA序列的有效方法,为该基因的后续可变剪接机制的研究奠定了基础.

  4. High-Throughput Plasmid cDNA Library Screening

    OpenAIRE

    Wan, Kenneth H.; Yu, Charles; George, Reed A; Carlson, Joseph W.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, Susan E.

    2006-01-01

    Libraries of cDNA clones are valuable resources for analysing the expression, structure, and regulation of genes, as well as for studying protein functions and interactions. Full-length cDNA clones provide information about intron and exon structures, splice junctions and 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs) derived from cDNA clones can be used to generate constructs allowing expression of native proteins and N- or C-terminally tagged proteins. Thus, obtaini...

  5. CLONING AND SEQUENCING OF THE GENE FOR A LACTOCOCCAL ENDOPEPTIDASE, AN ENZYME WITH SEQUENCE SIMILARITY TO MAMMALIAN ENKEPHALINASE

    NARCIS (Netherlands)

    Mierau, Igor; Tan, Paris S.T.; Haandrikman, Alfred J.; Kok, Jan; Leenhouts, Kees J.; Konings, Wil N.; Venema, Gerard

    1993-01-01

    The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-247 in lambdaEMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport syste

  6. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    International Nuclear Information System (INIS)

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease

  7. Cloning and Sequencing of a Full-Length cDNA Encoding the RuBPCase Small Subunit (RbcS)in Tea (Camellia sinensis)

    Institute of Scientific and Technical Information of China (English)

    YE Ai-hua; JIANG Chang-jun; ZHU Lin; YU Mei; WANG Zhao-xia; DENG Wei-wei; WEI Chao-lin

    2009-01-01

    This study was aimed to isolate ribulose-l,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) from tea plant [Camellia sinensis (L.) O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP (cDNA amplified fragment length polymorphism), we have isolated some transcript-derived fragments (TDFs) occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp (EF011075) cDNA was obtained via rapid amplification of cDNA ends (RACE). It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.

  8. Cloning of a NaCl-induced fructose-1, 6-diphosphate aldolase cDNA from Dunaliella salina and its expression in tobacco

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Xiaoning; (张晓宁); LIN; Changfa; (林长发); CHEN; Huoying; (陈火英); WANG; Hao; (王; 昊); QU; Zhicai; (曲志才); ZHANG; Hongwei; (张宏伟); YAO; Jianhong; (姚剑虹); SHEN; Daleng; (沈大棱)

    2003-01-01

    Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA encoding a NaCl-induced fructose-1, 6- diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%-73%) to chloroplast fructose-1, 6-diphos- phate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern, DsALDP was de novo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selected DsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco by Agrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated that DsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100-200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.

  9. Molecular Cloning and Sequence Analysis on cDNA of Cystatin Gene from Tea Leaves%茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    王朝霞; 李叶云; 江昌俊; 余有本

    2005-01-01

    对多种已知植物巯基蛋白酶抑制剂(cystatin)基因的氨基酸序列进行比对分析,根据其高度保守的氨基酸序列设计一对简并引物,并从茶树品种龙井43鲜叶中提取总RNA,用RT-PCR法扩增出-204 bp的cDNA特异片段,然后通过3'/5'RACE的方法,分别扩增出3'端和5'端的序列,从而获得茶树巯基蛋白酶抑制剂基因的cDNA全长序列,所得序列全长627 bp,编码101个氨基酸,分子量约11.062 KDa.该基因在推测的氨基酸序列中含有巯基蛋白酶抑制剂家族中高度保守的、与其活性有关的QXVXG结构,且经Blast分析表明,该基因序列与其他植物巯基蛋白酶抑制剂基因的氨基酸序列同源性为54%~77%.%Two degenerate primers were designed according to the conserved region among the known plant cystatins. A cDNA fragment of 204 bp was amplified by RT-PCR (reverse transcription polymerase chain reaction)of total RNA extracted from fresh leaves of Tea plant (Camellia sinensis cv Longjing43). A full-length cDNA of the cystatin gene was obtained by 3'/5'RACE (rapid amplification of cDNA ends). The cDNA sequence of this 627 bp clone contained an open reading frame encoding a polypeptide of 101 amino acid residues with a predicable molecular mass of 11.026 KDa. The deduced amino acid sequence contained the motif QXVXG conserved among most members of the cystatin superfamily. By using the program of Blast on GenBank database, the sequence presented a high match with the cystatin genes from other plants, such as European chestnut, Cassava, Cowpea,Tomato, Soybean et al. All researched out sequences were all cystatins, so we can conclude that the cloned sequence is a member of cystatin gene from Tea plant.

  10. A cDNA cloned from Physarum polycephalum encodes new type of family 3 beta-glucosidase that is a fusion protein containing a calx-beta motif.

    Science.gov (United States)

    Maekawa, Akinori; Hayase, Masato; Yubisui, Toshitsugu; Minami, Yoshiko

    2006-01-01

    The microplasmodia of Physarum polycephalum express three types of beta-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane beta-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane beta-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane beta-glucosidase 1 sequence and were highly homologous with the primary structures of fungal beta-glucosidases. Notably, the C-terminal half of membrane beta-glucosidase 1 contains two calx-beta motifs, which are known to be Ca(2+) binding domains in the Drosophila Na(+)/Ca(2+) exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane beta-glucosidase 1 differs from all previously identified family 3 beta-glucosidases. In addition to cDNA for membrane beta-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane beta-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other beta-glucosidase forms occurred in Physarum poycephalum. Thus, the membrane beta-glucosidase is a new type family 3 enzyme fused with the Calx-beta domain. We propose that Calx-beta domain may modulate the beta-glucosidase activity in response to changes in the Ca(2+) concentration.

  11. cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme a:anthocyanidin 3-o-glucoside-6"-o-malonyltransferase from dahlia flowers.

    Science.gov (United States)

    Suzuki, Hirokazu; Nakayama, Toru; Yonekura-Sakakibara, Keiko; Fukui, Yuko; Nakamura, Noriko; Yamaguchi, Masa-Atsu; Tanaka, Yoshikazu; Kusumi, Takaaki; Nishino, Tokuzo

    2002-12-01

    In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K(m), 18.8 microM) to pelargonidin 3-O-glucoside (K(m), 46.7 microM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k(cat) value of 7.3 s(-1). The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11-63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of beta

  12. Cloning of cDNA for a prolactin-inducible protein (PIP) and studies on the hormonal control of PIP gene expression in T47D human breast cancer cells

    International Nuclear Information System (INIS)

    Recently in this laboratory it was shown that in the human breast cancer cell line T47D, human prolactin of human growth hormone in the presence of hydrocortisone induced the synthesis and secretion of PIP's, a family of proteins which differed only in their degree of glycosylation. This finding represented the first demonstration of an inductin of specific proteins by prolactin in human target cells and has provided us with a unique model in which to study the molecular mechanism of multihormonal actions as well as the possible significance of prolactin in human breast cancer. In order to facilitate their studies the authors cloned PIP cDNA. The strategy chosen and the methods used are described in this article

  13. Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

    Science.gov (United States)

    Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

    1993-01-01

    Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

  14. Phenoloxidase in larvae of Plodia interpunctella (Lepidoptera: Pyralidae): molecular cloning of the proenzyme cDNA and enzyme activity in larvae paralyzed and parasitized by Habrobracon hebetor (Hymenoptera: Braconidae).

    Science.gov (United States)

    Hartzer, Kris L; Zhu, Kun Yan; Baker, James E

    2005-06-01

    Phenoloxidase (PO) is a major component of the insect immune system. The enzyme is involved in encapsulation and melanization processes as well as wound healing and cuticle sclerotization. PO is present as an inactive proenzyme, prophenoloxidase (PPO), which is activated via a protease cascade. In this study, we have cloned a full-length PPO1 cDNA and a partial PPO2 cDNA from the Indianmeal moth, Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae) and documented changes in PO activity in larvae paralyzed and parasitized by the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae). The cDNA for PPO1 is 2,748 bp and encodes a protein of 681 amino acids with a calculated molecular weight of 78,328 and pI of 6.41 containing a conserved proteolytic cleavage site found in other PPOs. P. interpunctella PPO1 ranges from 71-78% identical to other known lepidopteran PPO-1 sequences. Percent identity decreases as comparisons are made to PPO-1 of more divergent species in the orders Diptera (Aa-48; As-49; and Sb-60%) and Coleoptera (Tm-58; Hd-50%). Paralyzation of host larvae of P. interpunctella by the idiobiont H. hebetor results in an increase in phenoloxidase activity in host hemolymph, a process that may protect the host from microbial infection during self-provisioning by this wasp. Subsequent parasitization by H. hebetor larvae causes a decrease in hemolymph PO activity, which suggests that the larval parasitoid may be secreting an immunosuppressant into the host larva during feeding.

  15. cDNA cloning, characterization and expression analysis of a novel antimicrobial peptide gene penaeidin-3 (Fi-Pen3) from the haemocytes of Indian white shrimp Fenneropenaeus indicus.

    Science.gov (United States)

    Shanthi, S; Vaseeharan, B

    2012-03-20

    A new member of antimicrobial peptide genes of the penaeidin family, penaeidin 3, was cloned from the haemocytes of Indian white shrimp Fenneropeneaus indicus (F. indicus), by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE-PCR) methods. The complete nucleotide sequence of cDNA clone of Indian white shrimp F. indicus Penaeidin 3 (Fi-Pen3) was 243bp long and has an open reading frame which encodes 80 amino acid peptide. The homology analysis of Fi-Pen3 sequence with other Penaeidins 3 shows higher similarity with Penaeus monodon (92%). The theoretical 3D structure generated through ab initio modelling indicated the presence of two-disulphide bridges in the alpha-helix. The signal peptide sequence of Fi-Pen3 is almost entirely homologous to that of other Penaeidin 3 of crustaceans, while differing relatively in the N-terminal domain of the mature peptide. The mature peptide has a predicted molecular weight of 84.9kDa, and a theoretical pI of 9.38. Phylogenetic analysis of Fi-Pen3 shows high resemblance with other Pen-3 from P. monodon, Litopenaeus stylirostris, Litopenaeus vannamei and Litopenaeus setiferus. Fi-Pen3 found to be expressed in haemocytes, heart, hepatopancreas, muscles, gills, intestine, and eyestalk with higher expression in haemocytes. Microbial challenge resulted in mRNA up-regulation, up to 6h post injection of Vibrio parahemolyticus. The Fi-Pen3 mRNA expression of F. indicus in the premolt stage (D(01) and D(02)) was significantly up-regulated than the postmolt (A and B) and intermolt stages (C). The findings of the present paper underline the involvement of Fi-Pen3 in innate immune system of F. indicus. PMID:21885268

  16. Cloning and expression pattern analysis of a cDNA of pkc-2 gene in Caenorhabditis elegans%秀丽小杆线虫Caenorhabditis elegans pkc-2基因cDNA的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    钱雨; Laurent SEGALAT

    2009-01-01

    蛋白激酶C在秀丽小杆线虫中具有调节肌细胞渐进性萎缩的功能.为了揭示它的调节机制,本研究克隆了秀丽小杆线虫中蛋白激酶C pkc-2基因的cDNA pkc-2-c,构建了含该pkc-2 基因cDNA亚型的重组质粒pPD 118.20-pKG 63;揭示了该cDNA在秀丽小杆线虫体壁肌细胞中的定位.%Protein kinase C regulates the progressive muscle degeneration in Caenorhabditis elegans. In order to investigate the function of PKC involved in muscle degeneration, this paper cloned a cDNA isoform of pkc-2 gene of C. elegans and constructed the recombinant plasmids pPD118.20-pKG63 containing the isoform. The new isoform was then further studied for gene expression pattern. Immunocytochemistry experiment showed that this cDNA isoform expressed in body-wall muscle cells and located near the dense body.

  17. Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder;

    1993-01-01

    transformation and because peptide sequences were available in the microsequence database. The cDNA was cloned from a fibroblast cDNA library using degenerate oligodeoxyribonucleotides and expressed in AMA cells using the vaccinia virus expression system. Database searches indicated that the predicted protein......Treatment of proliferating and quiescent primary human keratinocytes with interferon-gamma (IFN-gamma) (100 U/ml, 23.5 h) followed by two-dimensional gel analysis revealed three proteins, IGUP I-3421 (M(r) = 48,200, pI = 6.06); IGUP I-3524 (M(r) = 56,900, pI = 5.92), a protein homologous to peptide......-chain-release factor and tryptophanyl-tRNA synthetase; and IGUP I-5111 (M(r) = 30,400, pI = 5.76) recorded in the keratinocyte protein database (Celis et al., 1991, 1992) that are highly and specifically up-regulated by IFN-gamma among several agents tested including 14 other cytokines, second messengers [dibutyryl c...

  18. Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants.

    Science.gov (United States)

    Gowri, G; Paiva, N L; Dixon, R A

    1991-09-01

    An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

  19. Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of Banana Fruit Ripening and Softening

    Institute of Scientific and Technical Information of China (English)

    LUWang-Jin; RyoheiNAKANO; YasutakaKUBO; AkitsuguINABAt; JIANGYue-Ming

    2004-01-01

    Xyloglucan endotransglycosylase (XET) is thought to be involved in fruit softening throughdisassembly of xyloglucan, which is the predominant hemicellulose of cell wall. To study the relationshipbetween fruit softening and XET during banana (Musa acuminata Colla cv. Grand Nain) fruit ripening, a fulllength cDNA (1 095 bp) encoding an XET, MA-XET1, was isolated from ripening banana fruit using RT-PCRand RACE-PCR (rapid amplification of cDNA ends) methods. Sequence analysis showed that the cDNAcontains 5' untranslated region of 66 bp, 3' untranslated region of 189 bp and ORF of 840 bp, encoding apredicted polypeptide of 280 amino acids, including DE|DFEFL motif, which is a presumptive catalyticdomain conserved in XETs. DNA gel blot analysis demonstrated that MA-XET1 is encoded by a multi-copyfamily in the banana genome. RNA gel blot analysis revealed that the level of MA-XET1 transcript in thepulp was undetectable, increased and decreased slightly at the preclimacteric, climacteric and postclimactericstages, respectively. In the peel, accumulation of MA-XET1 transcript was low, increased dramatically andthen decreased rapidly, at preclimacteric, climacteric and postclimacteric stages, respectively. Treatmentof fruit with propylene, an analog of ethylene, decreased the firmness and enhanced the accumulation ofMA-XET1 transcript in the peel and pulp. These results suggest that MA-XET1 is involved in softening ofthe peel and pulp during banana fruit ripening and its expression is regulated by ethylene at transcriptionallevel.

  20. Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

    Science.gov (United States)

    Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

    1993-01-01

    An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

  1. 一个新的枳NAC基因cDNA全长的克隆及其亚细胞定位分析%Cloning and Subcellular Localization Analysis of A New Gene NAC cDNA in Poncirus trifoliata

    Institute of Scientific and Technical Information of China (English)

    韩键; 王化坤; 宋长年; 上官林飞; 冷翔朋

    2012-01-01

    Taking the NACl cDNA sequence in Arabidopsis thaliana as the template, the homologous gene in the EST database of citrus was searched and screened, and the cDNA sequence of NACl gene in citrus was cloned by bioinformatics method. Taking the cDNA sequence in the flower of Poncirus trifoliata ( L. ) Raf. As the template, the specific primers were designed according to the a-bove cDNA sequence, and the 5' - end and 3' - end sequences of NAC1 gene were obtained by using 5'RACE and 3' RACE techniques, respectively. Based on these, the corresponding full length cDNA of NACl in Poncirus trifoliata was acquired through sequence splicing. This complete cDNA, designated as Pt - NACl, was 1351 bp in length, it contained a 1047 bp whole open reading frame (ORF) , its 5' - end included a putative translation start codon ( ATG) at the position of 25 bp, and the length of its 3' - end untranslated region was 280 bp. The deduced amino acid sequence of Pt - NACl was 348 residues, which showed 64. 8% , 57. 0% , 61.3% identical levels with that of Malus x domestica, Arabidopsis thaliana, Petunia xhybrida, respectively. Bioinformatics analysis showed that the cDNA of Pt - NACl had the recognition site of microRNA164, and it also had highly conserved NAC domains. Recom-binant plasmid 35S - GW - GFP - FJ619349 was transferred into onion' s epidermal cells by using the particle bombardment method, and the subcellular localization analysis results indicated that Pt - NACl was all localized at the cell membrane.%以拟南芥的NAC1 cDNA序列作为模板,对柑橘EST数据库进行同源检索筛选,利用生物信息学方法克隆了柑橘NAC1基因的cDNA序列.以枳(Poncirus trifoliata)花的cDNA为模板,根据以上cDNA序列设计特异性引物,利用5′RACE和3′RACE技术,分别获得了NAC1基因的5′和3′末端,序列拼接后获得枳的NAC1 cDNA全长,命名为Pt-NAC1.Pt-NAC1全长为1351 bp,含有1个1047 bp完整的开放读码框(ORF),5′

  2. Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA.

    Science.gov (United States)

    Richman, A M; Bulet, P; Hetru, C; Barillas-Mury, C; Hoffmann, J A; Kafalos, F C

    1996-08-01

    Larvae of the mosquito vector of human malaria, Anopheles gambiae, were inoculated with bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phylogenetically conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is induced in response to bacterial infection, in both adult and larval stages. In contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance. PMID:8799739

  3. Partial purification of the chloroplast ATP synthase from Chlamydomonas reinhardtii and the cloning and sequencing of a cDNA encoding the gamma subunit

    International Nuclear Information System (INIS)

    The chloroplast ATP synthase was partially purified from the green alga Chlamydomonas reinhardtii by extracting membranes with deoxycholate and KCl, followed by centrifugation and ammonium sulfate fractionation of the supernatant. The enzyme assay involved the reconstitution of such fractions with bacteriorhodopsin and soybean phospholipids to form vesicles capable of light-dependent [32P]-phosphate esterification. A cDNA for the gamma subunit from Chlamydomonas was isolated, expressed in vitro and sequenced. It contains the entire coding region for the gamma subunit precursor. A 35 amino acid long transit peptide resides at the NH2-terminus of a 323 amino acid long mature peptide that is 77% similar to the spinach gamma subunit. Six cysteines were found; three were conserved in Chlamydomonas and spinach

  4. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

    Science.gov (United States)

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-01-18

    Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN. PMID:9931470

  5. Cloning and sequence analysis of para sodium channel cDNA fragment from silkworm, Bombyx mori%家蚕Para钠通道cDNA片段克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    何琳; 刘丽花; 汪洋

    2011-01-01

    Previous studies have revealed that a point mutation of a target gene is related to insecticide resistance to pyrethroids. The para sodium channel in the insect central nervous system is the target of pyrethroid insecticides. We used the RT-PCR method to clone the para sodium ion channel in the silkworm, Bombyx mori L. (GenBank No. EF521818).The full length of this cDNA fragment is 4 882 base pairs and its partial ORF is 3 986 bp translated into 1 328 amino acids. BLAST analysis demonstrated that the cloned cDNA fragment is virtually identical to the para sodium channel a subunit gene amplified from other insects. Amino acid homology of the cloned fragment with para sodium channel a subunit genes from Heliothis virescens Fabricius, Aedes aegypti L. , Blattella germanica L. , Drosophila melanogaster Meigen and Musca domestica L. was 95%, 82%, 80%, 79% and 77% respectively.%昆虫神经系统para型钠离子通道是拟除虫菊酯类杀虫剂的主要靶标,已有的研究表明钠离子通道基因发生点突变与昆虫对菊酯类杀虫剂的抗性密切相关.本文通过RT-PCR方法克隆获得了编码家蚕Bombyx mori L.钠离子通道的cDNA片段(GenBank No.EF521818),该片段全长4 882 bp,部分ORF包含3 986 bp核苷酸,翻译成1 328个氨基酸.蛋白序列分析表明,PCR扩增获得的家蚕钠离子通道eDNA片段所编码的氨基酸与其他昆虫的para型钠离子通道α亚基的氨基酸具有很高的同源相似性,与棉铃虫Heliothis virescens Fabricius、埃及伊蚊Aedes aegypti L.、德国小蠊Blattella germanica L.、果蝇Drosophila melanogaster Meigen和家蝇Musca domestica L.的相似性分别为95%、82%、80%、79%、77%.

  6. 日本血吸虫原肌球蛋白cDNA的克隆及其在大肠杆菌中表达%Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    曹建平; 刘述先; 宋光承; 徐馀信

    2002-01-01

    Objective To perform cloning of the gene encod ing Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli.Methods SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. Japonicum . The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition. Results The RT-PCR product, cloned into a Tvector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1 % identical in deduced amino acid sequence to that of S. Mansoni tropomyosi n. The target DNA fragment was then subcloned into a prokaryotic vector pBV220 . Induced expression in E. Coli DH5α cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot rev ealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antise rum specific for native S. Japonicum tropomyosin (SjcTM). Conclusion The engineering of the cDNA encodingS. Japonicum tropomyosin and its bacterial expression was successfully made.%目的克隆日本血吸虫原肌球蛋白编码基因,并在大肠杆菌中表达.方法抽提日本血吸虫(大陆株)成虫总RNA,经逆转录-聚合酶链反应(RT-PCR)获得编码日本血吸虫原肌球蛋白的cDNA片段,该片段与全序列比较,缺氨基端14个氨基酸.该PCR产物克隆入T 载体并对插入片段进行序列测定后,亚克隆入表达载体pbV220,经琼脂糖凝胶电泳、限制性酶切反应和PCR鉴定后,选择克隆用于温控表达.结果 RT-PCR产物

  7. cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3.

    Science.gov (United States)

    Knight, L P; Primiano, T; Groopman, J D; Kensler, T W; Sutter, T R

    1999-07-01

    The aflatoxin B1 (AFB1) aldehyde metabolite of AFB1 may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction. Aflatoxin B1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reductases of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB1 dihydrodiol by forming AFB1 dialcohol. Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult human liver cDNA library. The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively. Recombinant rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia coli as hexa-histidine tagged fusion proteins. These proteins catalyzed the reduction of several model carbonyl-containing substrates. The NADPH-dependent formation of AFB1 dialcohol by recombinant human AKR7A3 was confirmed by liquid chromatography coupled to electrospray ionization mass spectrometry. Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein were shown to detect nanogram amounts of rat and human AKR7A protein. The amount of AKR7A-related protein in hepatic cytosols of 1, 2-dithiole-3-thione-treated rats was 18-fold greater than in cytosols from untreated animals. These antibodies detected AKR7A-related protein in normal human liver samples ranging from 0.3 to 0.8 microg/mg cytosolic protein. Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver and in several extrahepatic tissues, with relatively high levels in the stomach, pancreas, kidney and liver. Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB1 dihydrodiol at pH 7.4, the catalytic efficiency of this reaction (k2/K, per M/s) equals or exceeds those reported for other enzymes, for example cytochrome P450s and glutathione S-transferases, known to metabolize AFB1 in vivo. These findings indicate that, depending on the extent of AFB1 dihydrodiol formation, AKR

  8. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal, Quebec (Canada)]|[Kingston General Hospital, Ontario (Canada)] [and others

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  9. Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species.

    Science.gov (United States)

    Shimizu, Milton Massao; Melo, Geraldo Aclécio; Brombini Dos Santos, Adriana; Bottcher, Alexandra; Cesarino, Igor; Araújo, Pedro; Magalhães Silva Moura, Jullyana Cristina; Mazzafera, Paulo

    2011-09-01

    Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid. PMID:21530289

  10. Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA.

    Science.gov (United States)

    Wongchawalit, Jintanart; Yamamoto, Takeshi; Nakai, Hiroyuki; Kim, Young-Min; Sato, Natsuko; Nishimoto, Mamoru; Okuyama, Masayuki; Mori, Haruhide; Saji, Osamu; Chanchao, Chanpen; Wongsiri, Siriwat; Surarit, Rudee; Svasti, Jisnuson; Chiba, Seiya; Kimura, Atsuo

    2006-12-01

    alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I. PMID:17151473

  11. Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS

    Energy Technology Data Exchange (ETDEWEB)

    Onodera, Osamu; Oyake, Mutsuo; Takano, Hiroki [Niigata Univ. (Japan)] [and others

    1995-11-01

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by genetic anticipation and variable combinations of symptoms including myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a gene on the short arm of chromosome 12. We determined the consensus DRPLA cDNA sequence containing the complete coding region for 1,185 amino acids. The CAG repeat, which is expanded in DRPLA, is located 1,462 bp downstream from the putative methionine initiation codon and encodes a poly-glutamine tract. Although poly-serine and proline tracts exist near the CAG repeats, these poly-serine or proline tracts did not show any polymorphisms, which is in strong contrast to the high heterogeneity in the length of the CAG repeat. Northern blot analysis revealed a 4.7-kb transcript that is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. Reverse transcription-PCR analysis revealed that the expanded alleles are transcribed to levels comparable to those of normal alleles. These results indicate that there is no difference in transcriptional efficiency between expanded and normal alleles. Furthermore, mRNA from cerebellar hemispheres of DRPLA patients showed smaller sizes of CAG repeats compared with other regions of the brain, which reflects somatic mosaicism of the expanded alleles of the DRPLA gene. 49 refs., 6 figs.

  12. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

    Science.gov (United States)

    Weis, J J; Fearon, D T; Klickstein, L B; Wong, W W; Richards, S A; de Bruyn Kops, A; Smith, J A; Weis, J H

    1986-08-01

    Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2. PMID:3016712

  13. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin;

    2015-01-01

    the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral......RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high...... a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA...

  14. Hibernation-associated gene regulation of plasma proteins with a collagen-like domain in mammalian hibernators.

    OpenAIRE

    Takamatsu, N; Ohba, K; Kondo, J; Kondo, N; Shiba, T

    1993-01-01

    In mammals, hibernation is expressed by only a limited number of species, and the molecular mechanisms underlying hibernation are not well understood. Recently, we have found plasma proteins which disappear from blood specifically during hibernation in a mammalian hibernator, the chipmunk. Here, we report the cDNA cloning of these chipmunk hibernation-related proteins, HP-20, -25, and -27, and analyses of their expression. All three proteins contain a collagen-like domain near the N terminus ...

  15. Cloning and characterization of a cDNA encoding thioredoxin peroxidase from Trichomonas vaginalis%阴道毛滴虫硫氧还原蛋白过氧化物酶的cDNA克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    章家新; 傅玉才; 刘红; 郑晓红

    2004-01-01

    目的:获取阴道毛滴虫硫氧还原蛋白过氧化物酶的cDNA克隆,研究其还原烷基氢过氧化物和氢过氧化物的抗氧化作用,以及调节由氢过氧化物介导的信号转换. 方法:提取阴道毛滴虫的总RNA,用λTripIEx2噬菌体载体构建cDNA表达文库,阳性质粒cDNA克隆进行测序,并用生物软件RPS-Blast,NCBI Blast和ClustalW等对6个读码框架进行序列分析. 结果:获得一株开放阅读框为588对碱基的cDNA克隆,推测肽链有196个氨基酸.序列分析表明,该肽链与衣滴虫过氧化物氧化还原酶(Prx1蛋白)及人的自然杀伤增强因子B分别有56%和61%的同源性;除了两个高度保守的半胱氨酸作用基序外,还有蛋白激酶C磷酸化和酪氨酸激酶磷酸化作用基序,N-豆蔻酰化作用位点,脂质运载蛋白信号位点等. 结论:该蛋白有>56%的可能性位于胞浆,和典型2-Cys Prx亚家族有很高(56%~62%)的同源性,很可能是其中的一个新成员.%Objective:To clone and characterize the thioredoxin peroxidase gene of Trichomonas vaginalis for studying its functions involving not only in the reduction of alkyl hydroperoxide and hydrogen peroxide as an antioxidant, but also in regulating hydrogen peroxide-mediated signal transduction. Methods: T. vaginalis mRNA was isolated to construct a cDNA library using λTripIEx2 phage vector. Sequencing of the positive plasmid cDNAs was performed and the resulted sequences were translated in all six reading frames and compared with the nonredundant databases using the NCBI BlastX, RPS-Blast and ClustalW programs. Results: A cDNA clone was isolated and the sequence analysis revealed that the cDNA clone had an open reading frame with 588 bp. The deduced amino acid sequence contained 196 residues and shared 56% identity with a recently identified Chlamydomonas peroxiredoxin 1 (Prx1) and 61% identity with Human natural killing enhance factor B (NKEFB). The conserved sequence elements including the motif of

  16. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

    OpenAIRE

    Battey, J F; Way, J M; Corjay, M H; Shapira, H; Kusano, K; Harkins, R.; Wu, J M; Slattery, T; Mann, E.; Feldman, R I

    1991-01-01

    The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor i...

  17. A comparative study of 2',3'-cyclic-nucleotide 3'-phosphodiesterase in vertebrates: cDNA cloning and amino acid sequences for chicken and bullfrog enzymes.

    Science.gov (United States)

    Kasama-Yoshida, H; Tohyama, Y; Kurihara, T; Sakuma, M; Kojima, H; Tamai, Y

    1997-10-01

    In mammalian brain, two 2',3'-cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) isoforms, CNP1 and CNP2, are translated, respectively, from the two mRNAs, which have been transcribed and processed by alternative use of the two transcription start points and by differential splicing. In the present study, the cDNAs encoding chicken CNP2 and bullfrog CNP1, respectively, were isolated, and the amino acid sequences of chicken CNP2 and bullfrog CNP1 were deduced. Western blot analysis showed that chicken brain contains a major CNP2-type protein together with a minor unidentified isoform, and bullfrog brain contains only a CNP1-type protein. All available amino acid sequences of vertebrate 2',3'-cyclic-nucleotide 3'-phosphodiesterases were aligned and compared. Three conserved motif sequences were noted: (a) an ATP-binding site near the amino terminus, (b) an isoprenylation site at the carboxyl terminus, and (c) a probable catalytic site resembling the active site of beta-ketoacyl synthase (EC 2.3.1.41). The second and the third motifs are conserved also in goldfish RICH (regeneration-induced 2',3'-cyclic-nucleotide 3'-phosphodiesterase homologue), which has been shown recently to have 2',3'-cyclic-nucleotide 3'-phosphodiesterase activity. The third motif (probably catalytic site) was assigned for the first time in the present report. PMID:9326261

  18. 番茄GDP-D-甘露糖焦磷酸化酶cDNA的克隆、表达及定位%Cloning, Expression, and Mapping of GDP-D-mannose Pyrophosphorylase cDNA from Tomato (Lycopersicon esculentum)

    Institute of Scientific and Technical Information of China (English)

    邹礼平; 李汉霞; 欧阳波; 张俊红; 叶志彪

    2006-01-01

    GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled.The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1 086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato,tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.%GDP-D-甘露糖焦磷酸化酶催化GDP-D-甘露糖的合成,是植物抗坏血酸生物合成途径中上游的关键酶.以马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列为信息探针,在GenBank dbEST数据库中找到65条高度同源的番茄EST序列,通过序列拼接及RACE-PCR得到了番茄该基因的全长cDNA序列,命名为LeGMP.LeGMP与马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列一致率为96%,推导的氨基酸序列与马铃薯、烟草、紫苜蓿、拟南芥的GDP-D-甘露糖焦磷酸化酶基因的一致率分别为99%、97%、91%、89%.经Northern杂交分析,LeGMP在番茄根、茎、叶、花、果实中都有表达,但表达水平有差异.利用75个番茄远缘杂交重组系(IL系)将LeGMP定位在番茄第3

  19. 岩栖蝮蛇类凝血酶纯化、cDNA克隆和序列分析%Purification, cDNA cloning and sequence analysis of thrombin-like enzyme from Gloydius saxatilis

    Institute of Scientific and Technical Information of China (English)

    孙德军; 杨春伟; 杨同书; 颜炜群; 王伟

    2003-01-01

    Thrombin-like enzyme has great medical application in treating thrombus. A thrombin-like enzyme from Gloydius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on heparin-sepharose.SDS-polyacrylamide electrophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was 32000 dalton.Total RNAs were extracted from the venom gland of the G.saxatilis snake.Using degenerate primers,we amplified the cDNA of the thrombin-like enzyme gene in the venom gland of G.saxatilis using the reverse transcription-polymerase chainreaction (RT-PCR) method.The cDNA fragment was inserted into pGEMT vector,cloned and its nucleotide sequence was determined.Its open reading frame is composed of 774 nucleotides and codes a protein prezymogen of 258 amino acids,including a putative secretory signal peptide of 18 amino acids and a proposed pro-peptide of 6 amino acid residues.It contains 12 cysteine residues.The sequence analysis indicates that the deduced amino acid sequence of the cDNA fragment shares high identity with the thrombin-like enzyme genes of other snakes in the gene bank.The query sequence exhibits strong amino acid sequence homology of 88%,88% and 86% to the serine proteas of T.gramineus,thrombin-like defibrase Ⅰ of D.acutus and serine protease catroxase Ⅱ of C.atrox respectively.Based on the amino acid sequences of other thrombin-like enzymes,the catalytic residues and disulfide bridges of this thrombin-like enzyme are deduced as follows:catalytic residues,His65,Asp110,Ser%204;and six disulfide bridges Cys31-Cys163,Cys50-Cys66,Cys98-Cys256,Cys142-Cys210,Cys174-Cys189 and Cys200-Cys225.According to the possible linked glycosylation sites N-X-T (Asn-X-Thr) or N-X-S (Asn-X-Ser),its possible glycosylation sites are N44-S45-T46 and N251-T252-T253 residues [Acta Zoologica Sinica 49(6):878-882,2003].

  20. Cloning and SNPs analysis of CYP51 gene cDNA in sea urchin Strongylocentrotus intermedius%虾夷马粪海胆CYP51基因cDNA的克隆及其SNPs分析

    Institute of Scientific and Technical Information of China (English)

    仇雪梅; 吴领知; 郭晓黎; 张伟杰; 丛玉婷; 常亚青; 宋坚; 刘洋; 王秀利

    2012-01-01

    In this study,CYP51 gene was cloned and its single nucleotide polymorphisms (SNPs) was detected by reverse transcription polymerase chain reaction(RT-PCR) and polymerase chain reaction single-strand conformation polymorphism(PCR-SSCP) in sea urchin Strongylocentrotus intermedius. The results showed that the 1 536 bp sequence including a 1 491 bp open reading frame ( ORF) and encoding 496 amino acids was obtained from CYP51 gene. A SNP A→G was found in the 161st nucleotide of open reading frame in CYP51 cDNA. The analysis of the SNPs showed that there was a significant association (P<0. 05) between the SNP and growth traits such as gonad weight and body weight. The CYP51 cDNA cloning and its SNPs analysis was the first time conducted in sea urchin Strongylocentrotus intermedius. The findings provide basic theory of molecular marker-assisted breeding of the sea urchin.%采用RT-PCR、PCR-SSCP等分子生物学技术和关联分析等方法对虾夷马粪海胆Strongylocentrotus intermedius CYP51基因的cDNA序列进行了克隆和单核苷酸多态性分析.结果表明:虾夷马粪海胆的CYP51基因包括一个1491 bp的开放阅读框,编码496个氨基酸;其开放阅读框的第161个核苷酸处存在1个单核苷酸多态性,即核苷酸发生了A→G突变.关联分析结果表明,该单核苷酸多态性与虾夷马粪海胆的体质量、性腺质量之间存在显著相关性(P<0.05).本研究中首次克隆了虾夷马粪海胆CYP51基因的cDNA序列并进行了SNPs分析,研究结果可为虾夷马粪海胆的分子标记辅助育种提供参考.

  1. Molecular cloning of the sheep ARHGAP36 full length cDNA and the protein distribution in seminiferous tubules%绵羊 ARHGAP36全长 cDNA 的克隆及其蛋白在睾丸精细管中的分布检测

    Institute of Scientific and Technical Information of China (English)

    白雪; 王连庆; 邢万金

    2015-01-01

    ARHGAP36, a member of Rho GAP(GTPase-activating proteins) family, is over expressed in medulloblastoma , and thus may be implicated in regulation of cell proliferation .To elucidate its function in sheep spermatogenesis , the full length cDNA of sheep ARHGAP36 was cloned using RACE ( rapid amplification of cDNA ends ) technique from sheep testicular tissue .A cDNA of 2698 bp was obtained after joining the 5′-and 3′RACE fragments with the middle region cloned and sequenced independently .An ORF ( open reading frame) of 1593 bp encoding a peptide of 530 aa with a homology of 98%with the predicted cattle ARHGAP36 (A7MB27) in the UniProt was found in this cDNA .Analysis of the all predicted amino acid sequence of ARHGAP 36 and other ARHGAP members from various animals collected in the UniProt showed that this ARHGAP 36 lacked the conserved arginine residue ( it is replaced with a threonine)in the "arginine finger", which was a hallmark motif of the Rho GAP family , suggesting the catalytic function may not be required for ARHGAP36.By aligning the cDNA to the predicted sheep Arhgap36 gene sequence (Gene ID:100913153) in GenBank, a criticalshort gap was also found in the first exon which will led to incapability of deducing a correct sheep ARHGAP 36 amino acid se-quence.Thus, the results provided a correct sheep ARHGAP 36 cDNA for the further research on the function of this unique Rho GAP family member .Detection of the total protein of sheep testis by Western blot and tissue sections of sheep testis by Immunohistochemis -try using anti-ARHGAP36 polyclonal antibody as the primary antibody indicated that ARHGAP 36 protein might have three forms of va-rious lengths in testis and exists in nearly all types of the cells during spermatogenesis in sheep seminiferous tubules .%ARHGAP36是Rho GAPs家族的一员,在成神经管细胞瘤中超表达,可能参与调节细胞增殖。为了研究ARHGAP36在羊精子发生过程中的作用,采用RACE( rapid

  2. cDNA cloning and expression analyses of phytoene synthase 1, phytoene desaturase and ζ-carotene desaturase genes from Solanum lycopersicum KKU-T34003

    Directory of Open Access Journals (Sweden)

    Krittaya Supathaweewat

    2013-10-01

    Full Text Available We report on the cloning of Psy1, Pds and Zds cDNAs encoding the enzymes responsible for lycopene biosynthesis,namely phytoene synthase 1 (PSY1, phytoene desaturase (PDS and -carotene desaturase (ZDS, respectively, from high-lycopene tomato cultivar, Solanum lycopersicum KKU-T34003. DNA sequence analyses showed that the complete openreading frames of Psy1, Pds and Zds cDNAs were 1,239, 1,752 and 1,767 base pairs in length and encoded proteins of 412,583 and 588 amino acids, respectively. Phylogenetic and the conserved domain analyses suggest that PSY1, PDS and ZDSfrom S. lycopersicum KKU-T34003 potentially have similar structures and biological functions to the corresponding proteinsfrom other plants. Gene expression studies showed that Psy1 was expressed only in the petal and the breaker fruit, whereasthe expressions of Pds and Zds were observed in the petal, the breaker fruit and the leaf. The highest expression level for allgenes was detected in the breaker-stage fruit, suggesting that carotenoid accumulation was developmentally regulated inthe chromoplast-containing tissues.

  3. Modification of the full-length cDNA clone of Newcastle disease virus Isolated from an outbreak In the goose

    Institute of Scientific and Technical Information of China (English)

    LIU Yuliang; HU Shunli; ZHANG Yanmei; WU Yantao; LIU Xiufan; R(o)emer-Oberdoerfer Angela; Veits Jutta; Lange Martina

    2006-01-01

    A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length eDNA clone of the Newcastle disease virus (NDV) strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides (nts) into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length eDNA was substituted with the new one.The sequencing results showed that the three additional Gnts were successfully inserted and the mutant nts in the full-length eDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.

  4. 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.

  5. Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp.

    Science.gov (United States)

    Yu, E M; Liu, B H; Wang, G J; Yu, D G; Xie, J; Xia, Y; Gong, W B; Wang, H H; Li, Z F; Wei, N

    2014-08-01

    Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean-fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059-bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α-helixes, eight β-sheets, 16 regions of triple helical repeats, 21 low-complexity regions, 10 function domains and two zinc-binding sites; however, no calcium-binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi-quantitative RT-PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real-time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean-fed grass carp. PMID:24127725

  6. 菊花花瓣XTH基因cDNA序列的克隆与分析%Cloning and Analysis of the cDNA Sequence of XTH Gene in Chrysanthemum × morifolium Petal

    Institute of Scientific and Technical Information of China (English)

    孙悦; 苏媚; 王蕾; 姬筱雅; 郑必平; 谈建中

    2012-01-01

    [Objective] The aim was to study the structural characteristics of xyloglucan endotransglycosylase/ hydrolase ( XTH) gene in eth-ylene-insensitive feverfew. [ Method] The total RNA was extracted from Chrysanthemum x morifolium petal using Trizol reagent, and the cD-NA fragment of XTH gene was cloned by RT-PCR and T/A cloning, and then analyzed by agarose gel eleetrophresis and sequencing. [ Result] The cloned cDNA sequence was 911 bp. It was predicted to encode a polypeptide of 293 amino acids and had seven active sites of XTH family, and then named as CmXTH (gene accession number HM752243). In addition, the BLAST analysis showed that the deduced amino acid sequence of CmXTH showed high homology with other 19 chosen plant XTHs. Among these, CmXTH had closer genetic relationship with Ger-bera hybrid cuhivar XET, Solatium lycopersicum XTH7, whereas had relatively distant relationship with Populus euparatica XET, Fragaria ananassa XET1, Actinidia deliciosa XET, etc. [Conclusion] The cloned fragment was certainly cUNA sequence of XTH gene, which was associated with the petal, growth and senescence in Chrysanthemum x morifolium. The study lays foundation for further studying the structure and biological functions of XTH genes as well as the growth of Chrysanthemum X morifolium petals.%[目的]研究非乙烯敏感型菊科植物的木葡聚糖内糖基转移酶/水解酶(xyloglucan endo-transglycosylase/hydrolase,XTH)基因的结构特征.[方法]采用Trizol方法从菊花花瓣中提取总RNA,利用RT-PCR技术和T/A载体克隆XTH基因的cDNA序列,并对目的序列进行琼脂糖凝胶电泳和测序分析.[结果]克隆的cDNA片段大小为911 bp,编码293个氨基酸残基,具有XTH蛋白家族的7个活性位点,命名该基因序列为CmXTH(基因登录号为HM752243);其推导的氨基酸序列与其他19种植物的XTH具有较高的同源性,其中与非洲菊、蕃茄的亲缘关系最近,而与猕猴桃、草莓、胡杨等的亲缘关系较远.[结论

  7. Molecular cloning of cDNA of gonadotropin-releasing hormones in the Chinese sturgeon (Acipenser sinensis) and the effect of 17β-estradiol on gene expression.

    Science.gov (United States)

    Yue, Huamei; Ye, Huan; Chen, Xihua; Cao, Hong; Li, Chuangju

    2013-12-01

    The Chinese sturgeon, Acipenser sinensis, is a rare and large-sized anadromous bony fish and understanding of its reproductive regulation is a precondition for controlled reproduction. In this study, two gonadotropin-releasing hormone (GnRH) precursor cDNAs, AsGnRH1 (mammalian type) and AsGnRH2 (chicken type 2), were sequenced in A. sinensis. The precursor cDNAs of the AsGnRH1 and AsGnRH2 are 381 and 649 base pairs (bp), encoding signal peptide plus precursors of 92 and 86 amino acids, respectively. Multiple sequence alignment suggests that AsGnRH1 and AsGnRH2 decapeptides are highly conserved among vertebrates. Besides, AsGnRH1 had closer evolutionary relationship with tetrapods, while AsGnRH2 was conservatively grouped with teleosts in the phylogenetic analysis. Tissue distribution analysis shows that AsGnRH2 is exclusively transcribed in the brain, whereas AsGnRH1 exhibits more extensive tissue distribution including brain, liver, spleen and gonad. Furthermore, Chinese sturgeons were subcutaneously implanted with 17β-estradiol (E2) and the effect of E2 on brain GnRH mRNA levels was evaluated by real-time PCR. A significant increase in AsGnRH1 and AsGnRH2 mRNA levels is detected in fish receiving E2 implantation compared to controls after one month (P<0.05). These results indicate that E2 exerts positive feedback effects on the transcription of the two GnRHs in immature Chinese sturgeon. PMID:23994573

  8. Cloning and Analysis of a cDNA Encoding psbL and psbJ Gene in Rice Chloroplast Genome%水稻叶绿体基因组中一个编码psbL 和psbJ基因cDNA的克隆与分析

    Institute of Scientific and Technical Information of China (English)

    顾克余; 罗林广; 苏昌潮; 翟虎渠

    2001-01-01

    A 505 bp cDNA was cloned from the leaves of rice (Oryza sativaL.) Shanyou 63 combination. DNA sequence analysis showed that it is a part of rice chloroplast genome. Its homology comparison with those known in GenBank found that it encodes 38 amino acid peptide deduced from psbL gene and 40 amino acid peptide deduced from psbJ gene in rice chloroplast PSⅡ. Northern hybridization showed that the cDNA was differentially displayed in hybrid F1 and its parental lines.

  9. Cloning and Differential Expression of a 1-Aminocyclopropane-1-Carboxylate Synthase cDNA from Peach%桃ACC合酶基因的分离及其差异表达

    Institute of Scientific and Technical Information of China (English)

    金勇丰; 朱立成; 张耀洲; 张上隆

    2002-01-01

    The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach (Prunus persica (L.) Batsch) fruit ripening, we cloned a full-length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483-amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA-based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNA pacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that of pacs12. Pacs mRNA expressed in both green mature and ripening fruit, while pacs12 mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.%利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3

  10. Molecular Cloning of a Lectin cDNA from Alocasia macrorrhiza and Prediction of Its Characteristics%海芋凝集素cDNA的分子克隆及其性质预测

    Institute of Scientific and Technical Information of China (English)

    朱亚然; 王捷; 黄炳球; 侯学文

    2006-01-01

    用cDNA末端快速扩增-聚合酶链式反应(RACE-PCR)方法克隆了海芋(Alocasia macrorrhiza)凝集素的全长cDNA(GenBank检索号DQ340864),并用多种生物信息学工具对其性质进行了预测.根据来源于天南星科其他植物的凝集素和类似蛋白的保守区的DNA序列,设计了几个海芋凝集素基因aml特异引物(GSP).用RNeasy试剂盒从海芋块茎中提取出总RNA,并以此为模板,用SMARTTM RACE cDNA扩增试剂盒提供的经特殊设计的通用引物以及不同的基因特异引物,分别获得海芋凝集素5'-和3'-RACE-PCR扩增片段.这些PCR产物经0.8%琼脂糖凝胶纯化后,分别与T克隆载体pMD 18-T相连,筛选获得阳性克隆并提取质粒,经双酶切和特异引物的PCR验证无误后,进行序列分析.从5'-和3'-RACE-PCR测序结果拼接出全长海芋凝集素cDNA序列,并用新设计自5'-RACE-PCR 5'末端的引物GSP7进行全长3'-RACE-PCR反应,获得全长海芋凝集素cDNA克隆并再次测序验证.这一新克隆的海芋凝集素cDNA的长度为1124核苷酸,分析表明它是一个编码270个氨基酸残基的蛋白质,其等电点为pH 5.7,相对分子量为29.7 kD.同源性分析结果表明,海芋凝集素与其他来源于天南星科的甘露糖凝集素以及相似蛋白具有高度同源性.在海芋凝集素序列中发现了2个B型凝集素功能区域和3个甘露糖的结合位点.综合上述信息,认为这一新克隆的海芋凝集素cDNA是一个编码甘露糖识别凝集素的基因序列.%The cDNA of Alocasia macrorrhiza lectin(aml, GenBank accession number: DQ340864) was cloned by RACE-PCR and its characteristics were predicted by various bioinformatics tools. GSPs (Gene Specific Primers) were designed according to the conserved regions of the genes encoded for lectins and similar proteins from the same family Araceae. Total RNAs were extracted from the tubers of A. macrorrhiza by Qiagen RNeasy mini kit. The 3'- and 5'-RACE-PCRs were performed with the

  11. cDNA Clone of Prophenoloxidase for Litopenaeus Stylirostris and Sequence Structure Analysis%细角滨对虾酚氧化酶原cDNA 克隆及序列结构分析

    Institute of Scientific and Technical Information of China (English)

    许尤厚; 胡超群

    2015-01-01

    采用 RT-PCR 原理和长片段扩增技术克隆细角滨对虾酚氧化酶原基因。结果表明,细角滨对虾血淋巴细胞内存在2个 proPO 基因。 proPO gene 1的 cDNA 序列包含有372氨基酸,前190个氨基酸为一个M 家族血蓝蛋白,是一个铜结合位点区域,191-372为一个 C 家族的血蓝蛋白,是一个免疫球蛋白样的区域。proPO gene 2的2个功能位点之间的序列有重叠,proPO gene 2 cDNA 序列的6-935bp 包含了第一个功能位点,928-1464bp 则包含了第二个功能位点。系统进化树比对分析发现2个基因之间的序列差异非常大。细角滨对虾和凡纳滨对虾的 proPO gene 2同处于一个密切相关的群,proPO gene 1则和其他几种对虾的 proPO gene 处于一个群。 proPO gene 2与 proPO gene 1在对虾免疫活动中是否存在不同的功能还有待于进一步的研究。%Prophenoloxidase (proPO) is one of the important factors on humoral immunity of shrimp, so far there are no re-ports for Litopenaeus stylirostris. Depend on techniques of RT-PCR and long fragment amplification cloning, prophenoloxidase gene of L. stylirostris was cloned. The results show that, there are two proPO genes in the lymphocytes of L. stylirostris. ProPO gene 1 cDNA sequence contains 372 amino acids, the first 190 amino acids are a family of M hemocyanin, a copper binding site region, 191-372 is one of the C family of hemocyanin, is an immunoglobulin like region. There are sequence overlap between the 2 functional sites of proPO gene 2, which means that 6-935bp contains the first functional sites, while 928-1464bp contains sec-ond functional sites. The phylogenetic tree alignment analysis showed that sequence structures of two genes is very different. Pro-PO gene 2 of L. stylirostris and L. vannamei was in a closely related group; but proPO gene 1 of L. stylirostris and L. vannamei was in another group with other several shrimp. The function of ProPO gene 2 and proPO gene 1 in shrimp immune

  12. Cloning and Expression of Glucoamylase Genes from Aspergilus niger cDNA Library in Pichia pastrois%黑曲霉糖化酶基因在毕赤酵母中的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    汤斌; 钱鹏

    2012-01-01

    从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。%An expression cDNA library was constructed from high-yielding glucoamylase strains of A.niger and the glucoamylase gene was isolated,then the expression of the gene in Pichia pastoris was studied.The cDNA sequence of glucoamylase from A.niger was obtained by RT-PCR.The cDNA fragment was cloned into the expression vector pPIC9K and the linearized recombinant vector was transformed to Pichia pastrois GS115 by electroporation.The positive clones were analyzed subsequently.The recombined Pichia pastrois were cultured in the MM medium,using 1% methanol to induce the expression of recombinant gene.The results showed that the maximum activity of glucoamylase was 15.6 U/mL after it was fermented for 72 h.Sequence analysis revealed that glucoamylase had 1908 bp,which encodes a putative polypeptide of 636 amino acids.The expressed protein was purified from the fermented supernatant using DEAE column and determined by SDS-PAGE.The result of SDS-PAGE also showed that the molecular weights of the enzyme was 80 kDa.The expression vector of the glucoamylase gene was constructed successfully,and it could express glucoamylase in Pichia pastrois.

  13. Construction of cDNA Library from Populus euphratica

    Institute of Scientific and Technical Information of China (English)

    Yu Guangjun; Wang Yiqin; Shen Xin

    2003-01-01

    In order to isolate and clone salt-tolerance involved genes of Populus euphratica, we constructed a cDNA library from salt-treated leaves of P. euphratica. In the experiment, double strand cDNA were synthesized by a beads-based method. The syntheses of the first strand and the second strand cDNA, adapter ligation and restriction reaction for releasing cDNA were all conducted on the beads. The double strand cDNA were released from magnetic beads by digestion with NotI, and cDNA fragments smaller than 500 bp and residual adapters were removed through cDNA size fractionation columns. Finally, double strand cDNA were directionally cloned intoλExcell vector. The results show that the primary titer of the cDNA library is 7.46×106 pfu per mL and the packaging efficiency reaches 1.47×107 recombinants per μg DNA. λDNA extracted from two clones of plaque were digested by EcoR I and NotI, both of the clones contained inserts larger than 900 bp. These results show that the cDNA library of salt-treated P. euphratica leaves has been successfully constructed.

  14. 肝癌相关cDNA片段的快速克隆和表达%Rapid cloning and expression of hepatocellular carcinoma associated cDNA fragments

    Institute of Scientific and Technical Information of China (English)

    赵弘智; 梁平; 李靖; 李洪艳; 黄小兵

    2004-01-01

    目的:克隆原发性肝癌相关新基因.方法:利用抑制消减杂交法(suppression subtractive hybridization,SSH)已经发现了1条新的肝癌相关基因片断表达序列标签(EST),长447 bp,经Genebank检索,90%无同源性.在其保守序列区设计了2条用于3'Race扩增的寡聚核苷酸引物(3'GSP2:5'-CGCATAGTACCAGTATCGAC AAAGG-3',3'NGSP2:5'-TCCACATTACGGACC CGACGGATT-3'),利用cDNA末端快速扩增法(RACE)进一步克隆该基因的全长cDNA序列.人原发性肝癌细胞株HepG2,体外传代培养,培养基为RPMI1640培养基.提取HepG2总RNA,方法参照SV Total RNAIsolation System 的说明进行.RACE法扩增采用Clontech公司的Smart TM Race cDNA Amplification Kit.将3'RACE-PCR扩增的目的片段以Race自带的产物纯化试剂盒进行纯化、回收,然后将其克隆到PMD18-T Vector中,提纯质粒后进行酶切鉴定,确认质粒内有插入片段,由宝生物工程(大连)有限公司协助完成测序,将克隆所得cDNA片段用NCBI提供的BLASTN与GeneBank与dbEST、 nr数据库进行同源性比较,确认代表新基因的EST并且登录GeneBank(http://www.ncbi.nlm.nih.GOV/submission).3便病理标本取自西南医院肝胆科,病理证实均为原发性肝细胞肝癌,分别提取肝癌及远端正常肝组织总RNA.将酶切回收的克隆插入片段分别进行同位素标记获得cDNA探针,利用Clontech公司的 ExpressHYBTM杂交液通过RNA印记法检测克隆片段在肝癌及正常肝组织中的表达,方法参照ExpressHYBTM Hybridization Solution user manual说明进行.同时,利用一联网的基因表达分析序列数据库(http://www.ncbi.nlm.nih.gov/SAGE(series analysis of gene expression,SAGE)对基因的表达及其表达水平进行分析,从而确定其组织分布.结果:得到5条3'EST(694 447-3,724 447-3,697 447-3,711 447-3 692 447-3;大小500-550bp),5条3'EST均为登录GenBank(登录号:CK730344,CK730345,CK730346,CK730347,CK730348).对其中2条带有poly-A尾的3'EST(694 447-3 724 447

  15. Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

    Directory of Open Access Journals (Sweden)

    Douglas Carl J

    2008-01-01

    Full Text Available Abstract Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones

  16. Cloning and Bioinformatics Analysis of Toll-like Receptor 4 cDNA Full Length of Buffalo%水牛TLR4cDNA全长的克隆及其生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    匡文华; 祁超; 王凤阳; 张晓茹; 成鹰; 杜丽; 张冬琳; 郝永昌; 雷明; 焦寒伟; 刘涛

    2011-01-01

    The study aimed to clone Toll-like receptor 4 (TLR4) gene of buffalo and analysis the sequence of this gene. Buffalo TLRA gene was cloned by RT-PCR, the PCR products were sequenced and cloned into pMD20-T vector. Nucleotidesequence of this gene and structure of its encoding protein were predicted by using bioinformatics software. Sequence analysis results showed that the TLRi gene ORF was 2526 bp in length and encoded 841 amino acids, including a signal peptide of 25 amino acids(aa) at the N terminal. Relative molecular weight of the encoding protein was 95. 98 ku, and the isoelectric point (pI) was 6. 37. Homology analysis showed that the nucleotide sequence of cloned gene shared 99. 01% identities with the published sequence of buffalo TLR4 gene (DQ857349) in GenBank. The TLR4 gene sequence had a high homology compared with that of cattle, sheep, pig, horse, human and chimpanzee, which were over 80%, the homology with mouse and dog were 72. 17% and 61. 30% respectively, and that with chicken was only 53. 94%. The protein structure prediction showed that TLR4 was a transmembrane protein composed of extracellularC 1 to 634 aa), transmembrane (635 to 657 aa) and the intracellu-lar rejoins (658 to 841 aa) , which contained twelve LRRdeucine-rich repeats) tandem repeat ectodomains and a TIR (Toll-interleukinl-resistance) cytoplsmic domain. TLR4 of buffalo had been successfully cloned in the study, it lay a foundation for further researching the function of the gene and the protein characteristics.%本研究利用RT-PCR方法分段克隆水牛TLR4 cDNA后拼接成全长,并克隆于pMD20-T载体中,同时运用生物信息学软件对其核苷酸序列及编码蛋白质的结构进行分析和预测.结果表明,克隆的TLR4 cDNA ORF全长2526 bp,共编码841个氨基酸,N端具有由25个氨基酸组成的信号肽,该蛋白预测的分子质量为95.98 ku,等电点为6.37;水牛TLR4与GenBank中登录的水牛TLR4(DQ857349)核苷酸序列同源性达99.01%,与黄

  17. Isolation and Primary Expression Analysis of a Full-Length cDNA Clone Encoding Small GTP-Binding Protein Gene CaRab8 in Pepper%辣椒小G蛋白CaRab8基因全长cDNA的分离及表达特征的初步分析

    Institute of Scientific and Technical Information of China (English)

    赖燕; 肖翔; 林菁; 虞露; 陈桂信; 官德义; 何水林

    2011-01-01

    通过对辣椒均一化cDNA文库的筛选,分离获得了一个与烟草RAB8-2基因编码的蛋白有着高度同源性的cDNA阳性克隆,命名为CaR ab8.测序结果表明,该cDNA长度为961 bp,包含有651 bp的完整的开放阅读框,推测编码一个217个氨基酸的蛋白,该蛋白具有GTP/GDP结合活性必需的保守域和包括YYRGA在内的Rab家族成员特有的5个结构域.实时定量RT-PCR结果表明,CaRab8基因的表达水平在茉莉酸甲酯(MeJA)的诱导下在短时间内呈上调表达,在相应时间点受水杨酸(SA)诱导下调表达.%One 961bp full -length cDNA clone was isolated from pepper normalized cDNA library, which containing a complete 651 bp open reading frame and encoding a putative protein composed of 217 amino acids. Amino acid sequence deduced by this cDNA clone showed high similarity to NtRAB8-2 protein from tobacco. The full-length cDNA was named CaRab8. The protein encoded by this cDNA clone included conserved domains for guanine nucleotide binding and GTPase activities and five domains specific to Rab family including YYRGA domain. Real time quantitative PCR analysis showed the expression level of CaRab8 was induced by MeJA while was repressed by SA.

  18. cDNA Cloning and Expression Analysis of OsWOX4 in Rice%水稻基因OsWOX4cDNA克隆及表达模式分析

    Institute of Scientific and Technical Information of China (English)

    田夏; 张大兵

    2011-01-01

    WOX (WUSCHEL-related homeobox)是一类含有高度保守同源异型结构域的植物特有转录因子,部分WOX基因家族成员对于植物分生组织的维持发挥着重要的作用.分生组织发育和分化对于植物生长发育十分重要,然而目前人们对于水稻分生组织的维持的分子机制并不清楚.通过RACE(Rapid Amplification cDNA End)的方法我们成功克隆了水稻WOX基因家族成员OsWOX4全长cDNA,该基因编码区(Coding sequence,CDS)长度为711 bp碱基,编码一个236个氨基酸残基的蛋白,5’非翻译区(5'Untranslated region,5'UTR)位于起始密码子上游89碱基处,3'UTR位于终止密码子下游313碱基处.RT-PCR分析表明,该基因主要在茎顶端、花序第3时期的花序原基和成熟花器官中表达,暗示该基因可能参与花序和花分生组织的维持.%WUSCHEL-related homeobox (WOX) transcription factors belong to a large family specifically in plants. Some members of WOX family have been shown to be involved in regulation of meristem activities. Maintenance and differentiation of meristem cells are essential for plant growth and development, but the underlying mechanism of meristem activity control in plants particularly for rice remains largely unknown. In this study, we successfully cloned the full cDNA sequence of OsWOXA of rice by RT-PCR and RACE analyses. The coding sequence of OsWOXA is 711 base pairs in length and encodes a protein with 236 amino acid residues, and the length of 5' UTR (Untranslated Region) OsWOXA is 89 bases and 313 bases for its 3'UTR. Expression analysis indicated that OsWOXA is mainly expressed in rice shoot apical meristem (SAM),inflorescence meristem and flower by qualitative RT-PCR and quantitative RT-PCR,suggesting that OsWOXA may play a role in determining meristem activity in rice

  19. 日本白鲫IGF-1基因全长cDNA克隆及组织表达分析%The Cloning of IGF-1 cDNA and Tissue Distribution in Japanese Crucian Carp (Carassius cuvieri)

    Institute of Scientific and Technical Information of China (English)

    陶敏; 钟欢; 刘少军; 周毅

    2012-01-01

    In this investigation, we cloned the partial cDNA of IGF-1 in Japanese crucian carp by using a pair of degenerate primers based on the conservative sequence of obtained sequences from more species. The full length cDNA was then obtained by SMART RACE, which contained an ORF of 486 bp that encoded a protein of 161 amino acid residues containing signal peptide, B, C, A, D and E domains and six cysteine residues. Sequence alignment and phylogenetic analysis revealed that IGF-l of fishes is conservative in evolution. The study on tissue distribution indicated IGF-l of Japanese crucian carp had remarkably similar expression patterns in adult body tissues. The highest level of the expression occurred in liver. The highef level of the expression appeared in pituitary, heart, kidney and muscle, while the lower level of the expression occurred in brain, spleen, testis and ovary.%通过已获得的多个物种IGF-1基因的cDNA序列,设计合成简并引物,用日本白鲫的肝脏总RNA反转录获得的cDNA做模板,克隆获得了IGF-1中间序列.在此基础上,通过SMART RACE,获得了IGF-1全长cDNA序列.经过序列对比分析,所得到的序列是日本白鲫(Carassius cuvieri) IGF-1 cDNA全长,其中开放阅读框为486 bp,编码含161个氨基酸的蛋白质,该蛋白质含有保守的信号肽、B、C、A、D和E结构域和6个半胱氨酸残基.同时,系统进化分析表明,在进化过程中IGF-1基因在鱼类中保持着高度保守的进化特征.IGF-1在日本白鲫中广泛表达于多个组织,尤其在肝脏中表达量最高.在垂体、心脏、肾脏和肌肉中有较高的表达.而在脑、脾脏、精巢和卵巢中的表达量较低.

  20. Molecular cloning and characterization of a cDNA encoding the prophenoloxidase gene in Bombyx mandarina%野桑蚕酚氧化酶原基因cDNA的分子克隆及其特征

    Institute of Scientific and Technical Information of China (English)

    张永亮; 黄重; 李志清; 张春冬; 朱勇

    2008-01-01

    Phenoloxidase plays an important role in defense mechanisms in insect immunity.The complemental deoxyribonucleic acid(cDNA)of Bombyx mandarins prophenoloxidase(PPO)gene was cloned by means of reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE).The cDNA was 2 134 bp in length and contained an open reading frame(ORF)of 2 082 bp which eneoded 693 amino acid residues.The deduced amino acid sequence showed a high identity to the reported sequence of PPO2 from other Lepidopterous insects and shared the typical structural features of PPO from other inseets.The results of RT-PCR showed that PPO mRNA was expressed in the integument,haemocyte,head,testis,ovary,fat body and midgut of 5th instar larvae and at different developmental stages of B.mandarina.These results provide molecular basis for further studying the function of ppo gene in B.mandarina.%酚氧化酶在昆虫的免疫防御机制中起着重要作用.利用RT-PCR和RACE方法,克隆了野桑蚕酚氧化酶原基因,获得了其cDNA序列.该序列长2 134 bp,含有一个2 082 bp的完整开放阅读框,编码一个由693个氨基酸残基组成的蛋白质.推导的氨基酸序列与其他鳞翅目昆虫PP02基因相应氨基酸序列有较高的同源性,该序列具有它们的PPO基因所共有的典型特征.组织特异性表达分析表明了该基因在野桑蚕5龄幼虫的血细胞、体壁、头部、精巢、卵巢、脂肪体和中肠等组织及其不同的发育阶段均有表达.这些结果为进一步研究野桑蚕酚氧化酶原基因的功能提供了分子基础.

  1. Molecular cloning of transcripts induced by UV-radiation in rodent cells

    International Nuclear Information System (INIS)

    Several inducible DNA repair genes have been well characterized in bacteria. In eukaryotes including mammalian cells, there is increasing evidence that similar events may occur. Recently, the authors have shown that hybridization subtraction can be used to enrich for sequences induced only several fold by a particular cell treatment such as heat shock. Chinese hamster V79 cells were UV-irradiated with 17 Jm/sup -2/ and cDNA was synthesized from the polyadenylated (poly A) RNA. This ''UV'' cDNA was hybridized with a 3 fold excess of polyA RNA from unirradiated cells and the nonhybridizing cDNA was isolated. With this approach, UV-induced sequences were enriched over 20 fold. This enriched cDNA was cloned into a high copy number plasmid and a cDNA library was constructed. By RNA dot blot and northern analysis, 42 clones from this library were found to represent transcripts induced 3 to 25 fold by UV. The most common isolates were found to be metallothionein transcripts by DNA sequencing. The metallothionein transcripts were found to be induced 10 to 25 fold by UV with maximum induction at 4-8 h after 10 Jm/sup -2/. A similar approach was also used with a Chinese hamster ovary line which does not express metallothionein and multiple clones were isolated which represented transcripts induced 3-15 fold by UV. Except for the metallothionein clones, the other Chinese hamster cDNA clones have not been identified, but it is probable that the protein products of at least some of these transcripts play a role in the cellular response to UV damage

  2. Cloning, expression and bioactivity of BAFF from Petaurus breviceps.

    Science.gov (United States)

    Ren, Wen-hua; Xu, Ying; Zhang, Shuang-quan; Yang, Guang

    2010-10-15

    B-cell activating factor (BAFF), belonging to the TNF (tumor necrosis factor) family, is crucial for B-cell survival and maturation. In the present study, PbBAFF cDNA was amplified from the sugar glider Petaurus breviceps by RT-PCR and RACE (rapid amplification of cDNA ends) strategies. The open reading frame (ORF) of PbBAFF cDNA encodes a protein consisting of 287-amino acid. The deduced amino acid sequence contains a predicted transmembrane domain, a putative furin protease cleavage site, a potential N-glycosylation site and conserved cysteine residues similar to that identified in other mammalian BAFF. The soluble mature part of PbBAFF (PbsBAFF) showed 88-92% sequence identity with mammalian homologs. The predicted three-dimensional (3D) structural analysis of PbsBAFF analyzed by comparative protein modeling revealed that they are very similar to the 3D structure of human BAFF. Recombinant PbsBAFF fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, purified PbsBAFF co-stimulates the proliferation of human B-cells. These findings indicate PbBAFF, the first BAFF cloned from marsupial, plays an important role in proliferation of B-cells, and phylogenetic analyses reveal that the work is of value with respect to continued refinement of our understanding of mammalian phylogenetic relationships.

  3. 小麦NBS-LRR类抗病基因同源cDNA序列的克隆与表达分析%Cloning and expression analysis of a NBS-LRR resistance gene homology cDNA sequence from wheat

    Institute of Scientific and Technical Information of China (English)

    任晓娣; 刘彦慧; 李建螈; 张娜; 彭巧慧; 杨文香; 刘大群

    2013-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were applied in cloning disease resistance-associated gene with ESTs (Contig 914) as target sequence came from incompatible interaction cDNA library of TcLr19 to obtain wheat leaf rust resistance-associated gene. The full length cDNA of the aimed gene is 3 042 bp and containing an open reading frame of 2 739 bp. The sequence analysis showed that the gene belonged to the CC-NBS-LRR type gene, temporarily designated as TaNLR. TaNLR has continuous poly A tail and a typical poly adenylation signal. The ProtParam program puta-tively predicted that this gene encoded a protein of 912 amino acids. The phylogenetic tree analysis showed 89% identity of the protein encoded by TaNLR with that of Hordeum vulgare. Real-time PCR analysis revealed that TaNLR gene was induced and down-regulated expression during interaction between P. triticina and TcLr19. The resistance homology sequence was successfully obtained in TcLr19, which laid the foundation for understanding the function of NBS-LRR to wheat leaf rust resistance.%为获得小麦抗叶锈病相关基因,以小麦近等基因系TcLr19所构建的非亲和cDNA文库中获得的EST序列(Contig 914)为靶序列,用RT-PCR方法和cDNA末端快速扩增技术,分离克隆到片段为3 042 bp的全长cDNA序列.序列分析表明该序列符合典型单子叶植物的CC-NBS-LRR结构模式,命名为TaNLR.该基因包含一个完整的2 739 bp的开放阅读框(ORF),具有连续的Poly A尾和典型的加尾信号AATTAA.ProtParam程序预测表明该基因编码912个氨基酸.发育树分析显示该氨基酸序列与大麦的NBS-LRR类抗病基因蛋白同源性最高达89%.荧光定量PCR分析表明,在小麦与叶锈菌互作中,TaNLR基因受叶锈菌诱导下调表达.本研究在TcLr19小麦中成功获得了抗病同源基因,这为明确NBS-LRR在小麦抗叶锈病中的作用奠定了基础.

  4. cDNA cloning and expression analysis of hemocyanin gene in Exopalaemon carinicauda%脊尾白虾血蓝蛋白基因cDNA的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    于戈; 李健; 李吉涛; 李华

    2012-01-01

    In this study, a hemocyanin cDNA named EcHc was cloned from the hepato-pancreas of the ridgetail white prawn Exopalaemon carinicauda by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The full-length cDNA of EcHc was 2 158 bp and had an open reading frame(ORF) of 1 992bp encoding 663 amino acids which contained a signal peptide sequenced ~15 amino acid residues). The predicted molecular weight of the mature peptide was 75. 05kDa, and the theoretical pI was 5. 69. The homology a-nalysis of EcHc with other hemocyanin showed higher similarity with Macrobrachium nippon-ense (86%). Quantitative real-time RT-PCR analysis revealed that microbial challenge resulted in mRNA up-regulation, up to 6h post injection of Vibrio anguillarum or white spot syndrome virus (WSSV). The results indicated that the EcHc plays important roles in the immune defense system of E. carinicauda.%应用RACE技术克隆了脊尾白虾血蓝蛋白基因全长cDNA序列,并对该序列进行了分析.结果显示,该基因全长2 158 bp,开放式阅读框长1 992 bp,5’非编码区长26 bp,3’非编码区长140 bp,将该基因命名为EcHc.EcHc编码663个氨基酸,前15个氨基酸组成信号肽,推测成熟肽的分子量为75.05kDa.Blast比对结果显示,由脊尾白虾血蓝蛋白cDNA序列推导的氨基酸序列与日本沼虾、信号小龙虾血蓝蛋白氨基酸序列的同源性分别为86%、68%,由此推断该cDNA序列可能属于血蓝蛋白家族.利用Real-time PCR方法,分别研究了鳗孤菌和白斑综合征病毒(WSSV)感染脊尾白虾后,肝胰腺组织中EcHc基因在不同时间点的表达变化.实验结果表明,细菌或病毒感染后,EcHc基因在肝胰腺组织中的表达量显著增加,且均在感染后6h达到最高值,揭示脊尾白虾血蓝蛋白基因EcHc在脊尾白虾的免疫防御中具有重要作用.

  5. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

    Science.gov (United States)

    Chen, J J; Throop, M S; Gehrke, L; Kuo, I; Pal, J K; Brodsky, M; London, I M

    1991-01-01

    We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle. Images PMID:1679235

  6. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2. alpha. (eIF-2. alpha. ) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2. alpha. kinase

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.J.; Throop, M.S.; Kuo, I.; Pal, J.K.; Brodsky, M.; London, I.M. (Massachusetts Inst. of Tech., Cambridge (United States)); Gehrke, L. (Massachusetts Inst. of Tech., Cambridge (United States) Harvard Medical School, Boston, MA (United States))

    1991-09-01

    The authors have cloned the cDNA of the heme-regulated eIF-2{alpha} kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2{alpha} kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of {approx} 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2{alpha} kinase. This observation suggests that GCN2 protein kinase may be an eIF-2{alpha} kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

  7. 家蚕磷酸吡哆醛激酶cDNA的克隆和酶活表征%Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

    Institute of Scientific and Technical Information of China (English)

    石瑞君; 张剑韵; 江昌俊; 黄龙全

    2007-01-01

    Pyridoxal kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5'-phosphate (PLP), an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method (GenBank accession number: DQ452397). The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 kDa. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.%吡哆醛激酶(EC 2.7.1.35)在ATP和Zn2+的存在下,催化吡哆醛的磷酸化反应生成磷酸吡哆醛(PLP).在生物体内许多酶促反应中,PLP是一种重要的辅酶因子.家蚕和哺乳动物一样,需依赖食物中的维生素B6前体来合成PLP.文章描述了利用家蚕基因组数据库序列信息及使用PCR方法,克隆出编码家蚕吡哆醛激酶的cDNA(GenBank登录号:DQ452397).克隆到的cDNA含有一个894 bp的完整可读框,编码一条分子量为33.1 kDa,含298个氨基酸残基的蛋白质.序列比对显示此蛋白质序列与人类吡哆醛激酶蛋白序列具有48.6%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但其氨基酸残基数比哺乳

  8. Cloning and sequence analysis of cDNA encoding aquaporin (AQP) gene from Anopheles sinensis%中华按蚊水通道蛋白(AsAQP)cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    唐建霞; 张超; 白亮; 李菊林; Liu Kun; 周华云; 曹俊; 高琪

    2012-01-01

    目的 克隆中华按蚊水通道蛋白(AsAQP)基因的cDNA全长序列,分析其基因序列特征,为研究AsAQP的生物学功能提供分子基础.方法 根据已报道的昆虫水通道蛋白(AQP)氨基酸序列的保守区域,采用兼并引物从中华按蚊cDNA中获取AsAQP基因片段,在此基础上利用cDNA末端快速扩增(RACE)技术克隆该基因cDNA全长序列,并用生物信息学方法对获取的序列进行分析.结果 利用兼并引物从中华按蚊成蚊cDNA中分离到AsAQP基因片段,利用RACE技术克隆到该基因的全长cDNA.序列分析表明,该基因cDNA全长762 bp,编码253个氨基酸,蛋白分子量约为63.2 kD.生物信息学分析表明,AsAQP具有典型的6个跨膜区结构和2个天冬酰胺酸-脯氨酸-丙氨酸(NPA)结构,该结构是主要内在蛋白(MIP)家族典型的结构特征.AsAQP与致倦库蚊(Culex quinquefasciatus)AQP及埃及伊蚊(Aedes aegypti AQP蛋白的同源性分别为76%和78%.氨基酸序列聚类分析表明,AsAQP与其他蚊种的水通道蛋白遗传距离较近.结论 利用兼并引物结合RACR技术首次获得了编码AsAQP基因的cDNA全长序列,该基因属于MIP蛋白家族成员,具有典型的功能域,为进一步研究该蛋白的功能奠定了基础.%Objective To clone and analyze the full-length sequence of aquaporin gene of Anopheles sinensis (AsAQP) , so as to provide an insight into its biology functions. Methods The degenerate primers were used to amplify conserved region of AQP from An. Sinensis cDNA. After then, the full-length cDNA of AsAQP was obtained by rapid amplification of cDNA ends (RACE). Concurrently, the bioinformatics methods were applied to analyze the obtained sequence. Results The obtained full-length cD-NA of AsAQP consisted of 762 bp and 253 deduced amino acids with a predicted molecular mass of 63.2 kD. Bioinformatics analysis demonstrated that AsAQP had a typical structure with six membrane-spanning domains and an internal symmetry showing

  9. The cDNA Cloning and Analysis of Sequence Information and Quantitative Express of Chrysanthemum Rhythms Clock Output Gene CmGI (GIGANTEA)%菊花节律钟输出基因CmGI(GIGANTEA)的 cDNA 全长克隆、序列信息及定量表达分析

    Institute of Scientific and Technical Information of China (English)

    孙霞; 王秀峰; 郑成淑; 邢世岩; 束怀瑞

    2012-01-01

    chrysanthemum rhythms clock output gene GIGANTEA was cloned, and the bioinformatics of the sequence and the relative quantitative expression of mRNA were analyzed. [Method] Polymerase chain reaction (PCR) combined with 5'RACE, and 3'RACE technology were used to clone the full length cDNA of chrysanthemum rhythms clock output gene CmGI, analysts of sequence of nucleotides and code of protein was made by using the software of bioinformatics. Protein structure prediction of 3D modeling was made by using the online modeling software. The relative quantitative expression analysis of CmGl was conducted by real-time quantitative fluorescence PCR technology and 2-AACt method. [Result] The cDNA sequence of GIGANTEA was cloned from chrysanthemum 'Jniba', the full-length cDNA was 3 461 bp, open reading frame (ORF ) was 3 453 bp, and encoded 1 150 amino acids. Sequence analysis showed that the genetic code of protein was homologous with plant rhythms clock output gene GIGANTEA, named CmGl gene. The sequence was submitted to GenBank, and the registration number is JQ043439. Sequence alignment displayed that it was a similarity of 76% and 75% with GIGANTEA of Vitis vinifera, Ricinus communis, respectively. The phylogenetic tree showed that chrysanthemum CmGI and Arabidopsis thaliana GIGANTEA are closest in molecular evolution distance, followed by Brassica rapa GIGANTEA. It was speculated that CmGI protein has six transmembrane spiral across a cell membrane many times. They are transcription factors, located in the nucleus and it is a non-secretory protein. They do not have a signal peptide. CmGI 3D structure modeling projections show that the protein core structure accords with the transcription factors and the function of the common DNA combining domain HTH and HLH. Fluorescent relative quantitative analysis shows that the expression patterns of chrysanthemum CmGI are circadian rhythms expression. At different flower bud differentiation stage, the CmGI gene in the leaf blade mRNA level is

  10. Cloning and expression of xeroderma pigmentosum complementation group A cDNA and the identification of its recombinant protein%A组着色性干皮病基因的克隆、表达及重组蛋白的鉴定

    Institute of Scientific and Technical Information of China (English)

    杨勇; 卜定方; 汪科; 涂平; 朱学骏

    2003-01-01

    Objective: To clone and express xeroderma pigmentosum, complementation group A (XPA) cDNA and to identify its recombinant protein. Methods: Coding region of XPA cDNA was amplified from human tonsil cDNA by reverse transcription and nested PCR. The PCR product was cloned into pBluescript vector, confirmed by DNA sequencing, cloned in frame into a prokaryotic expression vector pTrcHis C and expressed in E. coli. TOP10. The recombinant fusion protein was identified by immunoblotting. Results: The entire coding region of XPA cDNA was cloned and expressed. The fusion XPA protein was identified by anti-XPA monoclonal antibody on western blot. Conclusion: Cloning of human XPA cDNA and successful expression of recombinant XPA protein will be useful for the construction of a viral gene transfer vector for the gene therapy of XPA.%目的:克隆、表达A组着色性干皮病基因,鉴定重组蛋白.方法:以人扁桃腺的cDNA为模板,RT-PCR及巢式PCR扩增A组着色性干皮病基因的全部编码区,克隆入pBluescript质粒,DNA测序鉴定.酶切并连接到pTrcHis C载体,在大肠杆菌TOP10中表达.免疫印迹鉴定表达产物.结果:克隆了A组着色性干皮病基因的完整编码序列,表达了预期分子量的融合蛋白,经免疫印迹法检测具有免疫原性.结论:成功克隆了A组着色性干皮病基因,表达了其编码蛋白.为构建用于基因治疗的重组病毒奠定了基础.

  11. Cloning, Structure Prediction and Function Analysis of Complete cD-NA of Tupaia belangeri ISG15%树鼩(Tupaia belangeri)ISG15分子全长克隆及分子生物学功能分析

    Institute of Scientific and Technical Information of China (English)

    夏幼辰; 吴珺; 高珊; 赵西平; 杨东亮

    2014-01-01

    目的:对丙型病毒性肝炎( hepatitis C virus , HCV )小动物模型树鼩的干扰素刺激基因15( in-terferon stimulated gene 15, ISG15)分子的全长cDNA序列进行克隆及分子生物学功能分析,为树鼩模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物,使用Smarter Race方法扩增树鼩ISG15全长序列。在序列两端设计特异性引物,引入酶切位点,进行全长片段扩增。全长 PCR 产物纯化回收后连接至 pMD18-T 载体,构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化,同时使用 SWISS MODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树鼩ISG15全长序列共687 bp,编码157个氨基酸。树鼩ISG15与其他哺乳动物ISG15高度同源,与人的核苷酸及氨基酸序列同源性分别高达72.99%及71.34%,核苷酸及氨基酸序列进化树分析均显示树鼩种系最为接近灵长类动物。结构域分析提示:树鼩ISG15主要由两个类泛素样结构域构成。软件预测所得树鼩ISG15三维结构与人ISG15三维结构高度相似,且具有类泛素样三维结构,动力学检测提示本试验预测的树鼩ISG15三维结构稳定,可信度高。结论对树鼩ISG15的克隆进一步完善了对树鼩模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。%Objective We aim to characterize the complete cDNA sequence and the function of Tupaia belangeri ISG15, to offer molecular biology information for the study of HCV in Tupaia be-langeri.Material and Methods Primers were designed according to the consensus sequence of mammalian ISG15, and the complete cDNA sequence was cloned by Smarter RACE meth-od.Specific primers including restricted enzyme site were designed for

  12. Evolution of the major histocompatibility complex: Molecular cloning of major histocompatibility complex class I from the amphibian Xenopus

    International Nuclear Information System (INIS)

    Class I major histocopatibility complex (MHC) cDNA clones have been isolated from an expression library derived from mRNA of an MHC homozygous Xenopus laevis. The nucleotide and predicted amino acid sequences show definite similarity to MHC class I molecules of higher vertebrates. The immunoglobulin-likeα-3 domain is more similar to the immunoglobulin-like domains of mammalian class II β chains than to those of mammalian class I molecules, and a tree based on nucleotide sequences of representative MHC genes is presented

  13. Homologue of mammalian apolipoprotein A-Ⅱ in non-mammalian vertebrates

    Institute of Scientific and Technical Information of China (English)

    Malay Choudhury; Shoji Yamada; Masaharu Komatsu; Hideki Kishimura; Seiichi Ando

    2009-01-01

    Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma highdensity lipoproteins(HDLs),it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-Ⅱ.We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa.Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database.Fish apo14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-Ⅱ.Nevertheless,phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-Ⅱ.In addition,in silico cloning of the TGI,Ensembl,or NCBI database revealed apoA-Ⅱs in dog,chicken,green anole lizard,and African clawed frog whose sequences had not so far been available,suggesting both apoA-Ⅰ and apoA-Ⅱas fundamental constituents of vertebrate HDLs.

  14. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification.

    Science.gov (United States)

    Tougan, Takahiro; Okuzaki, Daisuke; Nojima, Hiroshi

    2008-09-01

    Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 x 10(5) cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K(m)) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate. PMID:18603591

  15. FULL-LENGTH CDNA CLONING AND TISSUE EXPRESSION ANALYSIS OF ARGINASE GENE FROM HYRIOPSIS CUMINGII%三角帆蚌精氨酸酶基因的cDNA克隆与组织表达分析

    Institute of Scientific and Technical Information of China (English)

    刘巧林; 许宝红; 肖调义; 刘敏; 钟蕾; 苏建明

    2011-01-01

    Arginase (Arg) is a sign enzyme among organisms of urea cycle. It is not only related to many diseases in organisms, but also used to treat tumors and cancer as an important tool enzyme. According to the Hyriopsis cumingii (H. Cumingii) expressed sequence tags (EST) obtained by constructing subtractive hybridization cDNA library of H. Cumingii liver, the gene full-length cDNA sequence of arginase from H. Cumingii was cloned by RACE-PCR technique based on the designed gene-special primers. After analyzed by the software DNA Star and the bioinformatics technology, the results showed that the length of arginase gene cDNA sequence was 1720 bp, containing a complete open reading frame (65-1072) which was 1008 bp, encoding a peptide of 335 amino acid residues (aa), flanked by a 64 bp of 5' untranslated region (UTR) and a 648 bp of 3'-UTR. The deduced molecular weight of arginase was about 36.81 kD. At the transcriptional level, quantitative real-time PCR (qPCR) was used to detect the expression of the arginase gene in different tissues. The result revealed that H. Cumingii arginase gene could be expressed in seven kinds of tissue containing liver, stomach, intestine, gill, heart, mantle, axe foot collected from H. Cumingii, especially strongly expressed in digestive organs, such as liver, stomach and intestine, but weakly in heart and mantle. So it concluded that the arginase from the H. Cumingii which belongs to lower invertebrate could possess the same characteristics and functions of arginase type I and II from the higher animals. That means the arginase from the H. Cumingii may not onlyparticipates in urea cycle, but also plays an important role in the processes of physiology and pathology. And the deduction will be verified in the next experiment.%精氨酸酶(Arginase,Arg)是生物体尿素循环当中一种标志性的酶类,它不但与生物体许多疾病相关,而且是目前用于治疗肿瘤和癌症的一种重要的工具酶.根据三角帆蚌消

  16. 一种新的小鼠Gabarapl2 cDNA的克隆和鉴定%Cloning of a novel mouse Gabarapl2 cDNA and its characterization

    Institute of Scientific and Technical Information of China (English)

    陈政; 辛玉蓉; 蒋滢; 蒋菊香

    2001-01-01

    目的:克隆一个新的小鼠GABAA受体相关蛋白相似蛋白2基因(Gabarapl2),并对其功能进行初步分析.方法:将已知的人GABARAPL2 cDNA序列为信息探针筛选GenBank小鼠EST数据库,将获得的一系列互相重叠的同源度较高的EST序列进行拼接.经过实验验证,在小鼠中分离和鉴定了新基因.自行制备小鼠RNA印迹膜,用杂交方法分析该基因在不同组织中的表达情况.结果:新基因在GenBank注册,登录号AF190644.同源比较显示,该推导蛋白与人GABARAPL2基因编码的蛋白完全相同,被基因命名委员会命名为小鼠Gabarapl2.该基因的推导蛋白含多个蛋白激酶磷酸化位点.杂交显示,该基因在脑、胸腺等组织表达较高,转录本大小约1.35 kb.结论:克隆到一个新的小鼠Gabarapl2基因.%AIM: To clone a novel mouse GABAA-receptorassociated protein like 2 (Gabarapl2) gene, and to analysis its primary function.METHODS: With the aid of computer, the human GABARAPL2 cDNA was used as information probe to search mouse EST database of GenBank for mouse homolog. A series of overlapping EST were found and assembled into an EST contig using Genetics Computer Group (GCG) ASSEMBLY program.The existence of the gene was then identified by experiment. Northern blotting was performed to hybridize [α-32P]dATP labeled probe with mRNA of 11 different mouse tissues that had been transferred to the nylon membrane. RESULTS: The novel gene was deposited in GenBank under Accession No AF190644.Its cDNA contained an intact open reading frame and a canonical polyadenylation signal AATAAA followed by polyA.The deduced protein was completely identical to that of human GABARAPL2, and was termed Gabarapl2 by Mouse Gene Nomenclature Committee.The putative protein of Gabarapl2 has a calculated molecular weight of 13 700 and an isoelectric point of 8.56. It was also predicted to contain two protein kinase C phosphorylation sites and one tyrosine kinase

  17. On Cloning,Sequence Analysis and Tissue Expression of Ceruloplasmin Gene in Rare Gudgeon%稀有鮈鲫铜蓝蛋白基因 cDNA 克隆及组织表达分析

    Institute of Scientific and Technical Information of China (English)

    景致; 彭作刚; 张耀光

    2014-01-01

    铜蓝蛋白(Ceruloplasmin ,Cp)是一种重要的铜转运蛋白,合成于肝脏并参与生物体铁的代谢,在医学上是各种炎症、感染、中毒及癌症疾病的标志性蛋白.铜蓝蛋白的研究已在多种真骨鱼类中被报道,文中第一次在稀有鮈鲫(Gobiocypris rarus)中报道此基因.采用cDNA末端快速扩增技术(rapid amplification of cDNA ends ,RACE)克隆了稀有鮈鲫铜蓝蛋白基因,使用荧光定量PCR的方法构建了该基因组织表达谱.序列分析表明稀有鮈鲫铜蓝蛋白基因包含3264 bp全长编码序列,该序列编码1087个氨基酸,其核苷酸和氨基酸序列与斑马鱼同源性最高(分别为88.1%和90.3%).理论相对分子质量和等电点分别为124429.1 D和6.41.荧光定量PCR检测表明该基因在肝脏和脾脏中相对表达量最高,在肌肉和鳃中相对表达量最低.使用氨基酸序列进行蛋白结构保守域分析,结果表明铜蓝蛋白基因在脊椎动物中是相对保守的,推测其功能也与其他物种相似.这为进一步研究稀有鮈鲫该基因的功能及其应用奠定了基础.%Ceruloplasmin (Cp) ,which is the major copper-carrying protein synthesized in the liver ,plays a role in iron metabolism .It is a marker protein for inflammation ,infection ,poisoning and cancer .The Cp gene has been reported in several teleosts and here the gene in rare gudgeon (Gobiocy p ris rarus) has been first characterized . In this study , the Cp gene has been cloned by rapid amplification of cDNA ends (RACE) .Real-time PCR has been performed to demonstrate the expression pattern in different tissues . The CDS of Cp gene is 3 264 bp long ,which encodes 1 087 amino acids .BLAST result indicates that the most similar homologue of rare gudgeon Cp is from zebrafish ,with a homology of 88 .1% (DNA ) and 90 .3% (amino acid) .The predicted relative molecular mass of the protein is 124 429.1 D with an estimated PI of 6

  18. 茶树泛素活化酶基因全长cDNA克隆及序列分析%Cloning and Sequencing of UBA1 Gene Full-length cDNA from Tea Plant

    Institute of Scientific and Technical Information of China (English)

    邓婷婷; 吴扬; 李娟; 李银花; 黄建安; 刘仲华

    2012-01-01

    The cDNA-AFLP technology was applied to analyze gene expression during periodic albinism process of Anji Baicha. Some transcript-derived fragments (TDFs) were isolated occurring in both the albinistic and re-greening stage leaves. One of them showed a high similarity to ubiquitin-activating enzyme 1 (UBA\\) gene. Based on the fragment, the full length of UBAl gene with 3 764 bp (GenBank Accession No. JN180299) cDNA was obtained via rapid amplification of cDNA ends (RACE), named Camellia Sinensis UBA1 gene. It contained an open reading frame (ORF) encoding a polypeptide of 1 094 amino acid residues with a predicable molecular mass of 121 kD. Analysis of the nucleotide sequence and deduced amino acid sequence showed 82%, 81%, 79%, 79%, 77% homology with UBAl genes from Nicotiana tabacum, Ricinus communis, Oryza saliva subsp. Japonica, Triticum aestivum, Arabidopsis thaliana, respectively. Analysis by qRT-PCR showed that the transcript of UBAl was significantly up-regulated at the albinistic stage to 2.49-fold higher than that at the re-greening stage. This is a key enzyme in the ubiquitin-proteasome mediated protein degradation system. The clone and analysis of the tea plant UBAl gene establishes a good foundation for further study on the molecular mechanism of periodic albinism in Anji Baicha.%应用cDNA-AFLP技术分离安吉白茶阶段性返白过程中的差异表达基因,获得一白期表达上调片断TDF (transcript derived fragment,TDF).BLAST比对结果显示,该片段与其他物种的泛素活化酶基因有很高的相似性.通过SMART-RACE技术分别扩增出其3’和5’末端序列,成功获得该基因全长cDNA序列(GenBank登录号JN180299).所得序列全长3 764 bp,其开放阅读框编码1 094个氨基酸,蛋白分子量约为121 kD.该基因的氨基酸序列与烟草、蓖麻、水稻、小麦、拟南芥中的UBA1基因编码的氨基酸序列分别有82%、81%、79%、79%、77%的同源性.qRT-PCR分析表明,安吉白茶UBA1

  19. Cloning, characterization and prokaryotic expression of lactate dehydrogenase C cDNA in Tamias sibiricus%花鼠乳酸脱氢酶C基因cDNA的克隆、分析及原核表达

    Institute of Scientific and Technical Information of China (English)

    李昊; 韩崇选; 张冬辉; 周智敏

    2015-01-01

    In order to investigate the effects of lactate dehydrogenase C (LDH-C) on chipmunks,Tamias sibiricus,immune infertility control,the cDNA of chipmunks LDH-C was cloned from the chipmunks testis by RT-PCR,and its sequence was analyzed.The LDH-C gene was constructed into the prokaryotic expression vector,and this vector was transformed into E.coli BL21 (DE3) and was induced by IPTG.The SDS-PAGE and western-blot were conducted to identify the expression products.The results showed that the cDNA was 999 bp and encoded for a polypeptide of 332 amino acids,which contained complete open reading frame.The amount of negatively charged residues and positively charged residues were both 36 and the predicted molecular mass was around 37 kD.The theoretical isoelectric point (pI) was 7.04,and there was no signal peptide or transmembrane region.The LDH-C protein was predicted as non-secreted and hydrophobicity protein.Alpha helix,random coil and extended strand were the main components of the secondary structure of LDH-C.A 37 kD target protein with His-Tag was obtained from prokaryotic expression induced by IPTG.%为研究花鼠乳酸脱氢酶C(lactate dehydrogenase C,LDH-C)对花鼠免疫不育控制的影响,以花鼠睾丸cDNA为模板,通过RT-PCR技术得到花鼠LDH-C基因cDNA编码区,并进行序列分析,构建花鼠LDH-C的原核表达载体,导入到大肠杆菌BL21(DE3)中诱导表达,并采用聚丙烯酰胺凝胶电泳和免疫印迹法对表达产物进行鉴定.结果显示:扩增出的cDNA片段为999 bp,编码332个氨基酸,含有完整的开放阅读框;负电荷残基与正电荷残基均为36个;预测蛋白质分子量为37kD,理论等电点为7.04,无信号肽和跨膜区,推测其是一种非分泌、疏水性蛋白.α螺旋、无规则卷曲以及延伸链是sLDH-C蛋白二级结构的主要成分.重组菌在IPTG诱导下获得了约37 kD带有His-Tag的目的蛋白.

  20. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

  1. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1 and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Directory of Open Access Journals (Sweden)

    Oscar Rodríguez-Lima

    Full Text Available TATA-box binding protein (TBP is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1. Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5 and 2-Cys peroxiredoxin (Ts2-CysPrx gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  2. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  3. EFFECTS OF ESCHERICHIACOLI STRAINS AND TEMPERATURES ON STABILITY OF JAPANESE ENCEPHALITIS VIRUS CDNA CLONES%宿主菌株及培养温度对乙型脑炎病毒cDNA克隆稳定性的影响

    Institute of Scientific and Technical Information of China (English)

    郑旭晨; 郑浩; 童武; 单同领; 郭亦非; 童光志

    2013-01-01

    本研究旨在探讨宿主菌株及培养温度对JEV cDNA克隆在宿主菌中稳定性的影响。选取猪源乙型脑炎病毒基因Ⅰ型HEN0701株和基因Ⅲ型SH0601株为研究对象,分别将两毒株基因组克隆中不稳定的质粒转化不同大肠杆菌菌株,并改变转化菌的培养温度,将不同条件下培养的质粒进行序列分析。试验结果表明,不同宿主菌株对重组质粒不稳定性的影响不显著;低温培养可以克服克隆序列的不稳定。对获得的突变质粒的分析结果显示,基因组5'端的3个起始密码子和E蛋白可能与质粒的不稳定性相关。%To illustrate the effects of host strains and growth temperatures on genetic stability of Japanese encephalitis virus (JEV) cDNA clones, plasmids pTSHC1 from JEV genotype III (GIII) strain SH0601 and pTHENC1 from GI strain HEN0701 were transformed into different E. coli strains when grown at different temperatures. Both plasmids were unstable in host bacteria grown at 37 ℃ . At lower temperature, both plasmids propagated well in different hosts. However, some mutants with deletion and insertion of heteromorphic segments provided good growth for plasmids at 37℃. The sequence analysis of mutant plasmids showed that three start codons located at 5' terminus of genome and E gene might be relevant to unstable growth of plasmids.

  4. Molecular cloning and tissue expression analysis of immunoglobulin light κ chain cDNA from cobia Rachycentron canadium Linnaeus%军曹鱼Igκ轻链cDNA克隆及组织表达分析

    Institute of Scientific and Technical Information of China (English)

    侯月娥; 冯娟; 宁章勇; 茅莉娜; 郭志勋; 许海东; 孔小明

    2011-01-01

    The technique of homologous cloning and Rapid Amplification of cDNA Ends(RACE) was used to amplify full length cDNA gene of immunoglobulin light chain(κ chain) from cobia (Rachycentron canadium Linnaeus). The full length cDNA of κ in cobia is 969 bp, containing a 3′ untranslated region (UTR) of 188 bp, a 5′ UTR of 52 bp, and an open reading frame (ORF) of 729 bp, encoding 242 amine acids. The estimated molecular weight ofIg κ is 26.255 kD, and the theoretical isoelectric point is 7.52. The deduced Igκ amino acid sequences of cobia were compared with those of other teleost species. For the constant region of Igκ, higher percentage similarity was obtained from comparisons between R. canadium and Seriola quinqueradiata and between R. canadium and Salmo salar, which was higher than 77%. For the variable region, higher percentage similarity was obtained from comparisons between R. canadium and S. quinqueradiata, which reached 87%. By the phylogenetic tree of immunoglobulin light chain constant region, Igκ amino acid sequences of cobia were clustered with S. quinqueradiata (1,2,3) and Ictalurus punctatus G chain which belong to the type of κ chain, so IgL of cobia was supposed to type of κ. Salmo salar L2 chain, Danio rerios L2 chain, Cyprinus carpio L2 chain that belong to the type of λ chain were clustered together. The expression of Igκ gene in healthy cobia was initially measured by semi-quantitative RT-PCR. It was found that the expression of the Igκ existed more obviously in liver and gill than in other tissues, but they were hardly expressed in intestine and brain. The expression of the target gene in head kidney, spleen, intestine and gill increased obviously after cobia was immunized by intraperitoneal injection with Vibrio carchariae strain JT2, while the expression in liver decreased. The result indicated that head kidney, spleen, intestine and gill are main organs for Igκ production after stimulation, and play a critical role in host

  5. 建鲤催乳素基因全长cDNA的克隆及序列分析和组织表达%Full-length cDNA cloning and tissue expression of PRL from Cyprinus carpio var. Jian

    Institute of Scientific and Technical Information of China (English)

    王杰; 李冰; 张成锋; 朱健

    2012-01-01

    To reveal the gene sequences of prolactin and its expression in osmotic regulation tissue of Cyprinus carpio var.Jian,the full-length cDNA encoding prolactin was cloned from Cyprinus carpio var.Jian using homology cloning and RACE PCR approach.The prolactin was 1 028 bp in length,including 51 bp 5'terminal UTR,633 bp encoding region and 344 bp 3'terminal UTR.Phylogenetic analysis using MEGA 4 software showed that the putative prolactin amino acid sequence and other teleosts have the high similarity of 55.02%-94.76%.The prolactin amino acid sequence of Cyprinus carpio var.Jian and that of Ctenopharyngodon idella were close to each other in similarity of 94.76%.For other fishes,the similarity was 94.26%,90.00%,70.53%,70.53%,65.70%,63.90%,63.29%,62.93%,62.38%,62.32%,55.02% with Schizothorax prenanti,Danio rerio,Salmo salar,Oncorhynchus mykiss,Anguilla japonica,Oreochromis Niloticus,Epinephelus coioides,Acanthopagrus schlegelii,Paralichthys olivaceus,Sparus aurata,Takifugu rubripes,respectively.Real-time quantitative PCR results indicated that prolactin genes were expressed in the pituitary,brain,liver,kidney,intestines,gonad,spleen and gills.The prolactin transcript was detected at a high level in pituitary,moderate in brain,gills,spleen,liver,kidney and intestines,and low in gonad.This suggests that the pituitary gland is the main expression site of prolactin in Cyprinus carpio var.Jian.The prolactin transcript was detected in the gonads,liver,and spleen suggests that there may exist many physiological functions about prolactin in fish.%为了解催乳素(prolactin,PRL)的基因序列以及在建鲤渗透调节组织中的表达情况,采用同源克隆和末端快速扩增(rapidamplication of cDNA ends,RACE)的方法分离克隆了建鲤(Cyprinuscarpio var.Jian)PRL基因全长cDNA,得到1 028bp的全长cDNA,包括633bp的开放阅读框(ORF),51bp的5′末端非编码区(UTR)以及344bp的3′末端非编码区(UTR)。对该基因序列和

  6. THE TREE SHREW APOLIPOPROTEIN C-I cDNA: SEQUENCE AND ITS EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    王克勤; 吕新跃; 吴钢; 薛红; 陈保生

    2001-01-01

    A rabbit anti-serum to tree shrew apolipoprotein C-I (apo C-l) was used to screen an expression cDNA li-braDy constructed by us from tree shrew (TS) liver tissue. Two apo C-I cDNA clones were obtained. The longerone consists of 380 nucleotides, including 21 bp and 95 bp at the 5' and 3' end of the non-translated region srespectively, and a 2 64-bp fragment in an open reading frame encoding 88 amino acids prepropeptide which con-ta-ins 26 amino acids of signal peptide and a mature protein (62 amino acids). Comparing the amino-acid se-quence deduced from this cDNA with those of the published mammalian apo C-Is reveals that it shared some struc-tural similarity with zat, mouse and dog apo C-l, but it had 5 more amino acids than that of human and baboon.The expression of apo C-I mRNA in 8 different tissues were also assayed with Northern blot. The results demonstrat-ed that liver had the highest expression, intestine had much less expression and no expression in other tissues,which is much different from human and other species. This study has laid down a good foundation for further study-ing on the function and the stucture of tree shrew apo C-I gene.``

  7. Cloning and expression of Calreticulin full length cDNA in tissues of grass carp Ctenopharyngodon idella%草鱼钙网蛋白的克隆与组织表达

    Institute of Scientific and Technical Information of China (English)

    李旭威; 王海英; 吴锐全; 谢骏; 郁二蒙; 吴垠; 余德光; 王广军; 龚望宝

    2012-01-01

    钙网蛋白是一种高度保守的分子伴侣蛋白,作为内质网主要的钙结合蛋白,广泛存在于真核生物细胞中,在病毒、寄生虫感染以及温度、氧气胁迫等情况下对细胞起保护作用.本试验中采用RACE方法首次克隆获得草鱼Ctenopharyngodon idellus钙网蛋白cDNA全序列,其全长为1389 bp,开放阅读框为1263bp,编码为421个氨基酸.将获得的草鱼钙网蛋白编码氨基酸序列与其它物种分别进行同源性比较,发现草鱼与斑马鱼Danio rerio、虹鳟Oncorhynchus mykiss的同源性较高,分别为86.26%、76.78%,而与人Homo sapiens、小家鼠Mus musculus的同源性为69.67%、69.19%.通过半定量RT-PCR检测可知,钙网蛋白在草鱼的肌肉、肠道、皮肤、肝胰脏、肾脏、脾脏、鳃和鳍中均有表达,其中在肝胰脏、鳍条中的表达量最高,除与皮肤中的表达量无显著差异外(P>0.05),均显著高于其它5个器官组织(P<0.05).%Calreticulin (CRT) as a highly conserved molecular chaperone protein extensively exists in eukaryotic cells, and CRT on the surface of the cell can protect cells in temperature, and oxygen stresses, pathogens and parasitic infections. The full length cDNA of calreticulin was cloned from grass carp Ctenopharyngodon idellus by race and per methods to ecvaluate the expression in different tissues . The gene was founf to consist of 1 389 bp with an open reading frame of 1 263 bp, encoding 421 amino acids. The amino acid sequence analysis showed that the grass carp Calreticulin had higher homology with zebra fish (86. 26% ) , and rainbow trout (76. 78% ) , and had 69.67% amino acid identity to murine and 69.19% to human CRTs. Tissue expression studies showed that CRT gene was all detected but expression was different in the tested tissues including muscle, skin, intestine, hepato-pancreas, kidney, spleen, gills and rays, but the expression in the hepatopancreas liver and fins was found maximum, significantly higher

  8. 银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备%Gibel carp germ cell marker Vasa: cDNA cloning and its antibody preparation

    Institute of Scientific and Technical Information of China (English)

    徐红艳; 彭金霞; 桂建芳; 洪云汉

    2005-01-01

    种系细胞始自胚胎发育早期, 是动物生殖及生殖工程的基础.为研究鱼类的种系细胞提供标记分子, 我们克隆并鉴定了银鲫的vasa cDNA 即Cagvasa.Cagvasa cDNA全长2 771 碱基(nt), 编码的蛋白为银鲫Vasa即CagVasa, 全长701 个氨基酸(aa).CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列, 在C端Vasa所特有的8个功能域俱全.银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%, 89%, 61%-66%和50%的同源性.卵巢切片的RNA原位杂交揭示, Cagvasa 限于种系细胞, 且表达水平呈现出低-高-低的动态变化:即两头低 (卵原细胞跟Ⅳ期成熟卵子), 中间高(Ⅱ-Ⅲ期卵子).为分析鱼类种系细胞提供手段, 我们用310-aa 的N端序列产生细菌的重组蛋白来免疫大白兔, 获得了抗Vasa的多克隆抗体αVasa.Western免疫印迹表明, αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75 kD, 仅见于银鲫的性腺和卵子.卵巢切片的组织免疫荧光共聚焦显微分析表明, 抗体αVasa只对种系细胞染色:卵原细胞着色最深, 卵母细胞和早期的卵子都浓染, 成熟卵则浅染.类似情况亦见之于精子发生早期阶段的雄性种系细胞.卵巢和精巢的体细胞则不着色.因此, Cagvasa编码的当是Vasa同源蛋白, 为银鲫种系细胞的第一个标记分子.我们的研究表明, 抗体αVasa染色灵敏度高, 特异性好, 当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段%The basis for animal reproduction and reproductive biotechnology is germ cells that are segregated from the somatic lineage early in embryonic development and produce sperm and eggs for germline transmission between generations. To provide a germ cell marker, we cloned and characterized Cagvasa, a vasa homolog from the gibel carp Carassius auratus gibelio. The Cagvasa cDNA is 2 771 nt (nucleotide) and encodes a 701-aa(amino acid) protein

  9. Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

    Science.gov (United States)

    Yang, Dazuo; Zhou, Yibing; Zhao, Huan; Zhou, Xiaoxiao; Sun, Na; Wang, Bin; Yuan, Xiutang

    2012-11-01

    We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

  10. Cloning and sequence analysis of HMG-CoA reductase full-length cDNA from tea (Camellia sinensis)%茶树 HMG-CoA 还原酶基因全长 cDNA 克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    韩兴杰; 徐玲玲; 廖亮; 李同建; 邓辉胜; 樊启水; 徐小青

    2015-01-01

    )catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)to mevalonate,which is the committed step in the synthesis of isoprenoids via the MVA pathway.To help understand the molecular and genetic mechanisms underlying terpenoid synthesis of tea,a full-length cDNA encoding HMGR was cloned from tea (Camellia sinensis (L.)O.Kuntze)by using the RACE-PCR technique (designated as CsHMGR 1).It comprised 1 979 bp,with a 1 722 bp intact open read-ing frame encoding a 573-amino-acid protein.The deduced protein showed 80% to 82% similarities to homologs from rubber tree (Hevea brasiliensis ),common camptotheca fruit (Camptotheca acuminate ),ginseng (Panax ginseng ), litchis (Litchi chinensis ),American ginseng (Panax quinquefolius ),rooted salvia (Salvia miltiorrhiza ),Momordica grosvenori (Siraltia grosvenorii ),and longan (Dimocarpus longan).The phylogenetic tree,constructed with the cat-alytic domalned of CsHMGR1 and homologs from other species,indicated that CsHMGR1 belonged to the eukaryotic class I HMGR family.CsHMGR1 consisted of two transmembrane domalns,implying that it may be localized to en-doplasmic reticulum (ER)similarly to other eukaryotic homologs.It also contalned two HMG-CoA binding sites,two NADP(H)-binding sites,four conserved catalytic active residues and a phosphorylation site,indicating that phospho-rylation/dephosphorylation is likely a crucial mode of regulation of its biochemical activity.Tissue expression analysis indicated that CsHMGR 1 was expressed comparatively in the leaf buds of C .sinensis cv.Dayelong and in both leaf buds and floral buds of the mother plants.The regulation of expression and physiological activity of CsHMGR1 are likely to impact greatly on tea quality,and CsHMGR1 may provide a basis of the quality evaluation and breeding of tea given that its function is further resolved.

  11. 茶树 HMG-CoA 还原酶基因全长 cDNA 克隆及序列分析%Cloning and sequence analysis of HMG-CoA reductase full-length cDNA from tea (Camellia sinensis)

    Institute of Scientific and Technical Information of China (English)

    韩兴杰; 徐玲玲; 廖亮; 李同建; 邓辉胜; 樊启水; 徐小青

    2015-01-01

    )catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)to mevalonate,which is the committed step in the synthesis of isoprenoids via the MVA pathway.To help understand the molecular and genetic mechanisms underlying terpenoid synthesis of tea,a full-length cDNA encoding HMGR was cloned from tea (Camellia sinensis (L.)O.Kuntze)by using the RACE-PCR technique (designated as CsHMGR 1).It comprised 1 979 bp,with a 1 722 bp intact open read-ing frame encoding a 573-amino-acid protein.The deduced protein showed 80% to 82% similarities to homologs from rubber tree (Hevea brasiliensis ),common camptotheca fruit (Camptotheca acuminate ),ginseng (Panax ginseng ), litchis (Litchi chinensis ),American ginseng (Panax quinquefolius ),rooted salvia (Salvia miltiorrhiza ),Momordica grosvenori (Siraltia grosvenorii ),and longan (Dimocarpus longan).The phylogenetic tree,constructed with the cat-alytic domalned of CsHMGR1 and homologs from other species,indicated that CsHMGR1 belonged to the eukaryotic class I HMGR family.CsHMGR1 consisted of two transmembrane domalns,implying that it may be localized to en-doplasmic reticulum (ER)similarly to other eukaryotic homologs.It also contalned two HMG-CoA binding sites,two NADP(H)-binding sites,four conserved catalytic active residues and a phosphorylation site,indicating that phospho-rylation/dephosphorylation is likely a crucial mode of regulation of its biochemical activity.Tissue expression analysis indicated that CsHMGR 1 was expressed comparatively in the leaf buds of C .sinensis cv.Dayelong and in both leaf buds and floral buds of the mother plants.The regulation of expression and physiological activity of CsHMGR1 are likely to impact greatly on tea quality,and CsHMGR1 may provide a basis of the quality evaluation and breeding of tea given that its function is further resolved.

  12. Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

    OpenAIRE

    Kaibuchi, K; Mizuno, T; Fujioka, H.; Yamamoto, T; Kishi, K; Y. Fukumoto; Hori, Y.(University of Tokyo, Tokyo, Japan); Takai, Y.

    1991-01-01

    We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleoti...

  13. 杜仲肉桂醇脱氢酶基因全长cDNA克隆及序列分析%Cloning and Sequence Analysis of the Full-length cDNA of Cinnamyl Alcohol Dehydrogenase Gene from Eucommia ulmoides Olive

    Institute of Scientific and Technical Information of China (English)

    赵丹; 李晓毓; 陈建; 赵德刚

    2012-01-01

    以杜仲(Eucommia ulmoides Olive)4、5月份新长成的杜仲幼嫩叶片为材料,在克隆一段肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)基因的基础上,以杜仲cDNA为模板,采用cDNA末端快速扩增法(Rapid amplification of cDNA Ends,RACE)克隆了5'端828 bp和3'端798 bp cDNA序列,经5'RACE产物和3'RACE产物序列拼接,获得全长为1243 bp的杜仲CAD cDNA序列,开放阅读框编码243个氨基酸,命名为EuCAD(GenBank登录号:DQ142643).与GenBank中序列比对分析发现,该cDNA序列与苹果树、桉树、红橡树中的CAD基因序列同源性均为81%,预测编码的氨基酸序列与苹果树、桉树、红橡树的同源性分别为73%、70%和70%,因此认为是杜仲肉桂醇脱氢酶基因.该基因为首次从杜仲中克隆,为探索木质素的合成调控机理奠定基础.%Cinnamyl alcohol dehydrogenase ( CAD) plays an important role in the lignin biosynthesis. Cloning and sequence analysis of this gene ( CAD) from Eucommia ulmoides Olive were carried out by Rapid Amplification of cDNA Ends ( RACE) in the current work. The sequence analysis showed that the full-length cDNA of CAD contained 1243 bp, whose open reading frame ( ORF ) predicted a protein of 243 amino acids. The cDNA blast in GenBank showed 81% homology with Malus domestica, Eucalyptus gunnii, and Quercus suber, and amino acid blast demonstrated 73% , 70% , 70% homology with that of just-mentioned species, respectively , suggesting that full-length cDNA was authentic Eucommia CAD. The Cloning of Eucommia CAD may facilitate to unravel the synthetical mechanism of lignin in plant.

  14. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  15. A Seminar on Human Cloning: Cloning in Reproductive Medicine

    OpenAIRE

    Illmensee, Karl

    2001-01-01

    This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmacytical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

  16. STUDIES ON PHENOLOXIDASE-LIKE ACTIVITY OF HEMOCYANINS AND cDNA CLONING OF RELATED FUNCTIONAL UNIT IN SEPIELLA M41NDRONI%曼氏无针乌贼(Sepiella maindroni)血蓝蛋白酚氧化酶样活性研究和相关功能单元片段的克隆

    Institute of Scientific and Technical Information of China (English)

    常抗美; 杨刚; 刘慧慧; 迟长凤; 吕振明

    2011-01-01

    采用分光光度法对曼氏无针乌贼血蓝蛋白及相关功能单元(FUs)的酚氧化酶活性进行研究,并获得该功能单元的序列信息.结果表明,曼氏无针乌贼血蓝蛋白能氧化左旋多巴和邻苯二酚而不能氧化酪氨酸单酚,说明该血蓝蛋白可能具有酚氧化酶活性,并属于儿茶酚酶类;进一步研究显示,曼氏无针乌贼血蓝蛋白酶解片段FUg的酶活特性显著区别于其它功能单元,并与天然血蓝蛋白的酶活变化相一致,经同源克隆得到FUg可能的蛋白质编码区(CDS)区包含969个核苷酸,编码323个氨基酸,序列比对结果显示,该序列与商乌贼(Sepia officinalis)和水蛸(Dctopus dofleini)血蓝蛋白有很高的同源性.%Hemocyanins (Hcs) from haemolymph of the cuttlefish, Sepiella maindroni were studied by spectrophotometric method and a method for determination of phenoloxidase-like activity of related functional units was established and its cDNA cloning was also obtained. The results showed that L-DOPA and catechol had higher affinity with PO than tyramine which suggested that it belonged to the catehcol oxdiase-type. It was found that, comparing with other fragments and functional units exhibited phenoloxidase-like activity with L-DOPA as substrate, FUg was showed to be much better not only in intrinsic but also in proteolytically induced activity for the further research. Partial cDNA of FUg with possible CDS was cloned through homologous cloning method, which consisted of 969bp with encoded 323 amino acids. There were very high identities in cDNA cloning from FUg between this sequence of Sepia officinalis and Octopus dofieini.

  17. Poliovirus type 3: molecular cloning of the genome and nucleotide sequence of the region encoding the protease and polymerase proteins.

    OpenAIRE

    1983-01-01

    Overlapping cDNA clones representing the entire genome of poliovirus type 3 have been prepared in E. coli by two separate methods. Cloning of RNA . cDNA hybrids produced a more comprehensive set of clones with generally larger cDNA inserts than cloning of double - stranded cDNA. A restriction map of the entire genome and the nucleotide sequence of 2003 bases from the 3' terminus, comprising the region encoding the protease and polymerase proteins, are presented.

  18. Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases

    OpenAIRE

    Haas, Albert; Brehm, Klaus; Kreft, Jürgen; Goebel, Werner

    2011-01-01

    A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid se...

  19. [Characterization of cDNA of T-cell receptor beta chain in rainbow trout].

    Science.gov (United States)

    Partula, S; Fellah, J S; de Guerra, A; Charlemagne, J

    1994-08-01

    Using a two-step PCR strategy, we have cloned several cDNA segments encoding the T-cell receptor beta chain in a Teleost fish, the rainbow trout (Oncorhynchus mykiss). The nine clones analyzed encode identical N-terminal-truncated V beta regions which present limited sequence similarities with several mammalian TcR V beta chains, from residue Tyr-35 to residue Ser-95. These V beta regions are followed by V beta-D beta-J beta-like regions which are different in all the sequenced clones, and by identical C beta regions. The trout C beta domain (156 amino acids) is most related to the chicken and to amphibian (axolotl) C beta domains but no cysteine residue appears in the hinge region. Like in other vertebrate C beta s, the TM region carries a positively charged lysine residue (Lys-271). The intracytoplasmic domain is virtually absent. The possibility to analyze the structure, expression and diversity of a T-cell receptor chain in a Teleost fish model will be important for our future understanding of the evolution of specific immune recognition in vertebrates. PMID:7882160

  20. Molecular cloning and characterization of a mammalian excision repair gene that partially restores UV resistance to xeroderma pigmentosum complementation group D cells

    International Nuclear Information System (INIS)

    A hamster DNA repair gene has been isolated by cosmid rescue after two rounds of transfection of an immortalized xeroderma pigmentosum (XP) complementation group D cell line with neomycin-resistance gene (neo)-tagged normal hamster DNA and selection with G418 and ultraviolet irradiation. The functional length of the sequence has been defined as 11.5 kilobase pairs by measurement of the region of overlap between two hamster DNA-containing cosmids, cloned by selection for the integrated neo gene, that are able to confer an increase in resistance to ultraviolet irradiation on two XP-D cell line but not on an XP-A line. Detailed molecular characterization of the hamster repair gene has revealed no obvious similarities to two human excision repair genes (ERCC1 and ERCC2) that correct repair-defective hamster cells but have no effect on XP cells. Hybridization analyses of normal human and XP cell genomic DNAs and mRNAs, using a cosmid-clone probe from which repeated sequences have been removed, show that homologues are present and expressed in all cases

  1. 唐鱼热休克蛋白60基因cDNA克隆及表达%Cloning and expression of heat shock protein 60 cDNA of Tanichtys albonubes

    Institute of Scientific and Technical Information of China (English)

    刘海超; 陈辉辉; 覃剑晖; 马徐发

    2011-01-01

    Heat shock protein 60 (HSP60) functions as a molecular chaperon and plays an important role in protein folding,maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. To identify a sensitive biomarker of freshwater monitoring,the full-length cDNA of Tanichthys albonubes HSP60 (designated TaHSP60) was cloned by RT-PCR and RACE techniques. It was of 2 486 bp, including 5'UTR of 102 bp and 3'UTR of 656 bp. Its open reading frame contained 1 728 nucleotides which encoded a 575 amino acid peptide. The deduced amino acid sequence of T. Albonubes HSP60 had the highest similarity with Danio rerio (96. 2%). The similarity between T. Albonubes and Carassiusau-ratus,Paralichthys olivaceus, Salmo salari and Xenopus tropicalis was 93. 2%, 89. 7%, 88. 3% and 83. 8%, respectively. Clustal X analysis confirmed the existence of the typical mitochondrial signature sequence, ATP binding region and conserved GGM repeat motif at the C-terminal in T. Albonubes HSP60. Phylogenetic analysis placed T. Albonubes and the putative D. Rerio HSP60 into one separate cluster. The results from real-time PCR showed that the T. Albonubes HSP60 was ubiquitously expressed in different tissues such as liver,muscle,gill,fin clips,eye,ovary,intestine and brain. HSP60 expression levels in liver were the highest while extremely low in gill and fin clips. Statistical analysis indicated that the transcription of HSP60 in liver was significantly higher (P<0. 05) than in any other organs. After copper exposed,mRNA expression level of TaHSP60 in liver were significantly higher than those in control group in 48 h and 96 h(P<0. 05). The data would help design nucleotide probes for detecting HSP60 gene expressions as a biomarker in environmental monitoring.%采用RT-PCR和RACE技术,成功克隆了唐鱼(Tanichth ys albonubes)热休克蛋白60基因cDNA全长序列,命名为TaHSP60(GenBank登录号:HM234132).研究表明:该基因序列全长为2 486 bp,5

  2. Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC%以绵羊MHC 区段BAC 克隆酶切片段为探针杂交筛选绵羊混合组织cDNA 文库

    Institute of Scientific and Technical Information of China (English)

    杨小亮; 白大章; 邱巍; 董慧芹; 李大全; 陈芳; 马润林; Hugh T Blair; 高剑峰

    2012-01-01

    Under the premise what we have known bacterial artificial chromosome(BAC) clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.%在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC 文库MHC 区段的6 个BAC 克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA 文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC 克隆比对以及在NCBI Blastn 数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析.实验中,经过两轮杂交共筛选出27 个cDNA 阳性克隆(序

  3. Progress in interspecies cloning of mammals

    Institute of Scientific and Technical Information of China (English)

    WEN Duancheng; BI Chunming; CHEN Dayuan

    2004-01-01

    Interspecies mammalian cloning can be achieved by application of two key techniques, i.e.the technique of interspecies nuclear transfer and the technique of interspecies pregnancy.The general principles, problems and possible solutions, as well as the recent advances of interspecies mammalian cloning have been summarized in this review.

  4. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

    OpenAIRE

    Meyers, G; Tautz, N.; Becher, P; Thiel, H J; Kümmerer, B M

    1997-01-01

    After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7...

  5. Isolation and sequence of cDNA clones coding for a member of the family of high mobility group proteins (HMG-T) in trout and analysis of HMG-T-mRNA's in trout tissues.

    OpenAIRE

    Pentecost, B T; Wright, J. M.; Dixon, G H

    1985-01-01

    A specific oligonucleotide has been used to isolate a cDNA prepared from the mRNA for a trout High Mobility Group (HMG) protein closely related to trout HMG-T and bovine HMG 1 and 2 proteins. The sequence isolated more closely resembles bovine HMG-1 than the previously sequenced HMG-T protein in regions corresponding to the N terminal half of the protein. Northern blot analysis at low stringency indicated that 2 related sequences are expressed in total trout testis mRNA. Southern blots of tot...

  6. 华山松疱锈病的重寄生真菌(深绿木霉)中几丁质酶基因cDNA片段的克隆%Cloning of cDNA Fragment of Chitinase Gene from the Mycoparasite Trichoderma atroviride on Armandii Pine Blister Rust

    Institute of Scientific and Technical Information of China (English)

    马长乐; 李靖; 陈玉惠; 刘小烛

    2008-01-01

    [Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.

  7. cDNA Cloning of Vasoactice Intestinal Peptide Receptor-1 Gene (VIPR-1) and Its Expression Characteristics in Tissues of Black Muscovy Duck ( Cairina moschata)%黑番鸭血管活性肠肽受体-1基因(VIPR-1)的cDNA克隆及其组织表达特性分析

    Institute of Scientific and Technical Information of China (English)

    郑嫩珠; 陈晓燕; 陈晖; 朱志明; 缪中纬; 辛清武; 王正朝; 卢立志

    2012-01-01

    最近的研究表明,血管活性肠肽(VIP)与家禽的就巢习性密切相关.本研究采用反转录PCR方法从黑番鸭(Cairina moschata)母鸭下丘脑组织中克隆了血管活性肠肽受体(VIPR-1)基因的cDNA序列,长度为1125bp,编码355个氨基酸(GenBank登录号:JN625215).序列比对结果表明,该序列与家禽(鸡、火鸡、鹌鹑)和哺乳动物(人、小鼠、猪、牛)的基因序列分别有93%~94%和69%~71%的同源性,而相应氨基酸序列的同源性分别为96%和70%~72%.荧光定量PCR结果发现,黑番鸭VIPR-1基因的表达量在产蛋期、就巢期和休产期差异显著(P<0.05)或极显著(P<0.01),就巢期表达量最高,休产期次之,产蛋期表达量最低,表明VIPR-1基因与繁殖阶段变化密切相关;对不同组织VIPR-1的表达量分析发现,VIPR-1在垂体、下丘脑和卵巢中均有表达,其中垂体最多,其次是下丘脑,卵巢中的表达量最低,差异极显著(P<0.01).研究结果提示,VIPR-1基因具有高度保守性,参与下丘脑-垂体-卵巢轴(特别是垂体)对黑番鸭就巢行为的调控.%Recent studies have already indicated that vasoactice intestinal peptide (VIP) is closely related to the habit of birds' nesting. In this study, we first cloned VIP receptor gene and analyzed its characteristics of expression in different tissues of muscovy duck. Reversed transcript PCR was used to clone vasoactice intestinal peptide receptor-1 (VIPR-1) gene from the hypothalamus of black muscovy ducks(Cairirna moschata), the cDNA sequence of this gene consisted of 1 125 nucleotides, which coded 335 amino acids (GenBank accession No. JN625215). The sequence of VIPR-1 was highly conserved in avian species. The muscovy duck VJPR-] shared 96% amino acid identity to that of chicken (Gallus gallus), turkey (Meleagris gallopavo), and quail (Coturnix japonica), while it shared only 70%~72% amino acid identing to that of some mammalians like human(Homo sapiens), mice

  8. 真鲷天然抗性相关巨噬蛋白全长cDNA的克隆与序列分析%Cloning and sequence analysis of Nramp cDNA from Pagrus major

    Institute of Scientific and Technical Information of China (English)

    徐美瑜; 陈松林; 沙珍霞; 季相山

    2005-01-01

    Natural resistance associated macrophage protein (Nmmp) is an innate resistance protein to intracellular parasites, which is expressed plentifully in macrophage ceils. Nramp has been studied in mouse, human, cattle, rainbow trout and channel catfish.However, tittle was known about the structure of Pagrus major Nramp. In order to get the complete sequence of Pagrus major Nramp, a pair of primer is designed according to a 200bp known sequence of Pagrus major Nramp cDNA. By the use of SMART RACE, the full Nramp of Pagrus major cDNA about 5 000 bp was obtained, including about 200 bp 5' terminal region (UTR),complete encoding region and 3' terminal region. There were 3 ployA signals, which showed many possibilities of cutting at 3' terminal region. The character of Pagrus major Nramp nucleotide sequence and deduced amino acid sequence are analyzed. 12 putative transmembrane(TM) regions, a consensus transport motif (CTM), a predicted protein kinase C phosphroylation site and three predicted N-link glycosylation sites are indicated in its deduced amino acid sequence. The ‘consense transport motif' CTM is located etween TM8 and TM9. Furthermore, a protein kinase C phosphroylation site and three N-link glycosylation sites were predicted. The lignment of amino acid sequences between Pagrus major Nramp cDNA and several animals is analyzed and the deduced amino acid equence of Pagrus major Nrarnp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5%, 69.5% identity ith rainbow trout α(AAD20721), rainbow trout β(AAD20722), channel catfish(AF400108), fathead minnow (AAF01778),common carp (CABal96), mouse 1 ( AAA39838 ), mouse 2 ( AAC42051 ), human 1 ( D50403 ), human 2 ( NP - 0(106(18 ),respectively. The alignment reveals high conservation in TM and CTM regions. Analysis result makes us get familiar with the structure nd character of fish Nramp, furthermore, offers some infonnat/on for the enhancement of immunity of fish and genetic amelioration on fish breeding.

  9. Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    胡玉欣; 汪政科; 王永红; 包方; 李凝; 彭镇华; 李家洋

    2001-01-01

    We have systematically monitored brassinosteroid (BR) responsive genes in a BR-deficient mutant det2 suspension culture of Arabidopsis by using a cDNA array approach. Among 13000 cDNA clones arrayed on filters, 53 BR responsive clones were identified and designated BRR1-BRR53. Sequence analysis of 43 clones showed that 19 clones are novel genes, 3 clones are genes involved in the control of cell division, 4 clones are genes related to plant stress responses, 4 clones are transcriptional factor or signal transduction component genes, and 3 clones are genes involved in RNA splicing or structure forming. In addition, we also found that BR regulated the transcription of genes related to many physiological processes, such as photoreaction, ion transportation and some metabolic processes. These findings present molecular evidence that BR plays an essential role in plant growth and development.

  10. Full-length Cloning of Human Melanoma Antigen-encoding Gene D1 Using Rapid Amplification of cDNA Ends%采用RACE技术获得全长人新基因MAGE-D1

    Institute of Scientific and Technical Information of China (English)

    邢桂春; 张成岗; 魏汉东; 贺福初

    2001-01-01

    cDNA 末端快速扩增(RACE)技术是快速获得新基因5'和3'端未知序列的有效手段.鉴于新报导的人肿瘤基因MAGE家族成员之一MAGE-D1的cDNA序列不完全,从而拟用RACE技术获得MAGE-D1的全长cDNA序列.结果显示,MAGE-D1的cDNA全长为2 800个碱基,编码778个氨基酸.同时对RACE技术应用时的一些关键问题进行了讨论.

  11. Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Vianey-Saban, C;

    1996-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE......) based strategy to obtain the sequence of cDNAs encoding human VLCAD from placenta and fibroblasts. Alignment of the predicted amino acid sequence of human VLCAD with those of the other human ACD enzymes revealed extensive sequence homology. Moreover, human VLCAD and human acyl-CoA oxidase showed...... extensive sequence homology corroborating the notion that these genes are evolutionarily related. Southern blot analysis of genomic DNA from hybrid cell lines was used to localize the VLCAD gene to human chromosome 17p11.2-p11.13105. Using Northern and Western blot analysis to investigate the tissue...

  12. Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation

    International Nuclear Information System (INIS)

    PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, the authors described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, they have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G1 phase several hr prior to DNA replication

  13. 杜仲HDR基因全长cDNA克隆与序列分析%Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

    Institute of Scientific and Technical Information of China (English)

    刘攀峰; 杜红岩; 乌云塔娜; 杜兰英; 孙志强

    2013-01-01

    以杜仲叶片cDNA为模板,采用反转录RCR及RACE技术分离出HDR基因的cDNA克隆,命名为EuHDR.EuHDR基因cDNA全长1 653 bp,5'端非编码区长82 bp,3'端非编码区长188 bp,编码460个氨基酸,与喜树HDR基因序列相似性最高,达82%;推导EuHDR氨基酸序列中包含转运肽序列(A1-A33)及植物HDR蛋白多个保守的功能位点(A117,A208,A262,A345);EuHDR蛋白二级结构α-螺旋占35.65%,β-折叠占19.78%,螺环结构占44.57%;EuHDR蛋白三级结构为单体形式,呈不规则的三叶草形状;系统进化分析表明EuHDR蛋白与葡萄HDR蛋白的亲缘关系最为接近.%1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) synthesizes IPP and DMAPP in the last step of the plant 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.Homologous HDR gene cDNA was isolated from the leaves of Eucommia ulmoides by the method of reverse transcription polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) technique,and named as EuHDR.With the highest gene sequence similarity to Camptotheca acuminata (82%),the full-length cDNA of EuHDR was 1 653 bp including 5'non-coding region of 82 bp and 3' non-coding region of 188 bp and encoded 460 amino acids.The transit peptide sequence (A1-A33) and multiple conserved functional sites(A117,A208,A262,A345)of plant HDR protein were found in the deduced coding sequence of EuHDR.The secondary structure of EuHDR protein was predicted with proportion of α-helix to 35.65%,β-sheet to 19.78% and loop/coil to 44.57%.The calculated protein tertiary structure of EuHDR was formed as monomer,which in space displayed asymmetrical shamrock-like shape.Phylogenetic analysis revealed that the evolutionary relationship of EuHDR protein was the closest to Vitis vinifera HDR protein.

  14. Construction and characterization of a normalized whole-life-cycle cDNA library of rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA microarrays for functional genomic studies.

  15. 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

  16. 人脑红蛋白(NGB)全长cDNA序列的克隆%Full-length cDNA Cloning of Human Neuroglobin by Using Electronic Sequence Elongation Technique and RACE Technique

    Institute of Scientific and Technical Information of China (English)

    王春丽; 张成岗

    2002-01-01

    人脑红蛋白(neuroglobin,NGB)是新发现的神经系统特异的携氧蛋白,然而其全长cDNA序列一直未见报道.采用电子序列延伸技术和cDNA序列末端快速扩增技术(rapid amplification of cDNA ends,RACE)研究发现,人NGB全长cDNA序列为1 09 bp,5′非编码区为375 bp,编码区(456 bp)可编码151个氨基酸,3′非编码区为1 078 bp,其中含27bp的poly(A)(GenBank接受号:AF422797).综合采用电子序列延伸技术与RACE技术是获得全长cDNA序列的有效方法,为后续的功能研究提供了重要基础.

  17. Isolation and characterization of a cDNA clone encoding an auxin influx carrier in carnation cuttings. Expression in different organs and cultivars and its relationship with cold storage.

    Science.gov (United States)

    Oliveros-Valenzuela, María Del Rocío; Reyes, David; Sánchez-Bravo, José; Acosta, Manuel; Nicolás, Carlos

    2008-12-01

    Polar auxin transport (PAT) is necessary for the formation of adventitious roots in the base of leafy stem cuttings, as has been demonstrated in several studies in which the application of PAT inhibitors strongly inhibited the rooting of cuttings. However, unlike in the case of lateral roots, there is almost no information on the molecular mechanism that controls PAT in the formation of adventitious roots. A novel cDNA encoding an auxin influx carrier has been isolated and characterized from carnation (Dianthus caryophyllus) cuttings. The full length of DcAUX1 was obtained and the deduced aminoacid sequence revealed a high degree of identity with the corresponding auxin carrier proteins from several species. The expression of this gene depended on the organ, the carnation cultivar and the length of time cuttings had been stored in a cold chamber. As a rule, expression was higher in stem than in leaves, in the basal than in the first internode and in mature than in young leaves irrespective of the cultivar and the duration of the storage. This pattern of expression agrees with the results of a previous study showing that auxin from mature leaves was essential for rooting, while exogenous auxin applied to mature leaves was polarly transported in the stem and accumulated in the basal internode (the rooting zone). Variations in the expression observed during storage (depending of the cultivar) might be related to the variation in PAT and rooting reported in previous studies.

  18. Cloning and Expression of Heat Shock Protein 90 (HSP90) cDNA Sequence from Shaoxing Duck (A has platyrhynchos)%绍兴鸭热休克蛋白90基因(HSP90)cDNA克隆与组织表达分析

    Institute of Scientific and Technical Information of China (English)

    王德前; 卢立志; 田勇; 李进军; 沈军达; 陶争荣; 李国勤; 徐宁迎

    2013-01-01

    The heat shock protein(HSP) is closely related with thermal-resistance of organisms. This study was conducted to investigate the expression of HSP90 mRNA in examined tissues from Shaoxing duck under the different treatments and during the recovery period after heat challenge, in hope of elucidating the molecular mechanism of HSP90 gene. HSP90 cDNA were cloned and characterized from the livers of Shaoxing duck (Anas platyrhyncho) using RT-PCR. The full-length of HSP90 consisted of an open reading frame of 2 211 bp encoding a putative protein of 736 amino acids (GenBank accession No. JQ837244). Comparison of amino acid sequence of HSP90 revealed that Shaoxing duck shared 92.6%~99% amino acid identity to that of domestic poultry and shared 80%~88% amino acid identity to that of some mammalian. Fluorescent Real-time quantitative PCR was applied to determined HSP90 expression after exposure to different thermal shock. Long-term treatment at 30℃ up-regulated HSP90 transcription in pituitary; long-term treatment at 35℃ significantly enhanced HSP90 expression in heart, liver and pituitary; acute treatment at 40 ℃ increased HSP90 level at maximum in heart, liver, kidney, pituitary and pancreas, respectively. During the recovery period, HSP90 mRNA in liver and heart reached their highest levels at 1 h post challenge and then both decreased to pre-challenge levels from 3 h post challenge. We concluded that levels of HSP90 mRNA in heart, liver and pituitary were significantly associated with the intensity of heat shock. The study provided basic data for the prevention and control of heat shock in Shaoxing duck using HSP90 mRNA expression as an index.%热休克蛋白(HSP)与生物机体的耐热性能相关.为研究不同热应激模式下与热应激恢复过程绍兴鸭热休克蛋白90基因(HSP90) mRNA在不同组织中表达差异,阐明HSP90基因的热应激保护的分子机理.本研究采用RT-PCR方法从绍兴鸭(Anas platyrhynchos

  19. Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum upsilon chain deduced from cDNA sequence.

    Science.gov (United States)

    Fellah, J S; Kerfourn, F; Wiles, M V; Schwager, J; Charlemagne, J

    1993-01-01

    An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C upsilon) chain. C upsilon is 433 amino acids long and organized into four domains (C upsilon 1-C upsilon 4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C upsilon is most closely related to Xenopus C upsilon (40% identical amino acid residues) and C upsilon 1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C upsilon 1 and C upsilon 2 domains is consistent with an additional intradomain S-S bond similar to that suggested for Xenopus C upsilon and C chi, and for the avian C upsilon and the human C epsilon. C upsilon 4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C gamma 3, human C epsilon 4, and avian and anuran C upsilon 4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C upsilon 4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian upsilon chains, and mammalian epsilon chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions. PMID:8344718

  20. 猪SOSC1、SOCS3和SOCS4基因克隆及组织表达研究%Suppressor of cytokine signaling 1, 3 and 4 genes in pigs (Sus scrofa) :cDNA cloning and tissue expression

    Institute of Scientific and Technical Information of China (English)

    段庆梓; 何夏萍; 吕学斌; 李娟; 王亚军

    2012-01-01

    Using cDNA of Landrace pig brain as the template, the full-length cDNAs of SOCS-1, SOCS-3. and SOCS-4 genes were cloned. Moreover, a pseudo-SOCS-3 gene was cloned using Landrace genom-ic DNA as template. SOCS-1 cDNA encodes a protein precursor of 220 amino acids, which shares high amino acid sequence identity with that of human (94%), rat (91%), mouse (91%), cattle (94%). SOCS-3 cDNA (690bp) encodes a protein precursor of 229 amino acids, which shares high amino acid sequence identity (> 94%) with that of mammals. The full-length cDNA of SOCS-4 encodes a protein precursor of 441 amino acids which shares high amino acid sequence identity with that of human (97%) , rat (89%) and mouse (86%). Further structural analyses indicate that pig SOCS-1, SOCS-3 and SOCS-4 contain an SH2 domain and a C-terminal SOCS box domain; SOCS-4 contains a large amino-terminus of 285 amino acids with no identifiable protein-interaction motifs. Using RT-PCR assay, the mRNA expression of SOCS-1, SOCS-3, and SOCS4 genes were also examined in adult pig tissues. The results showed that they were widely expressed in all of the tissues examined.%本文以长白猪(Landrace)大脑cDNA为模板,克隆得到长白猪细胞因子抑制因子SOCS-3基因,并首次克隆得到长白猪SOCS-1和SOCS-4基因.此外,还利用长白猪基因组DNA为模板克隆得到SOCS-3假基因(pseudo-SOCS-3).序列分析显示:长白猪SOCS-1基因cDNA编码220个氨基酸的前体蛋白,与人、小鼠、大鼠和牛的氨基酸序列一致性分别为94%、91%、91%和94%;SOCS-3基因cDNA全长690 bp,编码229个氨基酸的前体蛋白,与人、大鼠和小鼠的氨基酸序列一致性高达94%以上;SOCS-4基因cDNA全长1404 bp,整个编码区没有内含子插入,编码441个氨基酸的前体蛋白,与人、大鼠和小鼠的氨基酸序列一致性分别为97%,89%和86%.氨基酸结构分析显示,长白猪SOCS-1,SOCS-3和SOCS-4基因都具有典型的中央SH2结构域和C末端有40