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Sample records for cdna clones encoding

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

    Science.gov (United States)

    Peduel, A D; Elizur, A; Knibb, W

    1994-01-01

    The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

  6. 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.

  7. 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.

  8. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  9. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    OpenAIRE

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-bindin...

  10. 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.

  11. 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

  12. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  13. 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

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

    Science.gov (United States)

    Venugopal, T; Mathavan, S; Pandian, T J

    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. 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.

  16. 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.

  17. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    Science.gov (United States)

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  18. 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.

  19. 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.

  20. 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.

  1. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    Science.gov (United States)

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  2. Cloning of a cDNA encoding the smallest neurofilament protein from the rat

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); K. Ramachadran; F.G. Grosveld (Frank)

    1985-01-01

    textabstractWe have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat neurofil

  3. 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.

  4. 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

    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...

  5. 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.

  6. A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

    Science.gov (United States)

    Kim, Y S; Nosaka, K; Downs, D M; Kwak, J M; Park, D; Chung, I K; Nam, H G

    1998-08-01

    We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

  7. 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...

  8. ISOLATION AND CLONING OF cDNA OF GENE ENCODING FOR METALLOTHIONEIN TYPE 2 FROM MELASTOMA AFFINE

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUT

    2009-01-01

    Full Text Available Metallothionein is an important protein for detoxifying heavy metal ions. h is research was conducted to isolate and clone cDNA of gene encoding for metallothionein type 2 from Melastoma affi ne . Total RNA was isolated from young leaves. Total cDNA was synthesized from the total RNA by reverse transcription. h e MaMt2 cDNA was successfully isolated by PCR technique. h e MaMt2 cDNA was inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into Escherichia coli DH5 α . DNA sequencing analysis showed that this cDNA is full length consisting of 246 pb encoding 81 amino acid residues. h is cDNA is identical to mRNA of AtMt2 from Arabidopsis thaliana. It does not contain any restriction sites found in the cloning sites of pGEM-T Easy. h e deduced protein of MaMT2 contains 14 cysteine residues distributed in the Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys motifs

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

    DNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha......'-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...

  14. 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.

  15. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    Science.gov (United States)

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  16. 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.

  17. 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.

  18. 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.

  19. Molecular cloning and characterization of cDNA encoding a putative stress-induced heat-shock protein from Camelus dromedarius.

    Science.gov (United States)

    Elrobh, Mohamed S; Alanazi, Mohammad S; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D

    2011-01-01

    Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B') from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77-91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80-94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species.

  20. 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.

  1. Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Sun, Li; Ouyang, Chao; Kou, Shanglong; Wang, Shenghua; Yao, Yunyi; Peng, Tong; Xu, Ying; Tang, Lin; Chen, Fang

    2011-01-01

    A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).

  2. Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera.

    Science.gov (United States)

    Zhang, Tian-Yi; Sun, Jiu-Song; Zhang, Liu-Bin; Shen, Jin-Liang; Xu, Wei-Hua

    2004-01-01

    Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.

  3. Cloning of a cDNA encoding cathepsin D from salamander, Hynobius leechii, and its expression in the limb regenerates.

    Science.gov (United States)

    Ju, B G; Kim, W S

    2000-01-01

    Cathepsin D is a major lysosomal aspartic proteinase participating in the degradation or modification of intra- and extracellular matrix molecules, and its activity is known to increase in the process of tissue reorganization during the early phase of salamander limb regeneration. Here, we report the cloning of a salamander cathepsin D cDNA from Hynobius leechii and its expression profile in normal and retinoic acid (RA) treated limb regenerates. The gene expression of cathepsin D increased notably during the dedifferentiation stage and decreased gradually thereafter. Furthermore, RA that enhances dedifferentiation of regenerating salamander limb caused the elevation of cathepsin D expression both in terms of level and duration. These results suggest that cathepsin D plays important role(s) in the dedifferentiation process, and enhancement of cathepsin D expression might be closely related to RA-evoked pattern duplication.

  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. Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase receptor in tropical periwinkle Catharanthus roseus.

    Science.gov (United States)

    Papon, N; Bremer, J; Vansiri, A; Glévarec, G; Rideau, M; Creche, J

    2006-09-01

    Signalling pathways involving histidine kinase receptors (HKRs) are widely used by prokaryotes and fungi to regulate a large palette of biological processes. In plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing transduction pathways. In this work, a full length cDNA named CRCIK was isolated from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205 amino acid protein that belongs to the hybrid HKR family. The deduced amino acid sequence shows the highest homology with AtHK1, an osmosensing HKR in ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK corresponding gene is either present in multiple copies or has very close homologues in the genome of the tropical periwinkle. The gene is widely expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly induced after exposure to low temperature, pointing to a putative role in cold-shock signal transduction.

  6. Molecular cloning and characterization of a cDNA encoding a laminin-binding protein (AhLBP) from Acanthamoeba healyi.

    Science.gov (United States)

    Hong, Yeon-Chul; Lee, Won-Myung; Kong, Hyun-Hee; Jeong, Hae-Jin; Chung, Dong-Il

    2004-01-01

    Adherence of Acanthamoeba to host tissue is believed to be crucial in the establishment of amoebic keratitis or GAE. We have isolated a cDNA from a GAE-causing gymnoamoeba, Acanthamoeba healyi, encoding a protein that binds laminin by screening with a peptide G-specific DNA probe. The cDNA clone (AhLBP) was identified on the basis of sequence homology to the nonintegrin mammalian metastasis-associated 67-kDa laminin receptor (67-LR). The predicted amino acid sequence is 256 residues long with a calculated molecular mass of 28.2kDa and a theoretical pI of 5.48. Southern and Northern blot analyses suggested the gene as a single copy in A. healyi genome and expressed as a single transcript of approximately 1.0kb. Virulent strains of Acanthamoeba revealed higher level of the AhLBP mRNA expression than soil isolates. Specific binding of the purified recombinant protein to laminin was confirmed by sandwich Western blot. The polypeptide encoded by AhLBP shared substantial identity with the acidic class ribosomal proteins involved in protein synthesis. Therefore, the AhLBP may be multifunctional in A. healyi, acting as a laminin-binding molecule but also playing a role in cell division and growth. AhLBP-EGFP fusion protein expressed in A. healyi was localized mainly at the cell membrane and nucleus and at cytoplasm with lesser degree. N-terminal 64 amino acids were important for the localization at the cell membrane. This is the first description of a cDNA encoding a laminin-binding protein from protozoan parasites.

  7. 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.

  8. Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

    Science.gov (United States)

    Santos, Mônica O; Pereira, Maristela; Felipe, Maria Sueli S; Jesuino, Rosalia Santos A; Ulhoa, Cirano J; Soares, Renata de Bastos A; Soares, Celia Maria de A

    2004-06-01

    A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

  9. 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.

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-06-10

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

  12. 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.

  13. Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes.

    OpenAIRE

    1993-01-01

    We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental sta...

  14. 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.

  15. 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.

  16. 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 ...

  17. Cloning and Sequencing of cDNA Encoding Islet Cell Autoantigen 69kD Protein from Chinese%国人 ICA69 基因 cDNA 的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: It is reported that cDNA encoding human islet cell autoantigen 69kD protein (hiICA69) has been cloned, so to confirm the nucleotide sequences from the insulinoma cells of Chinese. Methods: cDNA encoding hiICA69 has been amplificated by PCR, from the cDNA library of Chinese insulinoma cells. The PCR product was inserted into the pSPORT 1 vector, and was subcloned into the pUC18 plasmid. After the positive colony was screened by the blue/white colony and the restriction analysis, the nucleotide sequences of the full - length cDNA were analysed by means of the dideoxy chain termination method. Resalts: The results showed that the amplified fragment contained 1449bp, encoded 483 - amino acids. For the sequencing analysis of ICA69 gene from the insulinoma in Mongolian race, the nucleotide sequence of the recombinant was coincident with that reported by Miyazaki and that from EMBL data's bank in addition to one difference of only base on the codon. The change located in the 416th base (A→T), which led to the change of one amino acid (Gln→Leu) . Conclusion: The gene obtained by the method of gene engineering and identified by means of sequence analysis would be able to lay a foundation for follow - up research.%目的:克隆国人胰岛细胞自身抗原 69kD 蛋白基因 ( hiICA69 ) 并经序列分析予以确证。方法:采用聚合酶链式反应技术,从中国人胰岛细胞瘤 cDNA 文库中扩增出 hiICA69 编码序列cDNA,将基因片段插入 pSPORT 1 质粒,进一步亚克隆到 pUCl8 载体中,经蓝白斑和限制性酶谱分析得以初步筛选后,双脱氧末端终止法对其全部核苷酸序列予以确定。结果:证实了 hiICA69 基因全长为 1449bp、编码 483 个氨基酸。与 pietropaolo 等报道的序列比较,仅在编码第 139 位氨基酸的密码子由 CAA→CTA,即由谷氨酰胺→亮氨酸,其余均与文献报道和 EMBL 核酸数据库提供的序列相同。结论:这一基因的获得和

  18. 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.

  19. Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

    NARCIS (Netherlands)

    Visser, H.; Weijers, C.A.G.M.; Ooyen, van A.J.J.; Verdoes, J.C.

    2002-01-01

    Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The deduce

  20. Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease.

    Science.gov (United States)

    Kelley, M R; Venugopal, S; Harless, J; Deutsch, W A

    1989-03-01

    The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development.

  1. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2): a putative family of the Golgi autoantigen.

    Science.gov (United States)

    Kondo, M; Sutou, S

    1997-01-01

    The male-enhanced antigen-2 (Mea-2) gene was originally identified with a monoclonal histocompatibility Y (H-Y) antibody (mAb4VII). There is no report of the full length cDNA encode for Mea-2 product until this report. In this study, we isolated the full length mouse Mea-2 cDNA by screening a testis cDNA library with a PCR-amplified Mea-2 product, and direct PCR amplification of its upstream sequences from the cDNA library. The primary structure of the Mea-2 peptide, deduced from this nucleotide sequence, shows that it encode a 150 kDa protein, of 1325 amino acid residues, which contained five putative N-glycosylation sites and four leucine zipper motifs. A data bank search indicated that it has high homology with a human Golgi autoantigen (golgin-160) both in its nucleotides (78%) and amino acids sequence (83%). This suggests that Mea-2 gene product may encode a golgi structural protein. In situ hybridization analysis suggested that the Mea-2 gene is expressed in spermatids during spermatogenesis as already shown by Mea-1, suggesting that Mea-2 gene product as well as Mea-1 have also some role for spermatogenesis.

  2. Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

    Science.gov (United States)

    Maciejewski, J J; Vacchiano, E J; McCutcheon, S M; Buhse, H E

    1999-01-01

    The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

  3. 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.

  4. 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.

  5. 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.

  6. Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

    Science.gov (United States)

    Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

    2014-10-01

    Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future.

  7. 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.

  8. cDNA clone of hepatitis A virus encoding a virulent virus: induction of viral hepatitis by direct nucleic acid transfection of marmosets.

    OpenAIRE

    Emerson, S U; Lewis, M; Govindarajan, S. (Srinath); M. Shapiro(Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America); Moskal, T; Purcell, R. H.

    1992-01-01

    Direct inoculation of marmoset livers with an in vitro transcription mixture containing cDNA and full-length genomic RNA transcripts of hepatitis A virus resulted in acute viral hepatitis. Elevations in serum levels of liver enzymes were correlated with appearance of antibody to hepatitis A virus. Genomes of infectious hepatitis A virus isolated from the feces of transfected marmosets contained the same mutation as the cDNA template used for transfection. Liver biopsies confirmed that the vir...

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  14. Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds. Homology of the deduced amino acid sequence with members of alpha-amylase/trypsin inhibitor family.

    Science.gov (United States)

    Izumi, H; Adachi, T; Fujii, N; Matsuda, T; Nakamura, R; Tanaka, K; Urisu, A; Kurosawa, Y

    1992-05-18

    A cDNA clone of rice major allergenic protein (RAP) was isolated from a cDNA library of maturing rice seeds. The cDNA had an open reading frame (486 nucleotides) which coded a 162 amino acid residue polypeptide comprising a 27-residue signal peptide and a 135-residue mature protein of M(r) 14,764. The deduced amino acid sequence of RAP showed a considerable similarity to barley trypsin inhibitor [1983, J. Biol. Chem. 258, 7998-8003] and wheat alpha-amylase inhibitor [1981, Phytochemistry 20, 1781-1784].

  15. Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

    Science.gov (United States)

    Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

    1995-07-01

    Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

  16. Cloning and expression of a cDNA encoding ribosomal protein S7 in Mythimna separata%粘虫核糖体蛋白S7 cDNA的克隆和表达分析

    Institute of Scientific and Technical Information of China (English)

    李柯

    2012-01-01

    运用RT-PCR和RACE技术克隆了粘虫Mythimna separata(Walker)核糖体蛋白S7基因(RPS7)的全长cDNA序列(GenBank登录号:JN582331),并对其进行生物信息学分析.结果表明,粘虫RPS7全长cDNA序列为762bp,包括5′非编码区32bp和3′非编码区67bp.其开放阅读框(573bp)编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征.该肽链理论分子量为21.924ku,等电点为9.82,富含4种类型的特定功能位点.该蛋白序列与其他动物RPS蛋白序列具有96.8%~98.2%高度同源性.应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式.RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节.%A full-length cDNA of the ribosomal protein S7 (RPS7) gene was cloned from Mythimna separata (Walker) using RT-PCR and the RACE technique. The results show that the cloned cDNA ( GenBank accession number; JN582331) contained 762 base pairs, a 5'-untranslated region (32 bp ) and a 3'-untranslated region (67 bp ). The open reading frame (573 bp) encodes a 190 amino acid peptide with a predicted molecular weight of 21.924 ku and a PI value of 9. 82. The predicted protein is conserved in the coding region with 96. 8% -98.2% similarity at the amino acid level to RPS7s from other animals in which 4 typical ribosomal functional sites of the protein S7e family signature have been found. Real-time quantitative PCR revealed that RPS7 transcript levels were coincident with the remolding of the brain. The results suggest that RPS7 was developmentally regulated at the level of transcription.

  17. Cloning and bioinformatics analysis of a fragment cDNA encoding growth hormone-releasing hormone in Tibetan sheep%草地藏系绵羊生长激素释放激素基因部分 cDNA 的克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    王金玲; 王永; 刘鲁蜀; 陶永平

    2015-01-01

    为了克隆草地藏系绵羊生长激素释放激素基因,采用Trizol法从草地藏系绵羊下丘脑组织中提取总RNA,用反转录-聚合酶链式反应( RT-PCR)进行cDNA扩增并克隆测序,获得长度为207 bp的促生长激素释放激素基因( GHRH)的部分cDNA序列。结果表明获得的草地藏系绵羊GHRH部分cDNA序列与GenBank中注册的绵羊GHRH基因编码起始位置(86位)到292位区域高度同源,仅有1个碱基的差异,该cDNA序列编码69个氨基酸残基,其内含有信号肽序列,该氨基酸序列的31~57位具有典型胰高血糖素类似激素特征的GLUCA结构域。%Total RNA was extracted from the hypothalamus tissues of Tibetan sheep using Trizol method and cDNA was amplified by reverse transcription polymerase chain reaction ( RT-PCR) . The cDNA of Tibetan sheep GHRH gene was cloned from the amplified PCR product and sequenced. The cDNA was 207 bp in length and showed 99% homology with that of com-mon sheep registered in GenBank from the starting position (86) to 292 bp. The cDNA sequence encodes 69 amino acid resi-dues and contains a signal peptide sequence which has a GLUCA domain characterizing glucagon-like hormone at amino acid 31 to 57.

  18. 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...

  19. 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.

  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.

  1. 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.

  2. 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.

  3. Cloning, Southern blotting and RT-PCR analysis of a cDNA fragment encoding of 70 kDa heat shock cognate protein(Hsc70) from the oyster(Crassostrea ariakensis)%近江牡蛎Hsc70蛋白基因cDNA片段的克隆及Southern杂交和RT-PCR分析

    Institute of Scientific and Technical Information of China (English)

    张其中; 吴信忠; 高劲松; 潘金培

    2003-01-01

    In order to elucidate the molecular mechanisms of the oyster ( Crassostrea ariakensis) against adverse stimulating factors, we cloned and sequenced a partial cDNA encoding a 70 kDa heat shock cognate protein (Hsc70) from the oyster. The live oysters were obtained from Chengcun, Yangxi County, Guangdong Province, China. Various tissues, including mantle, gills, adductor muscle, heart and blood cells, were respectively collected from 5 untreated live oysters or treated ones at 36℃ for 1.5 hours, and immediately frozen in liquid nitrogen except for the blood cells which were suspended with Trizol Reagent after centrifugation (12 000 r/min for 30 s) and stored at - 20℃ . Total RNA was isolated using Trizol Reagent according to the manufacture's instructions. The first strand cDNA was synthesized using reverse transcriptase Superscript Ⅱ according to the manufacture's instructions. The primers were designed from a conserved region of C. gigas Hsc70 cDNA sequence (GeneBank accession No. AF144646). The polymerase chain reaction (PCR)was performed for 30 cycles with denaturation at 94℃ for 30 s, annealing at 49℃ for 40 s, and elongation at 72℃ for 30 s. The product was cloned to pGEM-T easy vector and sequenced. It is 509 base pairs (bp) and possesses 94 % identity with the cDNA encoding C. gigas Hsc70 using Blastn. This homology was strongly confirmed by amino acid sequence comparison using the Blastx (99 % ). The 509 bp fragment was labeled with α-32pdCTP and a random primer DNA labeling kit and employed as a probe to perform Southern blotting, the result demonstrated that the cDNA came from a partial mRNA transcript of C. ariakensis genomic DNA gene. The polymerase chain reaction (PCR) was carried out to investigate the expression of Hsc70, Using the cDNAs of several tissues, such as gills (heat shocked), mantle, adductor muscle (heat shocked), heart, blood cells (one sample with heat shock for 1.5 hours at 36℃ and another without any stimulus).The PCR

  4. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  5. 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.

  6. 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).

  7. cDNA cloning of the beta subunit of teleost thyrotropin.

    OpenAIRE

    Ito, M.; Koide, Y.; Takamatsu, N; Kawauchi, H.; Shiba, T.

    1993-01-01

    cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human fo...

  8. [cDNA cloning and sequence analysis of pluripotency genes in tree shrews (Tupaia belangeri)].

    Science.gov (United States)

    Wang, Cai-Yun; Ma, Yun-Han; He, Da-Jian; Yang, Shi-Hua

    2013-04-01

    In this paper, partial sequences of the tree shrew (Tupaia belangeri) Klf4, Sox2, and c-Myc genes were cloned and sequenced, which were 382, 612, and 485 bp in length and encoded 127, 204, and 161 amino acids, respectively. Whereas, their cDNA sequence identities with those of human were 89%, 98%, and 89%, respectively. Their phylogenetic tree results indicated different topologies and suggested individual evolutional pathways. These results can facilitate further functional studies.

  9. Molecular cloning of a cDNA encoding the porcine type XVII collagen noncollagenous 16 A domain and localization of the domain to the upper part of porcine skin basement membrane zone.

    Science.gov (United States)

    Xu, Luting; Olivry, Thierry; Chan, Lawrence S

    2004-06-01

    Bullous pemphigoid is an autoimmune blistering human skin disease mediated by immunoglobulin (Ig)G autoantibodies targeting skin basement membrane component type XVII collagen, a transmembrane protein. Also designated BP180 and BPAG2, type XVII collagen is an extracellular matrix element essential for the connection between the epidermis and the underlying dermis. In addition to being a target antigen in the human disease bullous pemphigoid, type XVII collagen is also targeted by autoantibodies of canine, feline, equine and porcine patients suffering from a similar blistering skin disease. Previously, enzyme-linked immunosorbent assay and Western blot analyses have shown that autoantibodies from pigs affected with bullous pemphigoid recognize the human NC16A domain of type XVII collagen. To facilitate the development of porcine model of bullous pemphigoid, we isolated cDNA encoding the porcine type XVII collagen NC16A domain using a reverse transcription-polymerase chain reaction technique. The amino acids deduced from the NC16A cDNA showed 61% identity with the sequence of human NC16A. An antibody generated against a 20-amino acid peptide within the porcine NC16A localized the NC16A epitope to the upper part of porcine skin basement membrane zone. Our data provide further information of the porcine bullous pemphigoid target antigen and may help investigators for their further studies of this disease.

  10. The full-ORF clone resource of the German cDNA Consortium

    Directory of Open Access Journals (Sweden)

    Michel Guenter

    2007-10-01

    Full Text Available Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen. A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available

  11. Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme.

    Science.gov (United States)

    Jones, A Lesley; Gane, Andy M; Herbert, Derek; Willey, David L; Rutter, Andrew J; Kille, Peter; Dancer, Jane E; Harwood, John L

    2003-03-01

    A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during

  12. 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.

  13. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C......-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed...

  14. 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.

  15. 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

  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

    We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I alpha chain (B-F) to screen lambda gt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemic B19 homozygous chickens. Eight clones were found to encode B-G molecules which...

  17. 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.

  18. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the [alpha]1 (XVIII) collagen gene to mouse chromosome 10 and human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Oh, S.P.; Warman, M.L.; Timmons, S.; Olsen, B.R.; Knoll, J.H.M. (Harvard Medical School, Boston, MA (United States)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Cheng, Sou-De (Children' s Hospital/Harvard Medical School, Boston, MA (United States))

    1994-02-01

    Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which the authors have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, they report the isolation of human cDNAs and genomic DNAs encoding the [alpha]1 (XVIII) collagen chain. Utilizing a genomic clone as probe, they have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, they have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2. 16 refs., 5 figs.

  19. 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.

  20. 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.

  1. 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

  2. Cloning and expression of murine immune interferon cDNA.

    OpenAIRE

    1983-01-01

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

  3. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    Science.gov (United States)

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  4. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    Energy Technology Data Exchange (ETDEWEB)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H. (Research Institutes, Postfach (West Germany))

    1988-06-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10{sup 6} independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

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

    Science.gov (United States)

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

    2008-11-01

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

  6. Cloning Full-Length cDNAs from Vascular Tissues and Cells by Rapid Amplification of cDNA Ends (RACE) and RT-PCR.

    Science.gov (United States)

    Shen

    1999-01-01

    The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA sequence encoding a protein, based on peptide sequences; 3) to obtain the sequence of a reported cDNA for functional analysis or expression studies; and 4) to define exon/intron boundaries of a cloned gene or determine transcription start site(s) of a promoter.

  7. Isolation of a cDNA Encoding a Protease from Perinereis aibuhitensis Grube

    Institute of Scientific and Technical Information of China (English)

    Rong-Gui LI; Dong-Meng QIAN; Dao-Sen GUO; Gui-Cai DU; Zhi-Yong YAN; Bin WANG

    2006-01-01

    The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMALPPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.

  8. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F

    DEFF Research Database (Denmark)

    Helin, K; Lees, J A; Vidal, M

    1992-01-01

    The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3,...... of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein....

  9. 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...

  10. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    WANG; Xin; (王鑫); WANG; Duncheng; (王敦成); CHEN; Xing; (陈兴),; HU; Meiru; (胡美茹); WANG; Jian'an; (王建安); LI; Yan; (黎燕); GUO; Ning; (郭宁); SHEN; Beifen; (沈倍奋)

    2001-01-01

    Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  11. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Our previous studies showed that some nuclear proteins that wereexpressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  12. Cloning of Mouse Enamel Matrix Serine Proteinase Encoding Mature Protein

    Institute of Scientific and Technical Information of China (English)

    MU Ya-bing; SUN Hong-chen; ZHANG Ze-bing; OUYANG Jie

    2003-01-01

    Objective: To clone cDNA of enamel matrix serine proteinase (EMSP1) encoding mature protein from mouse dental germs. Methods: Total RNA was isolated from developing incisors and molars of 7 days mouse pups and reverse-transcribed into cDNA. Two pairs of specific primers was designed to obtain the desired gene by Touchdown PCR and Nested PCR. The segment was inserted into Vector pMD-18T, and recombined vectors was transformed into E.coli JM109.The positive clone was chose and analysed by restriction endonuclease mapping and DNA sequencing. Results:700 bp of cDNA of mouse EMSP1 was sueccessfully cloned from mouse tooth germs tissue. The sequence was consistent with that displayed in PubMed. Conclusion:The mouse EMSP1 cDNA encoding mature protein is obtained for further study.%目的:克隆小鼠牙胚组织中釉基质丝氨酸蛋白酶(EMSP1)成熟肽编码区基因.方法:提取出生后7 d昆明种小白鼠切牙、磨牙牙胚总RNA,逆转录为cDNA,设计两对特异性引物,采用Touchdown PCR 和嵌套PCR方法,扩增出小鼠EMSP1起始密码子至终止密码子基因片段.将目的基因连入载体pMD-18T,转化入大肠杆菌JM109,通过蓝白筛选,挑选阳性克隆培养扩增,纯化重组质粒进行限制性酶切和核苷酸序列分析鉴定.结果:限制性酶切图谱和核苷酸序列分析均表明所克隆cDNA为小鼠700 bp的EMSP1成熟肽基因编码.结论:成功地克隆了小鼠编码EMSP1成熟肽基因片段.

  13. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

  14. 扩展莫尼茨绦虫蛋白激酶C相互作用蛋白(PICK1)基因的克隆及序列分析%Cloning and Sequence Analysis of the cDNA Encoding Protein Interacting with C Kinase 1 (PICK1) in Moniezia expansa

    Institute of Scientific and Technical Information of China (English)

    赵文娟; 康立超; 薄新文; 王新华

    2011-01-01

    [目的]分离和鉴定扩展莫尼茨绦虫(Monieziaexpansa)新基因,为进一步研究该基因的功能奠定基础.[方法]构建扩展莫尼茨绦虫成虫cDNA文库,随机挑取重组阳性克隆进行测序,对部分序列进行引物步移法测序,获取其全长cDNA序列;采用生物信息学等分析技术对该cDNA序列进行开放阅读框(ORF)的寻找、编码氨基酸的推导、核苷酸和氨基酸同源性比较及蛋白质二级结构的初步预测.[结果]获得了1个扩展莫尼茨绦虫新基因蛋白激酶C相互作用蛋白,全长1 527 bp,编码447个氨基酸,CDS预测存在明显的BAR,PDZ结构域.编码蛋白的理论分子质量为50.173 3 ku,等电点为5.22.[结论]获得了扩展莫尼茨绦虫蛋白激酶C相互作用蛋白的全长cDNA序列,为该基因功能的试验性鉴定工作奠定基础.%[Objective] The purpose of this program was to clone and identify novel genes from an adult Monieda expansa (M. Expansa) cDNA library, and provide a foundation for further research. [Method]A cDNA library was constructed from M. Expansa adult stage. Clones were selected randomly from the cDNA library and were sequenced by using the method of expression sequence tags (ESTs) . Novel genes were acquired by primer -walking. The cDNA sequence encoding M. Expansa PICK1 protein was analyzed, including searching the ORF, translating the nucleotide to protein sequence, similarity searches and secondary structure predication with bioinformatics analysis. [ Result ] PICK 1 genes, 1527 bp and coding for 447 amino acids, was cloned and sequenced, then the sequence was submitted to GenBank and got an accession number, GH291479. The theoretical pi was 5.22 and molecular weight was SO. 173 3 ku. [Conclusion]The full - length cDNA encoding M. Expansa PICK1 was obtained, which laid the foundation for further functional study of this gene.

  15. Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

    Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

  16. 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.

  17. 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.

  18. 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.

  19. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

    The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

  20. Isolation and characterization of a cDNA encoding a CBF transcription factor from E. globulus.

    Science.gov (United States)

    Gamboa, Maria C; Rasmussen-Poblete, Susana; Valenzuela, Pablo D T; Krauskopf, Erwin

    2007-01-01

    The transcription factors CBF/DREB play an important role during low temperature, drought and high-salt stress in higher plants. In this work, we isolated one full-length CBF cDNA clone from the angiosperm Eucalyptus globulus. The derived peptide sequence reveals that it encodes a transcriptional activator that has all the characteristic motifs present in CBF proteins previously described in Arabidopsis and tomato. RT-PCR analysis shows that EgCBF1 is transiently induced in E. globulus seedlings that had been exposed to low temperature within the first 15 min. These results suggest that the isolated CBF gene participates in the cold responsive pathway of E. globulus.

  1. 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.

  2. 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.

  3. 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).

  4. A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

    OpenAIRE

    1997-01-01

    Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones...

  5. 甘蓝型油菜(Brassica napus)中一个未知功能蛋白基因的cDNA克隆%The cDNA Cloning of a Gene Encoding an Unknown Protein from Brassica napus

    Institute of Scientific and Technical Information of China (English)

    邱峰; 王茂林; 刘阳; 周云涛; 赵云; 张帆

    2005-01-01

    随着现代分子生物学的发展,许多物种,特别是拟南芥等模式生物的整个基因组序列以及其中一部分基因的功能已经比较清楚,但仍然存在大量功能未知的基因有待进一步研究,从已知片段出发,利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术钓取基因全长,并结合生物信息学技术预测蛋白质结构,是研究未知基因功能的一种重要手段.

  6. 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.

  7. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

    OpenAIRE

    1991-01-01

    The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-t...

  8. Cloning and Sequence of cDNA Encoding ACC Synthase Specifically Expressed in Banana Fruit%香蕉果实特异性ACC合酶的cDNA克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    王新力; 彭学贤; 李宏

    2000-01-01

    根据ACC合酶高度保守区氨基酸序列设计两种兼并引物.通过RT-PCR,克隆了香蕉果肉ACC合酶1693bp的cDNA片段.再根据其序列测定结果进行5'RACE(Rapid amplification of cDNA ends).最终确定香蕉果肉中ACC合酶的mRNA全长为1752个核苷酸.其中5'非翻译区74个核苷酸,编码区1461个核苷酸,3'非翻译区217个核苷酸,编码产物为486个氨基酸.通过Northern杂交分析,证明此ACC合酶基因的表达具有果实特异性.

  9. 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

    2014-10-27

    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.

  10. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

  11. scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

    NARCIS (Netherlands)

    Brondijk, THC; Durand, R; vanderGiezen, M; Gottschal, JC; Prins, RA; Fevre, M

    1996-01-01

    A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degr

  12. Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor

    DEFF Research Database (Denmark)

    Nophar, Y; Kemper, O; Brakebusch, C

    1990-01-01

    found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable...

  13. Molecular Cloning and Sequence Analysis of Full-Length cDNA Encoding Human Bone Morphogenetic Protein-7%人骨形态发生蛋白-7全长基因cDNA的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    李新友; 刘淼; 李曙明; 姚煜; 王全颖; 杨广笑

    2003-01-01

    Objective: To clone the full-length human bone morphogenetic protein-7 ( BMP-7) gene and analyse its sequence, to aid in investigation of its function and structure. Methods: TotalRNA was isolated from Chinese fetal kidney by the acid guanidinium thiocyanate phenol-chloroformmethod. Two overlapping segments of human BMP-7 cDNA were obtained by reverse transcription(RT)-PCR. Fallowing application, the two segments were ligated to each other and subcloned intoPGEM-7 easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-ter-mination method was used to sequence the cDNA. Results: There was 750 bp fragment obtained RT-PCR using # 2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp frag-merit from 3' end was generated by RT-PCR using # 4 primer (PCR using # 3 and # 4). Full-lengthcDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 se-quence in Gene bank (XM30619) ,our full-length BMP- 7 cDNA has a G instead of a T at nucleotide862. This change results in valine substituting for phenylalanine in the protein. Conclusion: This isthe first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNAplays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an at-tractive target for various clinical applications.%目的:克隆人骨形态发生蛋白-7(BMP-7)全长基因,并进行测序及序列分析,研究其结构和功能.方法:采用异硫氰酸胍一步法从人胎儿肾中提取总RNA,利用逆转录-聚合酶链反应(RT-PCR)的方法,分段扩增出hBMP-7全长基因cDNA,与PGEM-T easy连接成PGEM-T easy/hBMP-7重组质粒,采用Sanger双脱氧链终止法测定基因序列.结果:以4号特异引物引导的RT-PCR扩增出BMP-7基因3'端540 bp片段,以2号特异引物引导的RT-PCR扩增出BMP-7基因5'端750 bp片段,重组连接两片段得到BMP-7全长基因cDNA.与Genebank上发表的hBMP-7序列(XM30619)

  14. 口虾蛄高血糖激素基因全长 cDNA 的克隆及表达分析%Molecular cloning and expression of a full length cDNA of encoding crustacean hyperglycemic hormone(CHH)in Oratosquilla oratoria

    Institute of Scientific and Technical Information of China (English)

    刘海映; 张秀芹; 隋宥珍; 李宏俊; 邢坤; 姜玉声; 杨贵福; 陈雷; 张娜

    2016-01-01

    Total RNA in eyestalk of Oratosquilla oratoria was extracted and cDNA sequence of crustacean hypergly-cemic hormone (CHH)were obtained by RACE for the first time.The result showed that the full length of the CHH gene is 2 421 bp,containing an 180 bp 5′-untranslated region (UTR),and 1 821 bp 3′UTR,and 420 bp open reading flame (ORF)which encoded 139 amino acids.Prediction of protein structure was composed of 23.74%α-helix,23.02% extension chain,53.24% random coil,and its molecular mass is 15 470 Daltons with an estimated pI of 7.049.The mature peptide contained six conserved Cys residues in CHH family.The C-terminus mature peptide was GK which has the same characteristics with ES-CHH.Based on protein similarity comparison,the separated CHH gene was classified into the type I CHH family of peptides.Phylogenetic analysis showed that CHH of O.oratoria and CHH of decapod species formed two subgroups on the same branch of the tree.The relative ex-pression of CHH gene in different organizations was tested by real time fluorescent quantitative PCR,the expres-sion was highest in the intestine,and then the eyestalk,antenna and brain,the expression is negligible in muscle and ovary,and no expression in testis.%本文首次采用 RACE 技术获得口虾蛄(Oratosquilla oratoria )眼柄部高血糖激素(crustacean hy-perglycemic hormone,CHH)基因 cDNA 全长序列,共2421 bp,5′UTR 长度为180 bp,3′UTR 为1821 bp,开放阅读框(ORF)长420 bp,编码139个氨基酸。预测蛋白二级结构α-螺旋23.74%,延伸链占23.02%,无规则卷曲占53.24%,预测分子量为15.47 kD,等电点(pI )为7.049。CHH 成熟肽包含6个 CHH 家族中位置保守的 Cys 残基,C 端为 GK,推断为 ES 型 CHH 基因。聚类分析表明:口虾蛄CHH 和十足目 CHH 聚为同一个分支的两亚群,具有较近的亲缘关系。荧光定量 PCR 分析结果表明,CHH 基因在肠中表达量最高,在眼柄、触角

  15. 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...... 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...

  16. cDNA Cloning of Two Novel T-superfamily Conotoxins from Conus leopardus

    Institute of Scientific and Technical Information of China (English)

    Wei-Hua CHEN; Yu-Hong HAN; Qi WANG; Xiao-Wei MIAO; Ling OU; Xiao-Xia SHAO

    2006-01-01

    The full-length cDNAs of two novel T-superfamily conotoxins, Lp5.1 and Lp5.2, were cloned from a vermivorous cone snail Conus leopardus using 3′/5′-rapid amplification of cDNA ends. The cDNA of Lp5.1 encodes a precursor of 65 residues, including a 22-residue signal peptide, a 28-residue propeptide and a 15-residue mature peptide. Lp5.1 is processed at the common signal site -X-Arg- immediately before the mature peptide sequences. In the case of Lp5.2, the precursor includes a 25-residue signal peptide and a 43-residue sequence comprising the propeptide and mature peptide, which is probably cleaved to yield a 29-residue propeptide and a 14-residue mature toxin. Although these two conotoxins share a similar signal sequence and a conserved disulfide pattern with the known T-superfamily, the pro-region and mature peptides are of low identity, especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5. 1a.The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution,the sequence diversity of T-superfamily conotoxins, the special gene structure and the evolution of these peptides.

  17. 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.

  18. Differential cDNA cloning by enzymatic degrading subtraction (EDS).

    OpenAIRE

    1994-01-01

    We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate th...

  19. 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...

  20. 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.

  1. Molecular cloning and sequence analysis of hamster CENP-A cDNA

    Science.gov (United States)

    Figueroa, Javier; Pendón, Carlos; Valdivia, Manuel M

    2002-01-01

    Background The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A) is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA. Results We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein. Conclusions We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural plasticity. PMID:12019018

  2. 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.

  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. Molecular Cloning of the Bovine Liver ADPRT cDNA

    Science.gov (United States)

    1988-12-13

    Sambrook. J. (1982) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, N. Y. Martinez, H (1988) Sequence Analysis Programs. Publ...Sambrook, J. (1982) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, N. Y. 1)..Messing, J. (1983) Methods in Enzymol. 101. 20-78. 1-Actin-S

  5. 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

  6. Molecular Cloning and Bioinformatical Analysis of a cDNA Encoding Mitochondrial 50S Ribosomal Protein L21 from Hevea brasiliensis Muell. Arg.%巴西橡胶树线粒体50S核糖体蛋白L21 cDNA的克隆与分析(英文)

    Institute of Scientific and Technical Information of China (English)

    邹智; 杨礼富

    2012-01-01

    [Objective] "Tapping panel dryness (TPD)", a syndrome known as tapping incision blocked partly or entirely during latex exploiting, has become the most important factor causing great losses for rubber production. Aiming to elucidate the molecular mechanism of tapping panel dryness occurrence, this study carried out molecular cloning and bioinformatical analysis of a mRPL21 cDNA sequence, a gene associated with TPD. [Method] In a preliminary study, an expressed sequence tag (EST) encoding a deduced protein homologous to mitochondrial 50S ribosomal protein L21 (mRPL21), which showed to be down-regulated in the latex of TPD-affected rubber trees, was isolated by suppression subtractive hybridization (SSH). After ESTs assembling and RT-PCR validation, an 853 bp cDNA sequence with an open reading frame (ORF) was cloned, which was named as HbmRPL21 under GenBank accession number of HM230670. [Result] Bioinformatical analysis suggests that HbmRPL21 encodes a deduced polypeptide of 271 amino acids with a theoretical molecular weight (Mw) of 30.52 kDa and isolectric point (pI) of 8.40, and HbmRPL21 is a mitochondrion-targeted protein with a conserved domain of Ribosomal_L21p involving translation. Homology analysis reveals high amino acid sequence identity of mRPL21 from plants, while diversity of that between plant and animal kingdom. [Conclusion] This study laid the basis for further revealing the biological functions of mRPL21 in TPD-affected rubber trees.%[目的]在橡胶生产中,一种叫做"死皮"的生理综合症严重制约了橡胶树(Hevea brasiliensis)单产的提高。为揭示橡胶树"死皮"发生的分子机理,研究对一差异表达的HbmRPL21进行了克隆,并在此基础上对其进行深入的生物信息学分析。[方法]在早期构建的差减文库中,筛选到一条在死皮植株中下调表达的基因片段,该片段编码的蛋白与线粒体50S核糖体蛋白L21(mRPL21)同源。通

  7. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    Science.gov (United States)

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L

    2004-07-01

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  8. Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding.

    Science.gov (United States)

    Huang, C F; Chuang, N N

    1998-12-11

    A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form.

  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. 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

  11. 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.

  12. Cloning and Expression Analysis of a Full-length cDNA Encoding Glucokinase from Pengze Crucian Carp (Carassius auratus var.Pengze)%彭泽鲫葡萄糖激酶基因全长cDNA克隆及表达分析

    Institute of Scientific and Technical Information of China (English)

    程汉良; 姬南京; 彭永兴; 申欣; 吴陈晨; 许建和; 董志国

    2011-01-01

    A full-length cDNA coding glucokinase (GK) was cloned from Pengze crucian carp ( Carassius au-ratus var. Pengze) by RT-PCR and rapid amplification of cDNA ends (RACE) methods. The cDNA sequence obtained is of 2 050 bp length with a 1 431 bp open reading frame (ORF) encoding 476 amino acids. The GK protein has a calculated molecular weight of 53. 78 ku and isolectric point of 5.14. The main domains of GK, such as ATP-binding domain, glucose-binding amino acids, hexokinases signature, N-linked glycosy-lation sites, cell attachment sequence and glycosaminoglycan attachment site for the Pengze crucian carp are basically conservative compared with other vertebrates. The amino acid sequence has a high similarity to GK of other species, and the percent identity compared with common carp, human and rat are 98.1% , 79. 8% and 79.1% , respectively. Tissue distribution of GK mRNA in brain, white muscle, spleen, mesenteric adipose tissue arid liver of Pengze crucian carp was analyzed by semi-quantitative RT-PCR method using β-actin gene as internal control. The result showed that the expression level of GK mRNA in liver was significantly higher than that in spleen, mesenteric adipose tissue and brain (P <0. 05). GK mRNA did not be detected in white muscle.%本试验采用RT-PCR和cDNA末端快速扩增(RACE)方法克隆获得了彭泽鲫(Carassius auratus var.Pengze)葡萄糖激酶(GK)基因全长cDNA序列.结果表明,该cDNA全长2 050 bp,含1个1 43l bp的开放阅读框(ORF),编码476个氨基酸,GK蛋白计算分子量为53.78 ku,等电点为5.14.彭泽鲫GK氨基酸序列的ATP结合位点、葡萄糖结合位点、己糖激酶标签序列、N-连接糖基化位点、细胞黏附序列和糖胺聚糖黏附位点等主要功能位点与其他脊椎动物相比基本保守.彭泽鲫GK氨基酸序列与建鲤、人和鼠GK氨基酸序列相似百分比分别为98.1%、79.8%和79.1%.以β-actin为内标,采用半定量RT-PCR方法对GK基因在彭泽鲫大脑、白

  13. 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.

  14. Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

    Science.gov (United States)

    Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

    1992-05-01

    A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

  17. 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.

  18. 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...

  19. cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

    Science.gov (United States)

    Perez, M; Ishioka, G Y; Walker, L E; Chesnut, R W

    1990-09-25

    The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.

  20. 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.

  1. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends.

    Science.gov (United States)

    Schaefer, B C

    1995-05-20

    Rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR)-based technique which was developed to facilitate the cloning of full-length cDNA 5'- and 3'-ends after a partial cDNA sequence has been obtained by other methods. While RACE can yield complete sequences of cDNA ends in only a few days, the RACE procedure frequently results in the exclusive amplification of truncated cDNA ends, undermining efforts to generate full-length clones. Many investigators have suggested modifications to the RACE protocol to improve the effectiveness of the technique. Based on first-hand experience with RACE, a critical review of numerous published variations of the key steps in the RACE method is presented. Also included is a detailed, effective protocol based on RNA ligase-mediated RACE/reverse ligation-mediated PCR, as well as a demonstration of its utility.

  2. 辽宁碱蓬胆碱单加氧酶基因克隆及转基因烟草的耐盐性%Cloning of cDNA Encoding Choline Monooxygenase from Suaeda liaotungensis and Salt Tolerance of Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    李秋莉; 刘大伟; 高晓蓉; 苏乔; 安利佳

    2003-01-01

    甜菜碱是生物体内普遍存在的一种很有效的渗透调节剂.在高等植物中,甜菜碱由胆碱经两步酶催氧化得到,即:胆碱→甜菜碱醛→甜菜碱,催化第一步反应的酶是胆碱单加氧酶(choline monooxygenase,CMO).用RT-PCR和RACE技术从盐生植物辽宁碱蓬(Suaeda liaotungensis Kitag)中分离了CMO cDNA全序列,其中包括5′端非编码区123 bp, 3′端非编码区368 bp, 开放阅读框1 329 bp,编码一个由442个氨基酸构成的多肽,与菠菜、甜菜和山菠菜CMO的氨基酸序列同源性分别为77%、72% 和74%.克隆了其编码区,构建了植物表达载体pBI121-CMO,根癌土壤杆菌(Agrobacterium tumefaciens)介导转化烟草(Nictiana tabacum L.cv.89),获得卡那霉素抗性植株.PCR和Southern杂交均证明外源CMO基因已整合到烟草基因组中,转基因烟草的甜菜碱含量明显高于对照,转基因烟草膜的相对电导率明显低于对照,说明盐胁迫下转基因烟草的膜结构所受损伤小于对照,转基因烟草具有一定的耐盐性,能在含250 mmol/L NaCl的培养基中正常生长.%Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline→betaine aldehyde→betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5′ UTR, a 368 bp 3′ UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco (Nicotiana tabacum L. cv. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated

  3. Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana.

    Science.gov (United States)

    Huang, W; Lin, Z; Sin, Y M; Li, D; Gong, Z; Yang, D

    2006-07-01

    Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two almost identical fragments with one varied gap region and one conserved spider fibroin-like C-terminal domain. Both in situ hybridization and immunoblot analyses have shown that it is specifically expressed in the tubuliform gland. Thus, it likely encodes the silk fibroin from the tubuliform gland, which supplies the main component of the inner egg case. Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an alpha-helical protein, which implies the distinct property of the egg case silk, though the fiber of tubuliform silk is mainly composed of beta-sheet structure. Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes.

  4. 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...

  5. 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.

  6. 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.

  7. 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.

  8. A cDNA encoding a cold-induced glycine-rich RNA binding protein from Prunus avium expressed in embryonic axes.

    Science.gov (United States)

    Stephen, John R; Dent, Katherine C; Finch-Savage, William E

    2003-11-27

    A cDNA clone encoding a presumed full-length glycine-rich ribonucleic acid (RNA) binding protein was isolated from a lambda-ZAP Express cDNA library generated from primarily nondormant Prunus avium (wild cherry) embryonic axes. The cDNA, designated Pa-RRM-GRP1 (Prunus avium RNA recognition motif glycine-rich protein 1), contains a single N-terminal RNA recognition motif (RRM) and single C-terminal glycine-rich domain. The glycine-rich domain is unusually long at 91 amino acids, 58 of which are glycines. The 534-base pair (bp) open reading frame (ORF) of this clone encodes a 178-amino-acid polypeptide with a predicted molecular weight of 17.33 kDa and pI of 7.84. Comparative sequence alignment of Pa-RRM-GRP1 reveals extensive homology to known and presumed glycine-rich RNA binding proteins from angiosperms and gymnosperms. Genomic Southern blot analysis suggests that this gene exists as a single copy in P. avium. Expression of this gene in P. avium embryonic axes during low-temperature dormancy-breaking treatments was studied and found to be induced by cold (3 degrees C) using real-time PCR of total cDNA supported by Northern blot analysis of total RNA. Expression dropped during prolonged storage at 3 degrees C and was reduced to control levels by interruption of cold treatment by warming to 20 degrees C.

  9. 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

    1995-04-03

    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.

  10. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜

    2004-01-01

    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  11. 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.

  12. Cloning of a Full-Length cDNA Encoding Insulin-Like Growth Factor I and Its Expression in Antler Tissue%梅花鹿IGF1全长cDNA克隆及在鹿茸组织的表达

    Institute of Scientific and Technical Information of China (English)

    胡薇; 孟星宇; 田玉华; 刘宁

    2011-01-01

    The study was to detect expression of Insulin-Like Growth Factor I(IGF1) from antler tip tissue to provide a theoretical basis for the molecular mechanism of antler growth. Total RNA was isolated from dermis, mesenchyme, pre-cartilage and cartilage layers in growing antler tip tissue respectively. A pair of primers was designed according to the homology region of IGF1 among other species. The cDNA sequence of 1GF1 gene was cloned by RT-PCR technology. Then, real-time PCR was used to detect the expression level of IGF1 in the antler tip. Results showed that the complete coding sequence of IGF1 gene was obtained (GenBank accession No: HQ890468). It has 465 base pairs in length, encoding a predicted protein of 154 amino acids. Bioinformatic analysis based on IGF1 genes of red deer, cattle, sheep, horse, pig, human and mouse showed that the homologies of ICF1 to red deer, cattle and sheep were 99.78% , 98.28% and 97. 85% , and the homologies of protein were 99. 35% , 98. 05% and 97. 40% respectively. Results of fluorescence quantification showed that IGF1 was expressed in all the tested organs in antler tip tissue, but its transcript level in cartilage and pre-cartilage layers was much higher than that in mesenchyme and dermis layers.%为了检测梅花鹿鹿茸组织IGF1基因的mRNA表达水平,利用TRIzol试剂法分别提取鹿茸真皮、间充质、前软骨和软骨组织总RNA,逆转录合成cDNA.根据GenBank已发表的相关序列设计梅花鹿IGFI基因特异引物并克隆IGFI基因,将IGF1基因DNA序列递交GenBank,并利用相对荧光定量Real-time PCR法检测IGF1在鹿茸顶端不同部位组织的转录表达丰度.研究结果表明:成功获得了梅花鹿鹿茸组织IGF1基因全长编码区cDNA(GenBank登录号为HQ890468),该基因长465 bp,编码154aa长度的多肽;与马鹿、牛、羊、马、猪、人和小鼠IGF1基因进行同源性分析显示,IGF1基因与马鹿、牛和羊同源性最高,核苷酸序列分别为99.78% 、98.28

  13. 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.

  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. 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.

  16. 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.

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

    Science.gov (United States)

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

    2004-05-01

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

  18. Molecular cloning and chromosomal localization of the ADH7 gene encoding human class IV ({sigma}) ADH

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hirokazu; Baraona, E.; Lieber, C.S. [Mount Sinai School of Medicine, Bronx, NY (United States)

    1996-01-15

    The ADH7 gene encoding human Class IV ({sigma}) alcohol dehydrogenase (ADH) was cloned from a Caucasian genomic DNA library and characterized. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of {sigma} ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs. 15 refs., 2 figs., 1 tab.

  19. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    Science.gov (United States)

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  20. Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

    Science.gov (United States)

    Bujaki, Erika

    2016-01-01

    The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

  1. 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.

  2. 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.

  3. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-07-03

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

  5. Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

    Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

  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. 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.

  8. Isolation and characterization of sequences homologous to the tobacco clone axi 1 (auxin independent) from a Vicia sativa nodule cDNA library

    NARCIS (Netherlands)

    Yalçin-Mendi, Y.; Çetiner, S.; Bisseling, T.

    2001-01-01

    In this research, partial nucleotide sequences of the axi 1 gene, which is related to auxin perception and transduction, isolated from Vicia sativa using cDNA library screening were investigated. Four V. sativa cDNA clones representing homologous of the tobacco axi 1 (auxin independent) cDNA clone w

  9. cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda.

    Science.gov (United States)

    Hao, Yan-Zhe; Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Peng, Zheng-Song

    2009-11-01

    RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first

  10. Cloning of the 5' mRNA for the 230-kD bullous pemphigoid antigen by rapid amplification of cDNA ends.

    Science.gov (United States)

    Elgart, G W; Stanley, J R

    1993-08-01

    The 230-kD bullous pemphigoid antigen (BPAG1), defined by autoantibodies in patient sera, is a hemidesmosomal plaque protein in the same gene family as the intracellular proteins desmoplakin I/II and plectin. We had previously isolated, from a lambda gt11 library, overlapping cDNA clones with 6921 bp of mRNA sequence for BPAG1. The coding sequence encoded by these clones included the 3' stop codon but not the 5' coding and non-coding region of the mRNA. To obtain these sequences we used the polymerase chain reaction (PCR) method called rapid amplification of cDNA ends (RACE). The PCR products were cloned into plasmids and sequenced. With five PCR primers we were able to obtain overlapping clones containing the 5' region of the mRNA. An upstream stop codon in frame with the rest of the coding sequence demonstrates that the full 5' coding sequence is obtained. Four different PCR products from two separate reactions had the same 5' end, suggesting that this 5' end is near, or at, the transcription start site. No alternatively spliced clones were found and no transmembrane site was predicted, confirming that BPAG1 is an intracellular hemidesmosomal plaque protein.

  11. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    Science.gov (United States)

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D

    1996-02-15

    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  12. Gibson assembly : an easy way to clone potyviral full-length infectious cDNA clones ex pressing an ectopic VPg

    OpenAIRE

    Bordat, Amandine; Houvenaghel, Marie-Christine

    2015-01-01

    Background Approaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyviruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce mosaic virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in...

  13. 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.

  14. Cloning and Characterization of Full Length cDNA of a CC-NBS-LRR Resistance Gene in Sweetpotato

    Institute of Scientific and Technical Information of China (English)

    CHEN Guan-shui; ZHOU Yi-fei; HOU Li-li; PAN Da-ren

    2009-01-01

    Conserved domain such as nucleotide binding site (NBS) was found in several cloned plant disease resistance genes.Based on the NBS domain,resistance gene analogues (RGAs) have been isolated.A full-length cDNA,SPRI was obtained by rapid amplification of cDNA ends (RACE) method.Sequence analysis indicated that the length of SPR1 was 3 066 bp,including a complete open reading frame of 2 667 bp encoding SPRI protein of 888 amino acids.Compared with known NBS-LRR genes,it presented relatively high amino acid sequence identity.The polypeptide has a typical structure of non TIR-NBS-LRR genes,with NB-ARC,CC,and LRR domains.The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting.Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues.The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato.The gene has been submitted to the GenBank database,and the accession number is EF428453.

  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. cDNA cloning and expression of the mRNA for cytochrome P-450kd which shows a fatty acid omega-hydroxylating activity.

    Science.gov (United States)

    Yokotani, N; Kusunose, E; Sogawa, K; Kawashima, H; Kinosaki, M; Kusunose, M; Fujii-Kuriyama, Y

    1991-03-28

    We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.

  17. 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.

  18. 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.

  19. Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

    Science.gov (United States)

    Guerrero, Consuelo; Martín-Rufián, M; Reina, José J; Heredia, Antonio

    2006-01-01

    A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.

  20. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  1. Cloning and Expression Analysis of a Prion Protein Encoding Gene in Guppy (Poecilia reticulata)

    Institute of Scientific and Technical Information of China (English)

    WU Suihan; WEI Qiwei; YANG Guanpin; WANG Dengqiang; ZOU Guiwei; CHEN Daqing

    2008-01-01

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

  2. Rainbow trout cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase). cDNA cloning, enzymatic properties and temporal pattern of ovarian P-450c17 mRNA expression during oogenesis.

    Science.gov (United States)

    Sakai, N; Tanaka, M; Adachi, S; Miller, W L; Nagahama, Y

    1992-04-13

    A cDNA clone encoding cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contained an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acid residues. The amino acid sequence of trout P-450c17 shows a much greater homology with chicken P-450c17 than with that of human, bovine and rat. The trout P-450c17 expressed in non-steroidogenic mammalian COS-1 cells showed both 17 alpha-hydroxylase and 17,20-lyase activities. The cDNA only hybridized to a single species of mRNA (2.4 kb) isolated from rainbow trout ovaries; the 2.4 kb transcripts were abundant in trout ovaries during the later stages of oogenesis.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  10. 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.

  11. 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.

  12. Molecular cloning and chromosomal localization of the nucleic acid sequences encoding the cerebrovascular and plaque amyloid peptide

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-05-01

    Amyloid deposits in vessels and neuritic plaques are found in large numbers in the brains of Alzheimer's Disease (AD) and adult Downs Syndrome (DS) patients. The partial amino acid sequence of the amyloid peptide has been determined. They used this amino acid sequence to synthesize an oligonucleotide probe specific for the amyloid peptide gene. Screening of a human brain cDNA library with this probe, yielded a clone which contained an insert 1.8 kb. This clone contains a long open reading frame including a region which encodes the 28 amino acids of the amyloid peptide. Northern blots of human brain mRNA detected a transcript of 3.3 kb long which hybridized to their cDNA clone. A similar mRNA was detected in the hamster, mouse, sheep and rabbit brains. Southern blots under stringent hybridization conditions detected sequences homologous to the amyloid gene in the genomes of hamster, mouse, sheep and rabbit suggesting that this gene has been conserved during mammalian evolution. Hybridization under reduced stringency revealed the presence of additional sequences related to the amyloid gene in the genome of the above organisms. Hybridization analysis of human x chinese hamster cell lines DNA showed that the gene encoding the amyloid peptide is located on chromosome 21, suggesting a genetic relationship between AD and DS.

  13. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

    Science.gov (United States)

    Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2015-12-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

  14. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2015-12-01

    Full Text Available Papaya leaf distortion mosaic virus (PLDMV is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV. The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA, was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

  15. [Construction of suppression subtractive hybridization (SSH) library of copepod Pseudodiaptomous annandalei and its ferritin cDNA cloning and differential expression under nickel stress].

    Science.gov (United States)

    Jiang, Jie-Lan; Wang, Gui-Zhong; Wu, Li-Sheng; Li, Shao-Jing

    2012-07-01

    To study the molecular response mechanisms of copepod to nickel stress, a suppression subtractive hybridization (SSH) cDNA library of Pseudodiaptomous annandalei under nickel stress was constructed by using SSH technique, and a total of 140 clones were randomly picked from the growing colonies and identified by PCR. The recombinant rate of the library was 98.6%, and the volume of the library was 1.12 x 10(6) cfu. After the recombinant plasmids were sequenced, a partial cDNA fragment of ferritin was recognized based on BLAST searches in NCBI, with a size of 859 bp and continuously encoding 170 amino acid residues. The semi-quantitative PCR results showed that the ferritin cDNA under 24 h nickel stress was distinctly up-regulated. The successful construction of the SSH library and the obtaining of ferritin cDNA fragment would supply basis for the further study of the molecular response mechanisms of copepod to nickel stress.

  16. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen

    Energy Technology Data Exchange (ETDEWEB)

    Federsppiel, B.; Melhado, I.G.; Delaney, A.; Clark-Lewis, I. (Univ. of British Columbia, Vancouver (Canada)); Duncan, A.M.V. (Queens Univ., Kinston, Ontario (Canada)); Jirik, F.R. (Hospital for Sick Children, Toronto, Ontario (Canada))

    1993-06-01

    A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E, was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin. 32 refs., 5 figs.

  17. Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro (National Institute of Agro-Environmental Sciences, Ibaraki (Japan) Univ. of Tokyo (Japan))

    1989-08-01

    A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M{sub r} of its subunit was 77,000. The cells converted ({sup 14}C)-L-phenylalanine into ({sup 14}C)-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M{sub r} 77,137), a 22-bp 5{prime}-noncoding region and a 207-bp 3{prime}-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.

  18. 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.

  19. 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.

  20. 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

  1. Purification, amino acid sequence, and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs.

    Science.gov (United States)

    Deshimaru, Masanobu; Watanabe, Akira; Suematsu, Keiko; Hatano, Maki; Terada, Shigeyuki

    2003-08-01

    Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

  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 of the immunoglobulin heavy chain genes in banded houndshark Triakis scyllium.

    Science.gov (United States)

    Honda, Yuka; Kondo, Hidehiro; Caipang, Christopher Marlowe A; Hirono, Ikuo; Aoki, Takashi

    2010-11-01

    In this study, cDNAs encoding the secreted forms of the immunoglobulin (Ig) heavy chains of IgM, IgNAR, and IgW were cloned from the banded houndshark Triakis scyllium. Two clones for the IgM heavy chains encoded 569 and 570 amino acids, whose conserved (C) region showed 47-70% amino acid identities to those reported in other cartilaginous fish. Four clones for the IgNAR encoded 673-670 amino acids with conserved Ig-superfamily domains. The IgNAR C region showed 56-69% amino acid identities to those so far reported. High-throughput sequencing revealed that in most of the IgNAR sequences, the two variable regions (CDR1 and CDR3) each possess a cysteine residue. Three types of IgW were identified; one contained Ig-superfamily domains that are in other cartilaginous fish, one lacks the 3rd domain in the constant region, and one lacks the 3rd to 5th domains. Despite these differences, the IgW isoforms clustered with IgWs of other cartilaginous fishes and the C regions showed 47-89% amino acid identities. mRNAs for IgM and IgNAR were detected in various tissues, while IgW mRNA was mainly detected in pancreas. The banded hounded shark also has IgM, IgW and IgNAR as well as the other cartilaginous fish with unique IgW isoform.

  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. Generation and characterisation of a full-length cDNA encoding murine myotonic dystrophy protein kinase from cardiac tissue

    Energy Technology Data Exchange (ETDEWEB)

    Carey, N.; Tongeren, T. van; Winchester, C. [Charing Cross & Wesminster Medical School, London (United Kingdom)] [and others

    1994-09-01

    The mutation underlying myotonic dystrophy (DM) is a CTG trinucleotide expansion in the 3{prime} untranslated region of a putative protein kinase gene (DMPK). We report the isolation of a full-length cDNA clone of the murine (DMPK) gene from a heart cDNA library. Sequence analysis shows that the clone is a splice isoform which has only previously been identified in brain, suggesting that there may be some flexibility of the splicing pattern in some tissues. We are currently analyzing the library for the presence of other isoforms. The full-length cDNA has been cloned into a bacterial expression system and the expressed protein is being used as an immunogen to generate both polyclonal and monoclonal antisera. These reagents will allow the analysis of the intracellular targets of the DMPK. Subclones of the cDNA have been generated for use as in situ hybridization probes, allowing investigation of the normal patterns of expression of the gene and the differential expression of the protein isoforms. These data will be essential for deciding on a rational use of rare patient material and will provide the necessary baseline for the analysis of transgenic and {open_quotes}knock-out{close_quotes} mice.

  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. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

    OpenAIRE

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex...

  8. cDNA cloning, characterization, and expression analysis of the Rac1 gene from Scophthalmus maximus.

    Science.gov (United States)

    Jia, Airong; Zhang, Xiao-Hua

    2009-09-01

    Rac1 is a small GTP-binding protein that belongs to the Rho small GTPases, which are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. The EST sequence of Rac1 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library previously. In this study, the full-length cDNA sequence of turbot Rac1 was obtained, which was 2420 nucleotides (nt) encoding a protein of 192 amino acids, with a putative molecular weight of 21.3 kDa. At the amino-acid level, turbot Rac1 was highly conserved to previously characterized GTPases of Rac sub-family, and was nearly identical to human Rac1 (95.3% identity). Quantitative real-time PCR demonstrated that the Rac1 was constitutively expressed in all tissues examined, but at different levels. Upon challenge with Vibrio harveyi, the expression level of Rac1 fluctuated in the liver at different time points. In the head kidney, its expression level decreased to the lowest at 4 h, and then increased to the background level at 24 h. The remarkable degree of evolutionary conservation observed in turbot Rac1 primary structure together with its changing in expression level upon challenge suggested a functionally important role for this Rho family member in the immune response.

  9. Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the Nsp2-encoding region.

    Science.gov (United States)

    Ran, Z G; Chen, X Y; Guo, X; Ge, X N; Yoon, K J; Yang, H C

    2008-01-01

    Non-structural protein 2 (Nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the most variable region and postulated to play an important role in cell and tissue tropism of PRRSV. To investigate the role of Nsp2 in the viability and growth of PRRSV in cells in vitro, two cDNA clones were constructed containing a deletion of 63 consecutive nucleotides (pWSK-DCBAd63) or 117 nucleotides (pWSK-DCBAd117) within the Nsp2-encoding region of PRRSV (BJ-4). The clone pWSK-DCBAd63 was infectious and produced viable recombinant virus, whereas clone pWSK-DCBAd117 could not be rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 cells and was stably propagated during sequential in vitro cell passages, like the virus recovered from the full-length cDNA clone of PRRSV BJ-4. In comparison to the parental virus (BJ-4) and the virus recovered from the full-length cDNA clone of the BJ-4 strain, the rescued virus from pWSK-DCBAd63 exhibited enhanced growth kinetics, reaching the peak progeny virus titer by 48 h postinfection. These observations suggest that the Nsp2-encoding region is necessary for productive virus infection, and partial deletion does not influence the viability and propagation of PRRSV in cell culture, which may provide a way to insert a foreign gene into the viral genome as a marker for differentiation.

  10. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    Energy Technology Data Exchange (ETDEWEB)

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O' Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    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 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent 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 nonprotein-coding RNA

  11. 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

  12. 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

  13. 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.

  14. Cloning and Analysis of cDNA Encoding Key Enzyme DXR in Diterpenoids Biosynthesis Pathway from Rabdosiae Rubescentis Herba%冬凌草1-脱氧木酮糖-5-磷酸还原异构酶(DXR)基因克隆与分析

    Institute of Scientific and Technical Information of China (English)

    苏秀红; 尹磊; 陈随清

    2016-01-01

    目的:为研究冬凌草二萜类合成的相关基因,在冬凌草转录组信息数据的基础之上,以冬凌草无菌苗为研究材料,克隆冬凌草二萜类合成的关键酶1-脱氧木酮糖-5-磷酸还原异构酶(l-deoxy-D-lxyluloses-phosphatereduetoisomerase,DXR)基因.方法:采用逆转录PCR技术克隆冬凌草DXR基因,实时荧光定量PCR法分析其组织表达模式.结果:DXR cDNA基因全长1 500 bp,DXR基因开放阅读框为1 422 bp,编码473个氨基酸组成的蛋白质序列,理论相对分子质量为51.39 kDa,等电点为6.09,是一种亲水性蛋白.DXR在茎中表达量相对较高,在愈伤组织中表达量最低.结论:研究结果为深入研究冬凌草DXR酶的活性和功能及为冬凌草二萜类化合物的生物合成机制、优良基因挖掘奠定基础.%Objective:To study the genes related to the synthesis of diterpenoid.Based on the data of transcriptome sequencing,cDNAs encoding 1-deoxy-D-xylulose-5-phosphatereduetoisomerase (DXR) were obtained from the leaves of aseptic seedlings of Rabdosiae Rubescentis Herba.Method:DXR was obtained by reverse transcription PCR.Real-time quantitative PCR was used to detect the relative expression patterns of DXR in different tissues of Rabdosiae Rubescentis Herba.Result:Sequence analysis showed that the full-length cDNA of DXR was 1 500 bp and contains gene open reading frame (ORF) of 1 422 bp encoding 473 amino acids.The theoretical molecular weight was 51.39 kDa and the isoelectric point was predicted as 6.09,suggesting it was a type of hydrophilic protein.The expression pattern of the gene in different tissues was analyzed by Real-time fluorescence quantitative PCR.The results showed the expression of DXR was relatively high in the stem and the lowest in callus.Conclusion:The results will provide a basis for studying the activity and function of DXR from Rabdosiae Rubescentis Herba,and lay a foundation for biosynthesis and gene mining of terpenoids.

  15. 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.

  16. ISOLATION, CLONING AND CHARACTERIZATION OF ACTIN-ENCODING cDNAs FROM Jatropha curcas L. IP-2P

    Directory of Open Access Journals (Sweden)

    Kinya Akashi

    2011-11-01

    Full Text Available Actin is a major component of the plant cytoskeleton, so all cells contain this protein. Actin is expressed constitutivelyand is involved in basic housekeeping functions required for cell maintenance. Because of this, it has been frequentlyused as an internal control to normalize changes in gene expressions analysis. Actually, the information of nucleotidesequence of actin gene of Jatropha curcas L. population IP-2P from Indonesia is not available yet. The objective of thisresearch was to isolate, clone and characterize cDNA of actin genes of J. curcas IP-2P. Three partial actin genesequences had been successfully isolated by PCR using total cDNA as template, and actin primer designed fromconserved region of Arabidopsis thaliana. Nucleotide sequence analysis showed that the length of JcACT fragment is610, 534, and 701 bp encoding 203, 177, and 234 amino acids respectively. Local alignment analysis based on mRNAsequences shows that JcACT fragment shares 98% similarity with actin mRNA of Hevea brasiliensis and 99% withactin mRNA of Ricinus communis. Based on deduced amino acid sequence, JcACT is 100% identical to actins fromPrunus salicina, Gossypium hirsutum, and Betula luminifera. Even though these clones of cDNA are not completed yet,they can be used as reference in J. curcas L. gene expression analysis.

  17. Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

    Institute of Scientific and Technical Information of China (English)

    周兆斓; 朱祯; 刘春明; 张海涛; 肖桂芳; 李向辉

    1996-01-01

    Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.

  18. 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.

  19. 兔TLR2、TLR3和TLR4部分cDNA序列的克隆及分析%Cloning and sequence analysis of cDNA encoding rabbit Toll-like receptor2,3,4

    Institute of Scientific and Technical Information of China (English)

    聂奎; 曾兴艳; 周作勇; 王裕文

    2012-01-01

    In this study, Toll-like receptor 2 (R TLR2 ), Toll-like receptor-3 (R TLR3) and Toll-like receptor-4 (R TLR4 ) gene were cloned from Japanese white rabbits spleen by reverse transcription-polymerase chain reaetion(RT-PCR). Sequence analysis indicated that the RTLR2,RTLR3 and RTLR4 eDNA cloned were 128,150 and 139 bp in length and that the nucleotide sequence of RTLR2 shared 99 % homology with the published sequence Oryctolagus cuniculus TLR2(NM_001082781),while RTLR3 and RTLR4 shared 100% homology with the published sequence TLR3 (NM_001082219)and TLR4 (NM_001082732). The predicted amino acid sequence of RTLR2,RTLR3 and RTLR4 gene was compared to that of the partial cDNA fragments of Oryctolagus cuniculus TLR2, TLR3 and TLR4, with 100% similarity. The comparison of the deduced amino acids sequence of R TLR2,RTLR3 and R TLR4 with that of horse,dog,cat, orangutan,human, cattle sheep and mouse showed that the amino acids homology similarity were 80%,78%,78%,78%,78%,76%,73% and 61% in TLR2,97%,97%,95%,95%,95%,95%,93%and 93% in TLR3, and 75 %, 75 %, 75 %, 73 %,71 %, 66 %,66 % and 62 % in TLR4, respectively. Based on the phylogenetic tree and alignment of predicted animo sequence,we concluded that R TLR2,RTLR3 and RTLR4 were partial eDNA fragments of Oryctolagus cuniculus TLR2,TLR3 and TLR4,individually. Likewise,it was suggested that there was species-specific of TLRs in different kinds of animials.%用RT-PCR技术从日本大耳白兔脾脏组织克隆出兔Toll样受体2、3、4基因(拟命名为RTLR2、R TLR3和RTLR4)的cDNA序列并进行测序,获得的3个Toll样受体基因序列长分别为128、150和139bp,并将其测序结果与GenBank中登录的穴兔(Oryctolagus cuniculus)的Toils核苷酸序列进行比对,发现本次克隆到的RTLR2与穴兔TLR2的基因序列(NM_001082781)相似性为99%,而RTLR3与TLR3(NM_001082219)和RTLR4与TLR4(NM_001082732)相似性均为100%。Protein Blast同源性结果显示,RTLR2、RTLR3

  20. Characterization of the cDNA encoding a BPI/LBP homologue in venom gland of the hundred-pace snake Deinagkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    Jianrao HU; Mingfu CAO; Jiong CHEN

    2009-01-01

    Bactericidal/permeability-increasing protein (BPI) and LPS-binding protein (LBP) play an important role in host defence. Current evidence shows that BPI/LBP may be widely existed in different cells and tissue types of animals. A full-length cDNA clone encoding a BPI/LBP homologue (dBPI), 1757 bp in size, was characterized in venom gland of the hundred-pace snake Deinagkistrodon acutus. Its deduced amino acid sequence of 417 residues had 13.8% - 21.5% identity to BPI like 1 (BPIL1) and BPI like 3 (BPIL3) of other animals. Conserved cysteine residues which are involved in disulfide bond formation between the final strand of the N-terminal beta sheet and the long alpha helix of BPI are identified as Cys146-Cys183 of dBPI. Phylogenetic tree analysis showed that the BPI/LBP homologues formed five large clusters and dBPl was in a large cluster including BPIL1 and BPIL3. dBPI mRNA shows a tissue specific expression in venom gland. This is the fast study to identify the cDNA encoding BPI/LBP homologues from reptiles [Current Zoology 55 (5) : 376 - 382, 2009].

  1. Characterization of the cDNA encoding a BPI/LBP homologue in venom gland of the hundred-pace snake Deinagkistrodon acutus

    Directory of Open Access Journals (Sweden)

    Jianrao HU, Mingfu CAO, Jiong Chen

    2009-10-01

    Full Text Available Bactericidal/permeability-increasing protein (BPI and LPS-binding protein (LBP play an important role in host defence. Current evidence shows that BPI/LBP may be widely existed in different cells and tissue types of animals. A full-length cDNA clone encoding a BPI/LBP homologue (dBPI, 1757bp in size, was characterized in venom gland of the hundred-pace snake Deinagkistrodon acutus. Its deduced amino acid sequence of 417 residues had 13.8%–21.5% identity to BPI like 1(BPIL1 and BPI like 3(BPIL3 of other animals. Conserved cysteine residues which are involved in disulfide bond formation between the final strand of the N-terminal beta sheet and the long alpha helix of BPI are identified as Cys146-Cys183 of dBPI. Phylogenetic tree analysis showed that the BPI/LBP homologues formed five large clusters and dBPI was in a large cluster including BPIL1 and BPIL3. dBPI mRNA shows a tissue specific expression in venom gland. This is the first study to identify the cDNA encoding BPI/LBP homologues from reptiles [Current Zoology 55 (5: –2009].

  2. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    Energy Technology Data Exchange (ETDEWEB)

    Matviw, Yu, G.; Young, D. (Univ. of Calgary Health Science Centre, Alberta (Canada))

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  3. Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV.

    Science.gov (United States)

    Huang, F F; Pierson, F W; Toth, T E; Meng, X J

    2005-09-01

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen. Increasing evidence indicates that hepatitis E is a zoonosis. Avian HEV was recently discovered in chickens with hepatitis-splenomegaly syndrome in the USA. Like swine HEV from pigs, avian HEV is also genetically and antigenically related to human HEV. The objective of this study was to construct and characterize an infectious cDNA clone of avian HEV for future studies of HEV replication and pathogenesis. Three full-length cDNA clones of avian HEV, pT7-aHEV-5, pT7G-aHEV-10 and pT7G-aHEV-6, were constructed and their infectivity was tested by in vitro transfection of leghorn male hepatoma (LMH) chicken liver cells and by direct intrahepatic inoculation of specific-pathogen-free (SPF) chickens with capped RNA transcripts from the three clones. The results showed that the capped RNA transcripts from each of the three clones were replication competent when transfected into LMH cells as demonstrated by detection of viral antigens with avian HEV-specific antibodies. SPF chickens intrahepatically inoculated with the capped RNA transcripts from each of the three clones developed active avian HEV infections as evidenced by seroconversion to avian HEV antibodies, viraemia and faecal virus shedding. The infectivity was further confirmed by successful infection of naïve chickens with the viruses recovered from chickens inoculated with the RNA transcripts. The results indicated that all three cDNA clones of avian HEV are infectious both in vitro and in vivo. The availability of these infectious clones for a chicken strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.

  4. 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.

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

    Science.gov (United States)

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

    2014-11-07

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

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

    Science.gov (United States)

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

    2012-06-01

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

  9. Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

    Science.gov (United States)

    Vanhooren, J C; Fransen, M; de Béthune, B; Baumgart, E; Baes, M; Torrekens, S; Van Leuven, F; Mannaerts, G P; Van Veldhoven, P P

    1996-07-15

    The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

  10. Molecular cloning of the feline thymus and activation-regulated chemokine cDNA and its expression in lesional skin of cats with eosinophilic plaque.

    Science.gov (United States)

    Maeda, Sadatoshi; Okayama, Taro; Ohmori, Keitaro; Masuda, Kenichi; Ohno, Koichi; Tsujimoto, Hajime

    2003-02-01

    Thymus and activation-regulated chemokine (TARC) is a member of CC chemokine and plays an essential role in recruitment of CC chemokine receptor 4 positive Th2 cells to allergic lesion. To investigate the association of TARC in allergic inflammation of cats, a TARC cDNA was cloned from feline thymus by RT-PCR with 3' rapid amplification of cDNA ends (RACE) method. The feline TARC clone contained a full length open reading frame encoding 99 amino acids which shared 80.8%, 72.5%, 65.6% and 67.8% homology with dog, human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lung, lymph node, kidney, small intestine, colon and skin of the normal cat tissues examined. Furthermore, it was found that TARC mRNA was strongly expressed in lesional skin of cats with eosinophilic plaque. The present results demonstrated that TARC might be involved in the pathogenesis of eosinophilic plaque in cats.

  11. Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein.

    Science.gov (United States)

    Huang, Wei; Fang, Xiao-Dong; Lin, Qi-Fen; Li, Guan-Yi; Zhao, Wen-Ming

    2003-03-01

    Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3' and 5' end of the KCTIP1 gene were obtained using the SMART RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3'end of this gene was obtained using the GSP1 primer, and a 690 bp fragment

  12. The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata.

    Science.gov (United States)

    Zafar, R S; Chow, L H; Stern, M S; Scully, J S; Sharma, P R; Vinogradov, S N; Walz, D A

    1990-12-15

    The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C

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

    Institute of Scientific and Technical Information of China (English)

    Shu-Juan Zhao; Zhi-Bi Hu; Di Liu; Frederick C. C. Leung

    2006-01-01

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

  14. Cloning of two genes encoding Rab7 in Paramecium.

    Science.gov (United States)

    Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2006-01-01

    Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.

  15. 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.

  16. 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.

  17. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  18. Isolation of cDNA encoding the catalytic site of phosphatidylinositol-specific phospholipase C from Coffea arabica L.

    Science.gov (United States)

    Sánchez-Cach, Lucila A; Ortiz-García, Matilde M; Minero-García, Yereni; Muñoz-Sánchez, J Armando; Hernández-Sotomayor, SM Teresa; Suárez-Solís, Víctor M

    2008-01-01

    A cDNA encoding the catalytic site of a phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from Coffea arabica suspension cells. The cDNA (designated CaPLC) encodes a polypeptide of 308 amino acids, containing the catalytic X and Y domains, and has 99% identity to the soybean gene. Recombinant CaPLC protein was expressed in Escherichia coli, purified, and used to produce a polyclonal antibody. The peptide has a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses. Immunoblots revealed the presence of PLC-like proteins in the tissues of different plant species. PMID:19513191

  19. 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

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

    OpenAIRE

    Gray, P W; Barrett, K; Chantry, D; Turner, M.; Feldmann, M

    1990-01-01

    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 surf...

  1. 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).

  2. Cloning and sequencing of murine T3 gamma cDNA from a subtractive cDNA library.

    Science.gov (United States)

    Haser, W G; Saito, H; Koyama, T; Tonegawa, S

    1987-10-01

    The coding sequences of the murine and human T3 gamma chains are of identical length (182 amino acids) and contain a remarkable conservation of residues. The most striking observation is the high degree of homology between the murine and human cytosolic domains (89%), suggesting that the effector function of the T3 complex may be extremely similar or identical within human and murine lymphocytes. Both murine and human T lymphocytes can express two T3 gamma mRNA transcripts, suggesting that a second polyadenylation signal is present downstream. A poly(A) tail is not found in the 3' untranslated region of the murine gamma presented here, indicating that the murine clones analyzed represent mRNA generated by reading through the overlapping poly(A) signals at position 850-860 and possibly terminating at a position that would produce the 1.0 kb transcript.

  3. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now...... obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions....... This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix...

  4. 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.

  5. 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.

  6. Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes

    DEFF Research Database (Denmark)

    Larsen, L K; Andreasen, P H; Dreisig, H

    1999-01-01

    We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features...

  7. 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.

  8. 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.

  9. 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

    Science.gov (United States)

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

    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.

  10. Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: trans-splicing may play an important role in the diversification of scorpion venom peptides.

    Science.gov (United States)

    Zeng, Xian-Chun; Luo, Feng; Li, Wen-Xin

    2006-04-01

    A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 5'-terminal region of BmKT' cDNA and the 3'-terminal region of an undocumented cDNA (named BmKu), was isolated from a cDNA library made from the venom gland of scorpion Buthus martensii Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse transcription. Genomic amplifications performed with three pairs of BmKT-u gene-specific primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single transcriptional unit. Genomic cloning for BmKT' showed that the BmKT' gene contains an intron of 509 bp inserted into the region encoding the C-terminal region of the signal peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT' revealed that the junction site of the hybrid molecule is located at the 5'-splicing site of the intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom peptides from venomous animals.

  11. PG25, a pineal-specific cDNA, cloned by differential display PCR (DDPCR) and rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Wang, X; Brownstein, M J; Young, W S

    1997-05-16

    Synthesis of melatonin in the mammalian pineal gland is regulated by the rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (SNAT) and other unknown factors. To screen for pineal-specific mRNAs potentially involved in melatonin synthesis and/or regulation, differential display PCR (DDPCR) was employed. We used 80 primer pairs and examined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochemical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the coding region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a recently identified human lung endothelial cell-specific gene, ESM-1. Interestingly, not only the amino acid sequences but also the cDNA sequences, including the long 3' untranslated regions, are highly similar. This suggests that the conserved 3' untranslated region may carry information to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) level compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potentially full-length cDNAs with restricted expression in anatomically complex regions of the brain. This protocol combining several existing methodologies is suitable for use with limited tissue sources and uses minimal amounts of isotopes.

  12. 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.

  13. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    Energy Technology Data Exchange (ETDEWEB)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  14. An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

    Science.gov (United States)

    Butterfield, Yaron S. N.; Marra, Marco A.; Asano, Jennifer K.; Chan, Susanna Y.; Guin, Ranabir; Krzywinski, Martin I.; Lee, Soo Sen; MacDonald, Kim W. K.; Mathewson, Carrie A.; Olson, Teika E.; Pandoh, Pawan K.; Prabhu, Anna-Liisa; Schnerch, Angelique; Skalska, Ursula; Smailus, Duane E.; Stott, Jeff M.; Tsai, Miranda I.; Yang, George S.; Zuyderduyn, Scott D.; Schein, Jacqueline E.; Jones, Steven J. M.

    2002-01-01

    We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites. PMID:12034834

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

    Institute of Scientific and Technical Information of China (English)

    Rong Long Jiang; Qiao Sheng Lu; Kang Xian Luo

    2000-01-01

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

  16. Cloning a cDNA Encoding Ribosomal Protein S25 from Amaranthus cruentus L.%籽粒苋(Amaranthus cruentus L.)核糖体蛋白S25基因(cDNA)的克隆及其表达分析

    Institute of Scientific and Technical Information of China (English)

    徐芳秀; 江树业; 等

    2001-01-01

    @@ Ribosomes, the agents of protein synthesis, consist of roughly equal amounts of RNA (rRNA) and protein (r-protein). Knowledge of the ribosome and its function mainly comes from the extensive work on 70S bacterial ribosomes. There are 21 proteins in the small (30S) subunit and 30 in the large (50S) subunit in E. coil ri bosomes. The 80S eukaryotic ribosomes are more com plex than the bacterial ones and contain at least 30 pro teins in the small (40S) subunit and 40 in the large (60 S) subunit. These r-proteins are named S1 to S30 and L1 to L40 according to whether they arise from the small or large subunit, and to their mobility in gels. In plants, several ribosomal protein genes and/or cDNAs have been isolated, such as the small subunit proteins S 11, S13, S14, S16, and S19 and the large subunit proteins L2, L7, L17, and L27. Here we report the r-protein S25 cDNA, Arps25, from Amaranthus cruentus L.

  17. 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.

  18. 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.

  19. 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.

  20. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA va

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 min. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable ehitinases so far isolated in fungi. Ca2+, Ba2+, Na2+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, Hg2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplieation. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.

  2. Cloning and Expressing of a Gene Encoding Cytosolic Copper/Zinc Superoxide Dismutase in the Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    HU Gen-hai; YU Shu-xun; FAN Shu-li; SONG Mei-zhen

    2007-01-01

    In this study, a gene encoding a superoxide dismutase (SOD) was cloned from senescent leaves of cotton (Gossypium hirsutum), and its expressing profile was analyzed. The gene was cloned by rapid amplification of cDNA ends (RACE)method. Northern blotting was used to show the profile of the gene expression, and the enzyme activity was mensurated by NBT deoxidization method in different growth periods. The full length of a gene of cytosolic copper/zinc superoxide dismutase (Cu/Zn-SOD) was isolated from cotton (GenBank Accession Number: DQ445093). The sequence of cDNA contained 682 bp, the opening reading frame 456 bp, and encoded polypeptide 152 amino acids with the predicted molecular mass of 15.03 kD and theoretical pI of 6.09. The amino acid sequence was similar with the other plants from 82 to 87%. Southern blotting showed that the gene had different number of copies in different cotton species. Northern blotting suggested that the gene had different expression in different tissues and development stages. The enzyme activity was the highest in peak flowering stage. The cotton cytosolic (Cu/Zn-SOD) had lower copies in the upland cotton. The copper/zinc superoxide dismutase mRNA expressing level showed regular changing in the whole development stages; it was lower in the former stages, higher in latter stages and the highest at the peak flowering stage. The curve of the copper/zinc superoxide dismutase mRNA expressing level was consistent with that of the Cu/Zn-SOD enzyme activity.The copper/zinc superoxide dismutase mRNA expressing levels of different organs showed that the gene was higher in the root, leaf, and lower in the flower.

  3. Characterization and expression of two cDNA encoding 3-Hydroxy-3-methylglutaryl coenzyme A reductase isoforms in coffee (Coffea arabica L.).

    Science.gov (United States)

    Tiski, Iris; Marraccini, Pierre; Pot, David; Vieira, Luiz Gonzaga Esteves; Pereira, Luiz Filipe Protasio

    2011-10-01

    In higher plants there are two independent pathways for isoprenoid biosynthesis, located in the cytosol (mevalonic acid or MVA pathway) or in the plastids [methylerythritol phosphate (MEP) pathway]. The 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) is the first committed step in the MVA pathway. Using the information available from the Brazilian Coffee Genome Project, we found 13 ESTs that originated two isoforms, CaHMGR1 and CaHMGR2, for the enzyme HMGR of Coffea arabica. A complementary DNA encoding the isoform CaHMGR1 was cloned, and its complete nucleotide sequence determined. The full-length cDNA of CaHMGR1 was 2,242 bp containing a 1,812-bp ORF encoding 604 amino acids. Bioinformatic analyses revealed that the deduced CaHMGR1 had extensive homology with other plant HMGRs and contained two transmembrane domains and two putative HMGR binding sites and two NADP(H)-binding sites. Under normal growth conditions, transcripts of isoform CaHMRG1 were detected in fruit tissues (pulp, perisperm, and endosperm) only at the initial stages of development, flower buds and leaves. CaHMRG2 was expressed in all tissues and during all fruit development stages examined. These results suggest a constitutive expression of isoform CaHMGR2, while the isoform CaHMGR1 shows temporal and tissue-specific transcriptional activation.

  4. Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Zheng, Hongying; Xiao, Caili; Han, Kelei; Peng, Jiejun; Lin, Lin; Lu, Yuwen; Xie, Li; Wu, Xiaohua; Xu, Pei; Li, Guojing; Chen, Jianping; Yan, Fei

    2015-11-01

    The complete 6243-nucleotide sequence of a cucumber green mottle mosaic virus (CGMMV) isolate from bottle gourd in Zhejiang province, China, was determined. A full-length cDNA clone of this isolate was constructed by inserting the cDNA between the 35S promoter and the ribozyme in the binary plasmid pCB301-CH. A suspension of an Agrobacterium tumefaciens EHA105 clone carrying this construct was highly infectious in Nicotiana benthamiana and bottle gourd. Another infectious clone containing the green fluorescence protein (GFP) reporter gene was also successfully constructed. This study is the first report of the efficient use of agroinoculation for generating CGMMV infections.

  5. cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients.

    Science.gov (United States)

    Heckel, D; Brass, N; Fischer, U; Blin, N; Steudel, I; Türeci, O; Fackler, O; Zang, K D; Meese, E

    1997-11-01

    There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.

  6. Cloning of genes encoding nonhost hypersensitive response-inducing elicitors from Phytophthora boehmeriae

    Institute of Scientific and Technical Information of China (English)

    LI Jun; ZHANG HaiFeng; ZHANG ZhengGuang; WANG YuanChao; ZHENG XiaoBo

    2007-01-01

    We have devised a high-throughput functional cloning method to isolate cDNAs from Phytophthora boehmeriae of which the products elicit a hypersensitive response (HR) in tobacco. The cDNAs were cloned into a binary potato virus X (PVX)-based expression vector and transformed into Agrobacterium tumefeciens (Mog101). 4100 colonies were individually toothpick-inoculated onto leaflets of Nicotiana benthamiana. 12 cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. 7 of these clones have different sequences. One of these clones PBC43 encodes specific elicitin. Clone PBC163 encodes a protein highly homologous to Rab; PBC241 encodes a prohibitin protein; PBN62 encodes a Heat Shock Protein 60 (HSP60). The other five cDNAs reveal no homology to known protein and are thus considered novel. These observations suggest that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode elicitors and other HR-inducing proteins.

  7. 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 fra...

  8. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

    Science.gov (United States)

    Santhanam, R; Naz, R K

    2001-09-01

    A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

  9. 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.

  10. 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.

  11. Completion sequence and cloning of the infectious cDNA of a chb isolate of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Zhong, M; Zhao, X; Liu, Y; Wang, Y; Cao, K

    2015-03-01

    Cucumber green mottle mosaic virus (CGMMV) is an important and widespread seed-borne virus that infects Cucurbitaceous plants. It is a member of the genus Tobamovirus in the family Virgaviridae with a monopartite (+) ssRNA genome. Here we report the complete genome sequence, construction and testing of the infectious clones of a chb isolate of CGMMV. Full-length CGMMV cDNA was cloned into the vector pUC19. The linearized vector containing full-length cDNA was used as template for in vitro transcription, and the synthesized capped transcript was highly infectious in Chenopodium amaranticolor and cucumber (Cucumis sativus). Inoculated plants showed symptoms typical of CGMMV infection. The infectivity was confirmed by mechanical transmission to new plants, RT-PCR and western blot. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first detailed report of a biologically active transcript from CGMMV.

  12. 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...

  13. Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

    Science.gov (United States)

    Mieszczanek, J; Kofta, W; Wedrychowicz, H

    2000-12-01

    The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

  14. Cloning a Chymotrypsin-Like 1 (CTRL-1 Protease cDNA from the Jellyfish Nemopilema nomurai

    Directory of Open Access Journals (Sweden)

    Yunwi Heo

    2016-07-01

    Full Text Available An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita or Hydrozoan (Hydra vulgaris. The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT and 3′ acceptor splice sequences (AG are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

  15. 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).

  16. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  17. Human Vascular Endothelial Growth Factor cDNA Cloning and Expression in Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Human vascular endothelial growth factor (VEGF) cDNA was amplified by nested polymerase chain reaction method from the HL60 cells. Then a pCD-hVEGF165 recombinant plasmid was constructed. Rabbit osteoblasts were transfected with pCD-hVEGF165 plasmid by lipofectin mediated gene transfer. The transient expressive results were detected by immunohistochemical method. It was observed that the expression of human VEGF gene was detected 72 h after transfecting distinctly.

  18. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

    Science.gov (United States)

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L

    1988-09-01

    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  19. [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.

  20. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

  1. 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.

  2. Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

    Science.gov (United States)

    Shin, Seung-Yong; Lee, Haeng-Soon; Kwon, Suk-Yoon; Kwon, Soon-Tae; Kwak, Sang-Soo

    2005-01-01

    Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

  3. Cloning and expression of AtPLC6, a gene encoding a phosphatidylinositol-specific phospholipase C in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    XU Xiaojing; CAO Zhixiang; LIU Guoqin; Madan K. Bhattacharrya; REN Dongtao

    2004-01-01

    A full-length eDNA clone corresponding to a putative phosphatidylinositol-specific phospholipase C (PIPLC) was isolated from Arabidopsis thaliana by screening a cDNA library and using RT-PCR strategy. The cDNA, designated AtPLC6, encodes a putative polypeptide of 578 amino acid residues with a calculated molecular mass of 66251.84 D and a pI of 7.24. The sequence analysis indicates that the polypeptide contains X, Y, EF-hand and C2 domains. The overall structure of putative AtPLC6 protein, like other plant PI-PLCs, is most similar to that of mammalian PLC& The recombinant AtPLC6 protein expressed in E. coli was able to hydrolyze phosphatidylinositol 4,5-biophosphate (PIP2) to generate inositol 1,4,5-trisphate (IP3) and 1,2-diacylglycerol (DAG). The protein hydrolyzes PIP2 in a Ca2+-dependent manner and the optimum concentration of Ca2+ is 10 μmol/L.These results suggested that AtPLC6 gene encodes a genuine PI-PLC. Northern blot analysis showed that the AtPLC6 gene is expressed at low level in all examined tissues, such as roots,stems, leaves, flowers, siliques and seedlings under normal growth conditions. The gene is strongly induced under low temperature and weakly induced under various stresses,such as ABA, high-salt stress and heat. These results suggested that AtPLC6 might be involved in the signal-transduction pathways of cold responses of the plants.

  4. Isolation and Expression of a cDNA Encoding Methylmalonic Aciduria Type A Protein from Euglena gracilis Z

    Directory of Open Access Journals (Sweden)

    Fumio Watanabe

    2013-02-01

    Full Text Available In animals, cobalamin (Cbl is a cofactor for methionine synthase and methylmalonyl-CoA mutase (MCM, which utilizes methylcobalamin and 5′-deoxyadenosylcobalamin (AdoCbl, respectively. The cblA complementation class of inborn errors of Cbl metabolism in humans is one of three known disorders that affect AdoCbl synthesis. The gene responsible for cblA has been identified in humans (MMAA as well as its homolog (meaB in Methylobacterium extorquens. Recently, it has been reported that human MMAA plays an important role in the protection and reactivation of MCM in vitro. However, the physiological function of MMAA is largely unknown. In the present study, we isolated the cDNA encoding MMAA from Euglena gracilis Z, a photosynthetic flagellate. The deduced amino acid sequence of the cDNA shows 79%, 79%, 79% and 80% similarity to human, mouse, Danio rerio MMAAs and M. extorquens MeaB, respectively. The level of the MCM transcript was higher in Cbl-deficient cultures of E. gracilis than in those supplemented with Cbl. In contrast, no significant differences were observed in the levels of the MMAA transcript under the same two conditions. No significant difference in MCM activity was observed between Escherichia coli that expressed either MCM together with MMAA or expressed MCM alone.

  5. Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

    Science.gov (United States)

    Muñoz, Rosario; García, José L.; Carrascosa, Alfonso V.; Gonzalez, Ramon

    2004-01-01

    Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy. PMID:15128507

  6. 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.

  7. Molecular cloning and expression analysis of cDNAs encoding androgenic gland hormone precursors from two porcellionidae species, Porcellio scaber and P. dilatatus.

    Science.gov (United States)

    Ohira, Tsuyoshi; Hasegawa, Yuriko; Tominaga, Satoshi; Okuno, Atsuro; Nagasawa, Hiromichi

    2003-01-01

    Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber AGH (Pos-AGH) and P. dilatatus AGH (Pod-AGH) were amplified by RT-PCR using degenerate oligonucleotide primers designed based on the amino acid sequence of A. vulgare AGH (Arv-AGH). Subsequently, full length cDNAs were obtained by 5'- and 3'-RACE. Both AGH precursors consisted of a signal peptide, B chain, C peptide and A chain, and exhibited the same organization as that of Arv-AGH. The amino acid sequences of the A and B chains, which comprise mature AGH peptide, were highly conserved among the three species, while that of the C peptide showed only low sequence similarity. In Northern blot analysis, each cDNA fragment used as a probe specifically hybridized with a single band (0.75 kb) in mRNA extracted from whole male reproductive organs. In analysis of the tissue-specific gene expression of these two AGHs by RT-PCR, it was revealed that both AGH transcripts were detected only in cDNA synthesized using total RNA from the testis carrying the androgenic glands, but not in that from testis only, seminal vesicle, vas deferens, or hepatopancreas.

  8. 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

    protein kinase phosphorylation sites were predicted using online software NetPhosK 1 and NetPhos 2. Sequence analysis results showed that the rainbow trout Ndufb2 cDNA was 899 bp in length,5'untranslated region(5'UTR) 1 52 bp,3'untranslated region(3'UTR)441 bp,open reading frame(ORF) 306 bp,encoding 101 amino acids.The protein molecular mass was 1 1.4 ku and isoelectric point was 5.31.This gene sequence had been submitted to the GenBank database (accession number:FJ534641).Mitochondrial target sequence of 50 amino acid motif appeared from the 1st to 50th amino acid site.Two antigenic sites (65-ILWHCWHDPD-74 and 23-QKIVIRK-29) of the Ndufb2 protein were found,and one serine phosphorylation site (aa49),two threonine phosphorylation sites (aa1 5,aa47) and seven kinase phosphorylation sites were also found.Sequence alignment exhibited 98% identity of amino acids between O.mykiss and Salmo salar ,and more than 55% identity of amino acids between O.mykiss and mammal.Phylogenetic tree showed that the Ndufb2 of rainbow trout had the closest relationship with those of Atlantic salmon and zebrafish,then the amphibians,birds,marsupials and mammals. The phylogenetic analysis showed that the constructed Ndufb2 protein phylogenetic tree was consistent with the traditional species classification tree. The above results indicate that the Ndufb2 gene is relatively conservative in the progress of evolution and play an important role in electron transport process of mitochondrial respiratory chain.The cloned full-length cDNA sequence of mitochondrial respiratory chain gene Ndufb2 of rainbow trout lays a theoretical foundation for the structure and function of clarifying fish mitochondrial complex I.%从虹鳟鱼(Oncorhynchus mykiss)的脑组织中提取 RNA,经逆转录聚合酶链反应及 cDNA 末端快速扩增技术克隆出 Ndufb2基因全长 cDNA 序列(GenBank 登录号:FJ534641),并对其序列进行分析。扩增结果表明:Ndufb2基因的 cDNA 序列全长899 bp

  9. 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.

  10. 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.

  11. Follicle cell trypsin-like protease HrOvochymase: Its cDNA cloning, localization, and involvement in the late stage of oogenesis in the ascidian Halocynthia roretzi.

    Science.gov (United States)

    Mino, Masako; Sawada, Hitoshi

    2016-04-01

    We previously reported that the sperm trypsin-like protease HrAcrosin and its precursor HrProacrosin participate in fertilization of the ascidian Halocynthia roretzi. The HrProacrosin gene is annotated in the H. roretzi genome database as Harore.CG.MTP2014.S89.g15383; our previously reported sequence of HrProacrosin gene appeared to include four nucleotides inserted near the 3'-end of HrProacrosin, resulting in a frame-shift mutation and a premature termination codon. The gene architecture of HrProacrosin and Harore.CG.MTP2014.S89.g15383 resembles that of Xenopus laevis ovochymase-1/OVCH1 and ovochymase-2/OVCH2, which encode egg extracellular polyproteases. Considering these new observations, we evaluated the cDNA cloning, expression, localization, and function of Harore.CG.MTP2014.S89.g15383, herein designated as HrOvochymase/HrOVCH. We found that HrOVCH cDNA consists of a single open reading frame of 1,575 amino acids, containing a signal peptide, three trypsin-like protease domains, and six CUB domains. HrOVCH was transcribed by the testis and ovary, but the majority of protein exists in ovarian follicle cells surrounding eggs. An anti-HrOVCH antibody inhibited elevation of the vitelline coat at a late stage of oogenesis, during the period when self-sterility is acquired. As trypsin inhibitors are reported to block the acquisition of self-sterility during oogenesis, whereas trypsin induces the acquisition of self-sterility and elevation of the vitelline coat in defolliculated ovarian eggs, we propose that HrOVCH may play a role in the acquisition of self-sterility by late-stage H. roretzi oocytes.

  12. Cloning, characterization and expression of $OsFMO_{(t)}$ in rice encoding a flavin monooxygenase

    Indian Academy of Sciences (India)

    Jicai Yi; Lanna Liu; Youpei Cao; Jiazuo Li; Mantong Mei

    2013-12-01

    Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of $OsFMO_{(t)}$, a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. $OsFMO_{(t)}$ was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a ‘G×××G’ characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO$_{\\text{(t)}}$ and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of $OsFMO_{(t)}$ in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO$_{\\text{(t)}}$ protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of $OsFMO_{(t)}$ was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by $OsFMO_{(t)}$ is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

  13. 蓝太阳鱼生长激素全长cDNA的克隆与序列分析%Cloning and sequencing of full length growth hormone cDNA from Lepomis cyanellus

    Institute of Scientific and Technical Information of China (English)

    曹运长; 李文笙; 叶卫; 林浩然

    2004-01-01

    The full length cDNA encoding growth hormone of a freshwater fish, Lepomis cyanellus, (LcGH) was cloned from pituitary RNA with RT-PCR, 3' and 5' RACE (rapid amplification of cDNA ends). The LcGH cDNA (Genbank No. AY530822), about 989nt (nucleotide) long, consisted of a open reading frame with 615nt long, 5'and 3'untranslated regions with 93nt and 224nt long respectively, and a 57nt poly (A) tail. The DNA sequence analysis showed that there are typical Kozak sequence and polyadenylation signal. The pregrowth hormone peptide of 204aa deduced from LcGH cDNA included a putative signal peptide (17aa) locating in its Nterminal. There exist a Asn-Cys-Thr glycosylation site at amino acid 201, and 4 cysteine residues (No. 69, 177, 194, 202) that are essential to construct two S-S bonds in this pregrowth hormone peptide. Homological comparision among LcGH and other species growth hormones showed that There is high homology (more than 85%) between growth hormone of Lepomis cyanellus and that of most perciformes fish, but low homology (less than 70%) in comparison with other species such as Siluriformes and Cypriniformes fish.

  14. 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.

  15. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

    OpenAIRE

    Jiang, H P; Serrero, G

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  16. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein

    OpenAIRE

    Jiang, Hui-Ping; Serrero, Ginette

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  17. Molecular cloning, characterization, and expression analysis of a gonadotropin-releasing hormone-like cDNA in the clam, Ruditapes philippinarum.

    Science.gov (United States)

    Song, Ying; Miao, Jingjing; Cai, Yuefeng; Pan, Luqing

    2015-11-01

    Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in vertebrates. In the present study, we have cloned and identified the cDNA sequence of the prepro-GnRH (rp-GnRH) in the Manila clam Ruditapes philippinarum. The open reading frame encodes a protein of 94 amino acids, which consists of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. These deduced peptides were highly homologous to that reported for other molluscs. We used temperature control to promote gonadal development of the clams. The mRNA expression of rp-GnRH in the visceral ganglia from clams at different reproductive stages was determined by quantitative RT-PCR. The levels of steroids progesterone (P), testosterone (T), and estradiol-17β (E2) in the hemolymph of the corresponding clam were measured by ELISA. The rp-GnRH mRNA was highly expressed at the gonadal early development stage. Concentrations of P, T, and E2 also increased at the early development stage in both sexes. Positive significant correlations between rp-GnRH expression and P content as well as between rp-GnRH expression and T content were observed throughout the gonadal maturation. The results from this study may help to better understand the physiological functions of the native forms of GnRH-like peptides and the actions of GnRH on sex steroids release in bivalve molluscs.

  18. A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

    Science.gov (United States)

    Finkelstein, A; Kostrub, C F; Li, J; Chavez, D P; Wang, B Q; Fang, S M; Greenblatt, J; Burton, Z F

    1992-01-30

    RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

  19. 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.

  20. Purification, characterization and cDNA cloning of a novel lipopolysaccharide-binding lectin from the shrimp Penaeus monodon.

    Science.gov (United States)

    Luo, Tian; Yang, Haijie; Li, Fang; Zhang, Xiaobo; Xu, Xun

    2006-01-01

    In invertebrates, C-type lectin plays an important role in innate immunity by mediating the recognition of pathogens to host cells and clearing microinvaders. A few C-type lectins have been identified from shrimps, but none of their gene or protein sequences is known to date. In this paper, a C-type lectin (named PmLec) specific for bacterial lipopolysaccharide was purified from the serum of the shrimp Penaeus monodon. The binding of PmLec to lipopolysaccharide was mainly mediated through the O-antigen. PmLec had a strong hemagglutinating and bacterial-agglutinating activity as well as an opsonic effect that enhances hemocyte phagocytosis. The PmLec cDNA sequence was obtained from the cDNA library of P. monodon by polymerase chain reaction with the degenerated primer designed according to the amino-terminal residue sequence of purified PmLec. A 546-bp open reading frame was found to encode a putative protein comprising 182 amino acids and containing a preceding signal peptide of 17 amino acids. A C-type lectin domain existed in PmLec, but no glycosylation site was found. The recombinant PmLec protein expressed in Escherichia coli also showed the same agglutinating activity and opsonic effect as that of the native protein. This is the first report of a lectin cDNA from the shrimp. PmLec functions as a pattern-recognition protein and an opsonin in the shrimp, and it provides a clue to elucidate the role of lectin in the innate immunity of aquatic invertebrates at the molecular level.

  1. CDNA CLONING OF FATHEAD MINNOW (PIMEPHALES PROMELAS) ESTROGEN AND ANDROGEN RECEPTORS FOR USE IN STEROID RECEPTOR EXTRAPOLATION STUDIES FOR ENDOCRINE DISRUPTING CHEMICALS

    Science.gov (United States)

    cDNA Cloning of Fathead minnow (Pimephales promelas) Estrogen and Androgen Receptors for Use in Steroid Receptor Extrapolation Studies for Endocrine Disrupting Chemicals. Wilson, V.S.1,, Korte, J.2, Hartig P. 1, Ankley, G.T.2, Gray, L.E., Jr 1, , and Welch, J.E.1. 1U.S...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum

    Directory of Open Access Journals (Sweden)

    Chengsheng Zhang

    2016-05-01

    Full Text Available Polygalacturonase inhibiting proteins (PGIPs are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum, hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5′-untranslated region (UTR, and 209 bp 3′-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP’s deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA, abscisic acid (ABA, salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3. Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.

  8. Cloning and sequencing of a DNA fragment encoding N37 apoptotic peptide derived from p53

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP,which could induce tumor cell apoptosis. To further explore the function of N37,we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database,the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apopto...

  9. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

    Science.gov (United States)

    Sung, Y C; Parsell, D; Anderson, P M; Fuchs, J A

    1987-06-01

    The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.

  10. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

    OpenAIRE

    Sung, Y C; Parsell, D; Anderson, P. M.; Fuchs, J A

    1987-01-01

    The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.

  11. Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli

    DEFF Research Database (Denmark)

    Harlow, Kenneth W.; Nygaard, Per; Hove-Jensen, Bjarne

    1995-01-01

    The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell...... analysis established the subunit Mr as 43,500. Primer extension analysis indicated the presence of an adequate Pribnow box and suggested that the transcript contained a 110-base leader sequence. Strains harboring the gsk gene on multicopy plasmids overexpressed both guanosine and inosine kinase activities....... N-terminal sequence and amino acid composition analyses of the 43,500-Mr polypeptide band confirmed the correct reading frame assignment and the identity of this band as the gsk gene product. Comparison of the amino acid sequence with the protein database revealed similarity to regions of other...

  12. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F. William (Stony Brook, NY); Dubendorff, John W. (Sound Beach, NY)

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  13. Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F. William (Stony Brook, NY); Dubendorff, John W. (Sound Beach, NY)

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  14. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F.W.; Dubendorff, J.W.

    1998-10-20

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  15. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F.W.; Dubendorff, J.W.

    1998-11-03

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

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

    Science.gov (United States)

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

    2008-02-01

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

  17. Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene

    Energy Technology Data Exchange (ETDEWEB)

    Tebbs, R.S.; Tucker, J.D.; Hwang, M. [Lawrence Livermore National Lab., CA (United States)] [and others

    1995-07-03

    The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to displatin and {gamma}-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is {approx} 2.5 kb and detects an {approx} 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels. 30 refs., 5 figs., 2 tabs.

  18. 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.

  19. Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.

  20. Cloning and Expression Pattern of a Gene Encoding a Putative Plastidic ATP/ADP Transporter from Helianthus tuberosus L.

    Institute of Scientific and Technical Information of China (English)

    Kun MENG; Tuan-Jie CHANG; Xiang LIU; Song-Biao CHEN; Yong-Qin WANG; Ai-Jun SUN; Hong-Lin XU; Xiao-Li WEI; Zhen ZHU

    2005-01-01

    Herein, we report the cloning and molecular characterization of a full cDNA encoding a putative plastidic ATP/ADP transporter, designated HtAATP, for Helianthus tuberosus L. The ATP/ADP translocator protein was isolated from the tuber-cDNA library of H. tuberosus for the first time. The predicted HtAATP protein was judged as a plastidic ATP/ADP translocator protein from its high homology at the amino acid sequence level to the two Arabidopsis thaliana plastidic ATP/ADP translocator proteins AATP1 and AATP2 (84.8% and 79.9% identity, respectively). Amino acid sequence analysis of the primary structure of HtAATP revealed that it belonged to the plastidic ATP/ADP transporter family. Hydropathy prediction indicated that HtAATP gene product is a highly hydrophobic membrane protein that contains 10 transmembrane domains to form a spanning topology. Southern blotting analysis showed that the HtAATP gene is a single-copy gene in the H. tuberosus genome. Tissue distribution analysis showed that the HtAATP gene is prominently expressed in sink tissues. A stable expression pattern in tubers at different developmental stages implies an active involvement of HtAATP during carbohydrate formation.

  1. 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

  2. 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.

  3. Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

    Science.gov (United States)

    1988-10-31

    fractionated by electrophoresis on an agarose gel. The cDNA fragments were cloned at the EcoRl site of pUCI 8 (Vieira and Messing, 1982...the viral RNA species of prototype dengue viruses. Amer. J. Trop. Med. Hyg. 29 (1980) 643-652. Vicira, J. and Messing, J.: The pUC plasmids , ain

  4. 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.

  5. Cloning and sequence analysis of gene encoding plasma aquaporin of Tamarix albiflonum

    Institute of Scientific and Technical Information of China (English)

    DONG Yuzhi; YANG Chuanping; ZHANG Daoyuan; WANG Yucheng

    2007-01-01

    Plant aquaporins are water-selected-channels in plants and are involved in seed germination,cell elongation,stoma movement,fertilization and so on.Some plant aquapotins also play an important role in drought stress response.In this paper,the gene encoding the Tamarix albiflonum Aquaporin (AQP) was amplified by 5'rapid amplification of cDNA end (RACE) on the basis of the sequence information obtained from the expressed sequence tag of the subtractive hybridization library constructed under PEG6000 stress.The cDNA of the T.albiflonum AQP gene is 1,043 bp long,encoding a protein of 287 amino acids with a predicted molecular mass of 30.9 kDa,has 6 transmembrane regions,and possessing the major intrinsic protein (MIP) family signal consensus sequence SGXHXNPAVT and the higher plant plasma membrane intrinsic protein (PIP) highly conservative sequence GGGANXXXXGY and TGI/TNPARSL /FGAA I/VI/VF/YN.A comparative molecular analysis of the nucleotide sequence in National Center for Biotechnology Information (NCBI) databases showed that it shared 95% homology with the gene ofArabidopsis thaliana (MIP-C),with a theoretical isoelectric point 8.84.

  6. cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula.

    Science.gov (United States)

    Rahman, Mohammad Matiur; Wang, Ling; Inoue, Akira; Ojima, Takao

    2012-10-01

    Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.

  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. 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.

  9. Properties and cDNA cloning of an antihemorrhagic factor (HSF) purified from the serum of Trimeresurus flavoviridis.

    Science.gov (United States)

    Deshimaru, Masanobu; Tanaka, Chie; Fujino, Kazuya; Aoki, Narumi; Terada, Shigeyuki; Hattori, Shosaku; Ohno, Motonori

    2005-12-15

    Habu serum factor (HSF) is a metalloproteinase inhibitor that is isolated from the serum of habu snake (Trimeresurus flavoviridis), and it can suppress snake venom-induced hemorrhage. In the present study, the inhibitory property and fundamental structure of HSF were analyzed in detail. HSF inhibited all the hemorrhagic and most of the non-hemorrhagic metalloproteinases tested from the venoms of T. flavoviridis and Gloydius halys brevicaudus. HSF was extremely stable in a broad range of temperature and pH, and the treatments with a temperature of 100 degrees C or pH ranging from 1 to 13 barely affects its reactivity against G. halys brevicaudus H6 protease. Gel filtration chromatography revealed that HSF binds to the H6 protease with a 1:1 molar ratio. A secondary structure profile of HSF that was monitored by circular dichroism spectrum remained unvaried up to 2 M urea. The activity of HSF was stoichiometrically abolished by chemical modification with 2,4,6-trinitrobenzene sulfonic acid and N-bromosuccinimide; this indicates that Lys and Trp residues in its sequence play a role in the inhibitory mechanism. In this study, the amino acid sequence of HSF that was obtained by cDNA cloning was identical to that reported previously, except for five substitutions. We concluded that these discrepancies reflect a difference in the places of capture of the snake specimens.

  10. The murine decorin. Complete cDNA cloning, genomic organization, chromosomal assignment, and expression during organogenesis and tissue differentiation.

    Science.gov (United States)

    Scholzen, T; Solursh, M; Suzuki, S; Reiter, R; Morgan, J L; Buchberg, A M; Siracusa, L D; Iozzo, R V

    1994-11-11

    Decorin, a proteoglycan known to interact with collagen and growth factors, may play key roles during ontogenesis, tissue remodeling, and cancer. We have deciphered the complete protein sequence of the murine decorin by cDNA cloning, elucidated its gene structure and chromosomal location, and investigated its expression in the developing embryo. The decorin protein and the gene were highly conserved vis à vis the human counterpart; however, the murine gene lacked a leader exon, exon Ib, which was found only in the human. Using interspecific backcrossing, we assigned the gene to chromosome 10 just proximally to the Steel gene locus. In situ hybridization studies of developing mouse embryos showed a distinct pattern of expression with a progressive increase of decorin mRNA during ontogenesis. At early stages (day 11 postconception), decorin was detectable only in the floor plate region. Subsequently (days 13-16 postconception), decorin expression was especially prominent in the meninges and mesothelial linings of pericardium, pleura, and coelomic cavity, as well as in the dermis and subepithelial layers of the intestine and urinary bladder. In contrast, the major parenchymal organs were only weakly positive for decorin mRNA. These findings suggest that decorin may play a role in epithelial/mesenchymal interactions during organ development and shaping.

  11. cDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli.

    Science.gov (United States)

    Han, Long; Cao, Min-Jie; Shi, Chao-lan; Wei, Xiao-Nan; Li, Huan; Du, Cui-Hong

    2014-02-01

    Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation.

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

    NARCIS (Netherlands)

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

    1986-01-01

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

  13. 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

  14. 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

  15. Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae.

    Science.gov (United States)

    Kawakami, K; Toma, C; Honma, Y

    2000-01-01

    A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16-amino acid signal peptide followed by a large propeptide. The N-terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS-PAGE, suggesting that further processing at the C-terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10-phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.

  16. Molecular cloning and characterization of an α-amylase cDNA highly expressed in major feeding stages of the coffee berry borer, Hypothenemus hampei.

    Science.gov (United States)

    Bezerra, C A; Macedo, L L P; Amorim, T M L; Santos, V O; Fragoso, R R; Lucena, W A; Meneguim, A M; Valencia-Jimenez, A; Engler, G; Silva, M C M; Albuquerque, E V S; Grossi-de-Sa, M F

    2014-12-10

    α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies.

  17. 佛手GRAS基因的克隆及表达分析%On cDNA cloning and expression analysis of GRAS gene in fingered citron

    Institute of Scientific and Technical Information of China (English)

    石瑞; 曹诣斌; 陈文荣; 郭卫东

    2011-01-01

    A fragment of EST was isolated from fingered citron by suppression subtractive hybridization. It was cloned the full-length cDNA of the EST by RACE. The result showed that this fragment was highly associated with GRAS genes of Arabidopsis and the sequence alignment showed that it had a conserved domain of GRAS. Therefore, the GRAS gene of fingered citron could be named as CmsGRAS ( GenBank accession number: JF440647) , which had an ORF of 1 236 bp and encoded 413 amino acids. Fluorescence quantitative PCR method was used to study the expression characteristics of GRAS gene in fingered citron under low temperature stress, the results showed that the gene expression significantly changed after injured by low temperature.%利用抑制消减杂交法从芸香科柑橘属不耐寒植物佛手中分离得到了一个表达序列标签(EST)片段,结合RACE技术克隆获得该基因的1 669 bp序列,编码区长1 236 bp,编码413个氨基酸.通过Blastn同源序列比对分析,结果显示该基因与拟南芥已知的GRAS基因同源性较高;与GenBank数据库比对分析,表明该基因具有GRAS特有的保守结构域.因此,命名该基因为:CmsGRAS(GenBank登录号:JF440647).用荧光定量聚合酶链式反应(qPCR)的方法研究了该基因在低温胁迫处理下的表达特性,结果显示该基因在低温胁迫后的表达量有明显的变化.

  18. 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.

  19. 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.

  20. Construction, cloning, and expression of synthetic genes encoding spider dragline silk.

    Science.gov (United States)

    Prince, J T; McGrath, K P; DiGirolamo, C M; Kaplan, D L

    1995-08-29

    Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.

  1. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

    OpenAIRE

    Gronwald, R G; Grant, F J; Haldeman, B A; Hart, C E; O'Hara, P J; Hagen, F S; Ross, R.; Bowen-Pope, D F; Murray, M. J.

    1988-01-01

    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 (approximately ...

  2. Identification and characterization of cDNA sequences encoding the HIS3 and LEU2 genes of the fungus Alternaria tenuissima

    Institute of Scientific and Technical Information of China (English)

    Ying Wan; Xuli Wang; Yun Huang; Dewen Qiu; Linghuo Jiang

    2008-01-01

    Alternaria tenuissima is a fungus widely present in the environment and could cause diseases in plants and humans.In this study,through a yeast genetic approach,cDNA sequences were isolated and characterized for the AtHIS3 and AtLEU2 genes.AtHIS3 cDNA encodes a protein of 238 amino acids,while AtLEU2 cDNA encodes a protein of 363 amino acids.Based on the phylogenetic analysis of amino acid sequences of AtHis3p and AtLeu2p,A.tenuissima is closely related to the plant pathogenic fungus Phaeosphaeria nodorum.This study provides two genetic markers for studies of functions of genes regulating development,morphology,and virulence of A.tenuissima.

  3. 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

  4. Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

    Directory of Open Access Journals (Sweden)

    Murphy Brian R

    2004-10-01

    Full Text Available Abstract Background A dengue virus type 2 (DEN-2 Tonga/74 isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30 in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. Methods A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2 and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice, in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. Results The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. Conclusions The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

  5. 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.

  6. Cloning and Characterization of upp, a Gene Encoding Uracil Phosphoribosyltransferase from Lactococcus lactis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1994-01-01

    Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced...... construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains...

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

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; SUN Xiuqin; ZHANG Jinxing; ZAN Jindong

    2008-01-01

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

  8. Expression in Escherichia coli of cDNA encoding barley beta-amylase and properties of recombinant beta-amylase.

    Science.gov (United States)

    Yoshigi, N; Okada, Y; Sahara, H; Koshino, S

    1994-06-01

    To express the cloned beta-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had beta-amylase activity that produced beta-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley beta-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn't change throughout the incubation. But Western blot analysis found that one beta-amylase having a molecular weight of about 56,000 was synthesized. The recombinant beta-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS-PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant beta-amylase lacked four amino acids at positions 2-5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley beta-amylase. Therefore, the recombinant beta-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant beta-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley beta-amylase) at positions 27-29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant beta-amylase were almost the same as those of barley beta-amylase except for the pI and the Km values for maltohexaose and maltoheptaose.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

    Science.gov (United States)

    Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Pan, Kehou; Pan, Jin; Yang, Guanpin

    2011-03-01

    A gene ( NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae). The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids (LCPUFAs), including eicosapentaenoic acid (20:5n-3, EPA). The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NANOC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid (LA) and α-linolenic acid (ALA).

  10. Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

    Institute of Scientific and Technical Information of China (English)

    SHEN Fafu; YU Shuxun; HAN Xiulan; FAN Shuli

    2004-01-01

    A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)+ trail, and included a putative 5′(98 bp) and a 3′(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pI of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.

  11. 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.

  12. 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.

  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. Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus derived cDNA expression vector.

    NARCIS (Netherlands)

    P.B.G.M. Belt; W. Jongmans; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude); P. van de Putte (Pieter)

    1991-01-01

    textabstractHuman cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this pr

  16. Cloning, Expression Profiling and Functional Analysis of CnHMGS, a Gene Encoding 3-hydroxy-3-Methylglutaryl Coenzyme A Synthase from Chamaemelum nobile

    Directory of Open Access Journals (Sweden)

    Shuiyuan Cheng

    2016-03-01

    Full Text Available Roman chamomile (Chamaemelum nobile L. is renowned for its production of essential oils, which major components are sesquiterpenoids. As the important enzyme in the sesquiterpenoid biosynthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS catalyze the crucial step in the mevalonate pathway in plants. To isolate and identify the functional genes involved in the sesquiterpene biosynthesis of C. nobile L., a HMGS gene designated as CnHMGS (GenBank Accession No. KU529969 was cloned from C. nobile. The cDNA sequence of CnHMGS contained a 1377 bp open reading frame encoding a 458-amino-acid protein. The sequence of the CnHMGS protein was highly homologous to those of HMGS proteins from other plant species. Phylogenetic tree analysis revealed that CnHMGS clustered with the HMGS of Asteraceae in the dicotyledon clade. Further functional complementation of CnHMGS in the mutant yeast strain YSC6274 lacking HMGS activity demonstrated that the cloned CnHMGS cDNA encodes a functional HMGS. Transcript profile analysis indicated that CnHMGS was preferentially expressed in flowers and roots of C. nobile. The expression of CnHMGS could be upregulated by exogenous elicitors, including methyl jasmonate and salicylic acid, suggesting that CnHMGS was elicitor-responsive. The characterization and expression analysis of CnHMGS is helpful to understand the biosynthesis of sesquiterpenoid in C. nobile at the molecular level and also provides molecular wealth for the biotechnological improvement of this important medicinal plant.

  17. Cloning and characterization of a pathogen-induced chitinase in Brassica napus

    DEFF Research Database (Denmark)

    Rasmussen, U.; Bojsen, K.; Collinge, D.B.

    1992-01-01

    A chitinase cDNA clone from rapeseed (Brassica napus L. ssp. oleifera) was isolated. The cDNA clone, ChB4, represents a previously purified and characterized basic chitinase isozyme. The longest open reading frame in ChB4 encodes a polypeptide of 268 amino acids. This polypeptide consists of a 24...

  18. Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato.

    Science.gov (United States)

    Becker, T W; Caboche, M; Carrayol, E; Hirel, B

    1992-06-01

    A full-length cDNA encoding glutamine synthetase (GS) was cloned from a lambda gt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C- and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression

  19. Cloning and sequence analysis of complete gene encoding an alkaline lipase from Penicillium cyclopium.

    Science.gov (United States)

    Zhang, H M; Wu, M C; Guo, J; Li, J F

    2011-01-01

    The complete gene (PG37 lipI) encoding an alkaline lipase (PG37 LipI) was cloned from the genomic DNA of Penicillium cyclopium PG37. The cloned PG37 lipI is 2020 bp in length, consisting of 632 bp of the 5' flanking promoter region and 1388 bp of the downstream fragment that contains 6 exons and 5 short introns. The promoter region harbors putative TATA box, CAAT box and several transcription factor binding sites. The open reading frame (ORF) encodes a PG37 LipI of 285 amino acid residues, which was predicted to contain a 20-aa signal peptide, a 7-aa propeptide and a 258-aa mature peptide with a conserved motif Gly-X-Ser-X-Gly. However, PG37 LipI shows only 32%, 30%, 28% and 26% identity with lipases of Aspergillus parasiticus, Penicillium camembertii, Thermomyces lanuginosus and Rhizomucor miehei, respectively. It was predicted that the main secondary structures of PG37 LipI are alpha-helix and random coil. Three amino acid residues, Ser132-Asp188-His241, compose the enzymatic active center in the tertiary structure.

  20. Cloning and characterization of an endo-b-1,3(4) glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938

    DEFF Research Database (Denmark)

    Villadsen, Ingrid; Bang, M_L; Sandal, T.

    1999-01-01

    We describe the identification and expression cloning of two novel enzymes, a P-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-beta-glucanase (bg1......) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bgl cDNA encodes a 424-residue precursor protein with a putative signal peptide. The prl cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified...

  1. Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

    Directory of Open Access Journals (Sweden)

    Souza Paulo R Eleutério de

    2001-01-01

    Full Text Available The zinc finger motifs (Cys2His2 are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

  2. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber.

    Science.gov (United States)

    Hinman, M B; Lewis, R V

    1992-09-25

    Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account for the amino acid composition of dragline silk. We have isolated a partial cDNA clone for another dragline silk protein (Spidroin 2), demonstrating that dragline silk is composed of multiple proteins. The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1. Spidroin 2 is predicted to consist of linked beta-turns in proline-rich regions which alternate with beta-sheet regions composed of polyalanine segments. This structure for Spidroin 2 provides a model for dragline silk structure and function.

  3. Cloning of full-length cDNA of Microsporum canis membrane protein PQ-loop repeat protein gene%犬小孢子菌膜蛋白PQ-LRP基因全长cDNA的克隆

    Institute of Scientific and Technical Information of China (English)

    庞娟; 祝逸平; 杨国玲

    2012-01-01

    Objective To clone the full-length cDNA of Microsporum canis membrane protein PQ-loop repeat protein (PQ-LRP) gene,so as to investigate the roles of PQ-LRP in the pathogenesis of tinea capitis.Methods A Microsporum canis strain (A518) from a patient with tinea capitis served as the experimental strain.Rapid cDNA end amplification (RACE) was performed to clone the full length cDNA sequence of PQLRP gene.Bioinformatics methods were used to make a preliminary functional analysis of the gene.Results The cDNA of PQ-LRP gene was obtained with a full length of 1522 bp,including the 5' untranslated region (49 bp),coding region (1080 bp) and 3' untranslated region (393 bp).The coding region encoded a protein precursor including 359 amino acid residues.The cloned cDNA of PQ-LRP gene shared an 81% nucleotide identity with that of Trichophyton tonsurans and a 79% nucleotide identity with that of Trichophyton rubrum.Conclusions The full-length cDNA of Microsporum canis membrane protein PQ-LRP gene has been successfully cloned,which will provide an important basis for further researches into the roles of PQ-LRP in Microsporum canis-associated diseases.%目的 克隆犬小孢子菌膜蛋白PQ-LRP(PQ-loop repeat protein)基因全长cDNA,探讨在头癣发病机制中的作用.方法 选用犬小孢子菌头癣株(A518)为实验株,采用cDNA快速末端扩增法(RACE),克隆PQ-LRP基因的全长序列.结合生物信息学方法对获得的序列进行初步功能分析.结果 获得犬小孢子菌PQ-LRP全长序列为1522 bp,拥有一个1080 bp的开放阅读框,编码359个氨基酸,5 '非编码区为49 bp,3 '非编码区为393 bp;同源性比对与断发毛癣菌的PQ-LRP同源性达到81%,与红色毛癣菌PQ-LRP同源性达到79%.结论 克隆出犬小孢子菌膜蛋白PQ-LRP cDNA全长序列,为研究膜蛋白PQ-LRP基因在犬小孢子菌病中的功能奠定基础.

  4. (+)-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.

  5. Properties and cDNA cloning of antihemorrhagic factors in sera of Chinese and Japanese mamushi (Gloydius blomhoffi).

    Science.gov (United States)

    Aoki, Narumi; Tsutsumi, Kadzuyo; Deshimaru, Masanobu; Terada, Shigeyuki

    2008-02-01

    An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein-designated Chinese mamushi serum factor (cMSF)-suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Molecular cloning, characterisation, and tissue distribution of oestrogen receptor alpha in eelpout (Zoarces viviparus)

    DEFF Research Database (Denmark)

    Andreassen, Thomas K; Skjødt, Karsten; Anglade, Isabelle;

    2003-01-01

    A cDNA encoding the eelpout (Zoarces viviparus) oestrogen receptor alpha (eERalpha) has been isolated from eelpout liver, cloned and sequenced. The cDNA contains a complete open reading frame encoding 570 amino acid residues (mw: 63.0 kDa). The amino acid sequence of eERalpha showed a high degree...

  11. A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect alpha-amylase: cDNA cloning and chromosomal location of the gene.

    Science.gov (United States)

    Mena, M; Sanchez-Monge, R; Gomez, L; Salcedo, G; Carbonero, P

    1992-11-01

    A 14.5 kDa barley endosperm protein that is a major allergen in baker's asthma disease, as previously shown by both in vitro (IgE binding) and in vivo tests, has been identified as a glycosylated monomeric member of the multigene family of inhibitors of alpha-amylase/trypsin from cereals. A cDNA encoding this allergen (renamed BMAI-1) has been isolated and characterized. The deduced sequence for the mature protein, which is 132 residues long, is identical in its N-terminal end to the 20 amino acid partial sequence previously determined from the purified allergen, and fully confirms that it is a member of the multigene family of cereal inhibitors. Southern-blot analysis of wheat/barley addition lines using the insert in the BMAI-1 cDNA clone as a probe, has led to the location of the allergen gene (Iam1) in barley chromosome 2, while another related member of this protein family, the barley dimeric alpha-amylase inhibitor BDAI-1 gene (Iad1) has been located in chromosome 6. Iam1 is the first member of this inhibitor family in cereals to be assigned to chromosome group 2, thus extending the dispersion of genes in the family to five out of the seven homology groups of chromosomes in wheat and barley (chromosomes 2, 3, 4, 6 and 7).

  12. 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.

  13. RecA-mediated multistrand formation for cloning PCR products into vectors: simplified process for 5'-rapid amplification of cDNA ends.

    Science.gov (United States)

    Shigemori, Yasushi

    2005-06-01

    I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5'-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.

  14. 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.

  15. 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.

  16. Development and characterization of an infectious cDNA clone of the modified live virus vaccine strain of equine arteritis virus.

    Science.gov (United States)

    Zhang, Jianqiang; Go, Yun Young; Huang, Chengjin M; Meade, Barry J; Lu, Zhengchun; Snijder, Eric J; Timoney, Peter J; Balasuriya, Udeni B R

    2012-08-01

    A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals.

  17. 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.

  18. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

    Science.gov (United States)

    Jaye, M; de la Salle, H; Schamber, F; Balland, A; Kohli, V; Findeli, A; Tolstoshev, P; Lecocq, J P

    1983-04-25

    A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

  19. Isolation of cosmid and cDNA clones in the region surrounding the BTK gene at Xq21.3-q22

    Energy Technology Data Exchange (ETDEWEB)

    Vorechovsky, I.; Zhou, J.N.; Hammarstroem, L. [Karolinska Institute, Huddinge (Sweden)] [and others

    1994-06-01

    A regional physical and transcription map involving yeast artificial chromosomes (YACs), cosmids, and cDNAs has been constructed for Xq21.3-q22 around the gene BTK (formerly atk or BPK) defective in X-linked agammaglobulinemia (XLA). With a positional cloning strategy employing direct cDNA selection, novel cDNAs were found to cluster in the region of approximately 100 kb flanking the XLA and {alpha}-galactosidase A loci. While these widely expressed transcripts are in the area known to contain CpG islands, a less evolutionarily conserved gene, located more than 130 kb distal of DXS178, maps to cosmid clones that could not be digested with rare-cutting restriction enzymes. The presence of transcribed sequences flanking the BTK allowed investigation of their involvement in complex XLA phenotypes. Southern blot analysis using cDNA clones isolated from this region permitted exclusion of a contiguous deletion syndrome as an underlying defect in three patients with XLA and associated growth hormone deficiency. A single XLA patient with torsion dystonia and cosegregating X-linked deafness has been found with a deletion in the 3{prime} part of BTK extending centromerically into the flanking expressed sequence DXS1274E. This suggests a possible involvement of the DXS1274E in this phenotype. The GenBank accession numbers for novel cDNA sequences are as follows: DXS1269E (L20773), DXS1271E (UO1923), DXS1273E (UO1925), and DXS1274E (UO1922). 51 refs., 4 figs., 1 tab.

  20. 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...

  1. 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.

  2. 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.

  3. Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii.

    Science.gov (United States)

    Bae, Jung-Hoon; Sohn, Jung-Hoon; Park, Chang-Seo; Rhee, Joon-Shick; Choi, Eui-Sung

    2004-04-15

    Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P. ciferrii. A positive clone was confirmed by nucleotide sequencing. A syringomycin-E resistance phenotype of a S. cerevisiae sur2-null mutant was complemented by expression of the cloned P. ciferrii SUR2 gene. Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S. cerevisiae SUR2. .

  4. 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.

  5. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Geneva Univ. Medical School (Switzerland); Morris, M.A.; Rossier, C. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  6. Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

    Institute of Scientific and Technical Information of China (English)

    REN Xueying; SUI Zhenghong; ZHANG Xuecheng

    2006-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene (gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  7. Cloning, Bioinformatics Analysis of RERG Gene cDNA in Guizhou Black Goat%贵州黑山羊RERG基因cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    朱冠群; 杨红文; 罗卫星; 韩勇; 刘若余; 陈志

    2013-01-01

    选择与羊同源性较高的牛RERG基因组序列设计特异性引物,提取贵州黑山羊脾脏总RNA,通过RT-PCR技术对RERG基因进行克隆测序及序列分析.结果:首次克隆了贵州黑山羊RERG基因cDNA序列629 bp,GenBank登录号为JN672576,编码199个氨基酸;贵州黑山羊RERG基因蛋白与牛的同源性高达98.5%;聚类分析表明RERG基因编码区适于构建种间系统进化树.%Cow RERG genome sequence with high homology with sheep was selectde and one specific primers had been designed. Total RNA was extracted from the Guizhou black goat spleen and the cDNA encoding RERG was obtained by the reverse transcription PCR (RT-PCR). The purified RT-PCR product was cloned into T vector, and then the sequence was analyzed. The results demonstrated that the 629 bp product was the Guizhou black goat RERG cDNA, and GenBank accession number was JN672576. It encoded 199 amino acids. RERG protein in Guizhou black goat had 98.5% homology with cow. Cluster analysis revealed that probably mammals, birds and amphibians, each of these belonged to different categories separately. The result of phylogenetic clustering was identical to the genetic distance and zoological classification, which indicated that the RERG gene was also fit to construct molecular phylogenetic tree among different species.

  8. 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.

  9. Purification, characterization and cDNA cloning of an analgesic peptide from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU2).

    Science.gov (United States)

    Zhang, R; Yang, Z; Liu, Y F; Cui, Y; Zhang, J H

    2011-01-01

    In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 (GenBank Acc. No. AF288608) and BmK alphaTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.

  10. 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.

  11. Divergence of host range and biological properties between natural isolate and full-length infectious cDNA clone of the Beet mild yellowing virus 2ITB.

    Science.gov (United States)

    Klein, Elodie; Brault, Véronique; Klein, Delphine; Weyens, Guy; Lefèbvre, Marc; Ziegler-Graff, Véronique; Gilmer, David

    2014-01-01

    Plant infection by poleroviruses is restricted to phloem tissues, preventing any classical leaf rub inoculation with viral RNA or virions. Efficient virus inoculation to plants is achieved by viruliferous aphids that acquire the virus by feeding on infected plants. The use of promoter-driven infectious cDNA is an alternative means to infect plants and allows reverse genetic studies to be performed. Using Beet mild yellowing virus isolate 2ITB (BMYV-2ITB), we produced a full-length infectious cDNA clone of the virus (named BMYV-EK) placed under the control of the T7 RNA polymerase and the Cauliflower mosaic virus 35S promoters. Infectivity of the engineered BMYV-EK virus was assayed in different plant species and compared with that of the original virus. We showed that in vitro- or in planta-derived transcripts were infectious in protoplasts and in whole plants. Importantly, the natural aphid vector Myzus persicae efficiently transmitted the viral progeny produced in infected plants. By comparing agroinoculation and aphid infection in a host range assay, we showed that the engineered BMYV-EK virus displayed a similar host range to BMYV-2ITB, except for Nicotiana benthamiana, which proved to be resistant to systemic infection with BMYV-EK. Finally, both the BMYV-EK P0 and the full-length clone were able to strongly interfere with post-transcriptional gene silencing.

  12. 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.

  13. 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.

  14. 大腹园蛛Avg1 cDNA的克隆和序列分析%Cloning and Sequence Analysis of Araneus ventricousus Avg1 cDNA

    Institute of Scientific and Technical Information of China (English)

    任洪林; 柳增善; 卢士英; 潘风光

    2004-01-01

    Quantity and activity of cathepsin B (CB) are increased during the progress of carcinoma cells' invasion and metastasis. So, CB is being applied to the diagnosis of cancers and inhibiting their diffusion. The complete Avgl eDNA was randomly cloned from the cDNA library of major ampullate gland of Araneus ventricousus. It is 1 253 bp and encode 334 amino acids in its 1 002 bp encoding region. The molecular weight of the protein is 36 953.00.GenBank accession number is AY302573. The 3′ non-coding region is composed of 179 bp with a polyadenylation signal AATAAA sequence appearing at the position 129 nt and the poly(A) tail is at the position 153 nt downstream of stop codon TAA. The signal peptide cleavage site of its deduced protein is between codon 16 and 17. Two glycosylation sites of AsnThrThr and AsnValSer, respectively, appear at codon 23 and 202. The high homology genes are not found in all genes known in NCBI, but the typical conserved domain of peptidase-C1 has been detected in NCBI BLASTp, and high homologies with CB of some kinds of creatures are shown. The objective function of the Avgl has not been studied in the spide silk gland yet.

  15. Isolation of a cDNA encoding thymic shared antigen-1. A new member of the Ly6 family with a possible role in T cell development.

    Science.gov (United States)

    MacNeil, I; Kennedy, J; Godfrey, D I; Jenkins, N A; Masciantonio, M; Mineo, C; Gilbert, D J; Copeland, N G; Boyd, R L; Zlotnik, A

    1993-12-15

    We have previously characterized a novel mouse thymocyte marker, defined as thymic shared Ag-1 (TSA-1), present on both immature thymocytes and a subset of thymic medullary epithelial cells. MTS 35, a mAb specific for TSA-1, alters T cell differentiation when added to fetal thymic organ cultures, suggesting TSA-1 may be important for T cell development in the thymus. In this study, we describe the isolation of a cDNA encoding TSA-1 using transient expression of COS-7 cells and selection with MTS 35. The predicted amino acid sequence of this cDNA encodes a 15 to 17-kDa protein and the expressed protein is linked to the membrane via a phosphatidylinositol moiety. TSA-1 is transcriptionally active at various levels in all organs examined, suggesting that its role is not solely intrathymic. TSA-1 shares amino acid sequence homology to the mouse Ly6 multigene family, epidermal growth factor-like receptors, and to cobra venom neurotoxin. The Tsa-1 locus is located on chromosome 15 linked to Ly6 on the mouse genome. We also examined the effects of MTS 35 in fetal thymic organ cultures repopulated with two subsets of thymocytes representing defined stages of T cell development. Our results suggest that TSA-1 may play a role during positive selection and the transition from CD4+CD8+ thymocytes to the mature CD4+CD8- and CD4-CD8+ subsets.

  16. Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus

    NARCIS (Netherlands)

    Nakajima, H.; Hagting, A; Kunji, E.R S; Poolman, B.; Konings, W.N

    1997-01-01

    The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpT(LH)) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coil E1772. Functional expression of the peptide

  17. Molecular cloning, sequence analysis and expression of genome segment 7 (S7) of Antheraea mylitta cypovirus (AmCPV) that encodes a viral structural protein.

    Science.gov (United States)

    Chavali, Venkata Ramana Murthy; Ghosh, Ananta K

    2007-10-01

    The Genome segment 7 (S7) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus (AmCPV) was converted to cDNA, cloned and sequenced. The nucleotide sequence showed that segment 7 consisted of 1789 nucleotides with an ORF of 530 amino acids and could encode a protein of approximately 61 kDa, termed P61. The 5' terminal sequence, AGTAAT and the 3' terminal sequence, AGAGC of the plus strand was found to be the same as genome segment 10 of AmCPV encoding polyhedrin. No sequence similarity was found by searching nucleic acid and protein sequence databases using BLAST. The secondary structure prediction showed the presence of 17 alpha-helices, 18 extended beta-sheets along the entire length of P61. The ORF of segment 7 was expressed in E. coli as His-tagged fusion protein, purified through Ni-NTA chromatography, and polyclonal antibody was raised in rabbit indicating that P61 is immunogenic. Immunoblot analysis using this antibody on viral infected cells as well as purified polyhedra showed that P61 is a viral structural protein. Motif scan search showed some similarity of P61 with Inosine monophosphate dehydrogenase (IMPDH) cystathionine-beta-synthase (CBS) domain at the C-terminus and it was hypothesized that by binding to single stranded viral RNA through its CBS domain P61 may help in virus replication or transcription.

  18. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

    OpenAIRE

    Decai Tuo; Wentao Shen; Pu Yan; Xiaoying Li; Peng Zhou

    2015-01-01

    Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length ...

  19. Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Shan Qin; Hong Tang; Lian-San Zhao; Fang He; Yong Lin; Li Liu; Xiao-Mei He

    2003-01-01

    AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind Ⅲ/Not Ⅰ followed by ligation with pRc/CMV, or BamHI/EcoR Ⅰ followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSGSUTPL/Flag plasmid vectors with T4DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418.RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preSl-preS2S and preSlS encoding genes,determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins.Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen.CONCLUSION: Eight recombinant plasmids expressing S,M, L or preSlS proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells

  20. Functionally cloned pdrM from Streptococcus pneumoniae encodes a Na(+ coupled multidrug efflux pump.

    Directory of Open Access Journals (Sweden)

    Kohei Hashimoto

    Full Text Available Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance-nodulation-cell division (RND, small multidrug resistance (SMR, major facilitator (MF, ATP binding cassette (ABC, and multidrug and toxic compounds extrusion (MATE. We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4',6-diamidino-2-phenylindole (DAPI in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na(+- or Li(+-dependent manner. Moreover, Na(+ efflux via PdrM was observed when acriflavine was added to Na(+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na(+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.

  1. Cloning and sequencing of the gene encoding LipL21 in the vaccinal leptospira serovars

    Directory of Open Access Journals (Sweden)

    Rasoul Hoseinpur

    2016-01-01

    Full Text Available Background: Leptospirosis is a zoonotic disease in humans and animals, caused by the bacterium Leptospira interrogans. Gene expressing LipL21 is one of the genes identified in the bacterium, existing only in the pathogenic strains. The aim of this study was to cloning and analyzing the sequence of the gene encoding surface lipoprotein, LipL21, in five vaccinal leptospira serovars in Iran. Material and Methods: Pathogenic Leptospira interrogans serovars were cultured in EMJH medium with 10% rabbit serum. After genomic DNA extraction, PCR with specific primers was employed and the resulting product inserted in a vector then transferred into E. Coli DH5&alpha. The recombinant plasmids were finally sent for sequencing. Results: The analysis of gene lipL21 in domestic vaccinal serovars and comparison of them with other serovars in the GenBank database revealed that three vaccinal serovars serjo hardjo, canicola and pomona had 100% similarity with each other and grippotyphosa serovar had the highest difference with the vaccinal serovars. In general, the results showed that this gene is a highly conserved gene in the domestic vaccinal serovars and serovars in the GenBank database with more than 95.7 percent similarity. Conclusion: These results showed that the gene, lipL21, is highly conserved in the vaccinal serovars (similarities > 96.4 %. Therefore, the gene encoding surface protein LipL21 can serve as a useful serologic test with high specificity and sensitivity for diagnosis of leptospirosis in clinical samples and in future as an effective subunit vaccine candidate to be used.

  2. 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.

  3. Cloning and Sequence Analysis of Ma-14-3-3d Encoding a Homologue 14-3-3 Protein from Banana%香蕉14-3-3蛋白基因Ma-14-3-3d的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    李美英; 徐碧玉; 杨小亮; 刘菊华; 张建斌; 金志强

    2008-01-01

    [Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.

  4. Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

    Directory of Open Access Journals (Sweden)

    Bharat Bhusan Patnaik

    Full Text Available BACKGROUND: Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. METHODOLOGY/PRINCIPAL FINDINGS: Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4, at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC. SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC. The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp. In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR. The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps involved in plant development and defense. CONCLUSIONS/SIGNIFICANCE: The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was

  5. Cloning of the Gene Encoding Urease Subunit A in Helicobacter Pylori

    Institute of Scientific and Technical Information of China (English)

    施理; 张宜俊; 陈劼; 候晓华

    2004-01-01

    Summary: The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined.Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet.0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44 % G+ C content. The DNA sequence was 98 % homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99 % homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  7. cDNA cloning, characterization and expression analysis of DTX2, a human WWE and RING-finger gene, in human embryos.

    Science.gov (United States)

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

    The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions.

  8. Cloning and sequence analysis of the gene encoding 19-kD subunit of Complex I from Dunaliella salina.

    Science.gov (United States)

    Liu, Yi; Qiao, Dai Rong; Zheng, Hong Bo; Dai, Xu Lan; Bai, Lin Han; Zeng, Jing; Cao, Yi

    2008-09-01

    NADH:ubiquinone oxidoreductase (complex I ) of the mitochondrial respiratory chain catalyzes the transfer of electrons from NADH to ubiquinone coupled to proton translocation across the membrane. The cDNA sequence of Dunaliella salina mitochondrial NADH: ubiquinone oxidoreductase 19-kD subunit contains a 682-bp ORF encoding a protein with an apparent molecular mass of 19 kD. The sequence has been submitted to the GenBank database under Accession No. EF566890 (cDNA sequences) and EF566891 (genomic sequence). The deduced amino-acid sequence is 74% identical to Chlamydomonas reinhardtii mitochondrial NADH:ubiquinone oxidoreductase 18-kD subunit. The 19-kD subunit mRNA expression was observed in oxygen deficiency, salt treatment, and rotenone treatment with lower levels. It demonstrate that the 19-kD subunit of Complex I from Dunaliella salina is regulated by these stresses.

  9. Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an actin-capping protein from the Z line of skeletal muscle.

    OpenAIRE

    Casella, J F; Casella, S J; Hollands, J. A.; Caldwell, J E; Cooper, J A

    1989-01-01

    cDNA encoding the alpha chain of Cap Z has been isolated by screening a lambda gt11 library with affinity-purified antibodies. A single cDNA insert (designated CE2) of 2153 base pairs (bp) contains an open reading frame of 836 bp, which is incomplete at its 5' end. The technique of "rapid amplification of cDNA ends" has been used to extend the 5' end of this open reading frame to a potential transcription initiation site that is preceded by 320 bp of an apparently untranslated region. The pro...

  10. Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance.

    Directory of Open Access Journals (Sweden)

    Vinay Kumar

    Full Text Available Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.. Dihydroflavonol 4-reductase (DFR and anthocyanidin reductase (ANR are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.

  11. Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

    Institute of Scientific and Technical Information of China (English)

    MA Lifang; ZHANG Shicui; ZHUANG Zhimeng; LIU Zhenhui; LI Hongyan; XIA Jianjun

    2005-01-01

    In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.

  12. Evaluation of the immune response of male and female rats vaccinated with cDNA encoding a cysteine proteinase of Fasciola hepatica (FhPcW1).

    Science.gov (United States)

    Wesołowska, Agnieszka; Norbury, Luke J; Januszkiewicz, Kamil; Jedlina, Luiza; Jaros, Sławomir; Zawistowska-Deniziak, Anna; Zygner, Wojciech; Wędrychowicz, Halina

    2013-06-01

    Not only do males and females of many species vary in their responses to certain parasitic infections, but also to treatments such as vaccines. However, there are very few studies investigating differences among sexes following vaccination and infection. Here we demonstrate that female Sprague-Dawley rats vaccinated with cDNA encoding a recently discovered cysteine proteinase of Fasciola hepatica (FhPcW1) develop considerably lower liver fluke burdens after F. hepatica infection than their male counterparts. This is accompanied by differences in the course of their immune responses which involve different eosinophil and monocyte responses throughout the study as well as humoral responses. It is evident that host gender influences the outcome of parasitic infections after vaccination and research on both sexes should be considered when developing new treatments against parasites.

  13. Human mitochondrial HMG CoA synthase: Liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal (Canada)] [and others

    1994-10-01

    Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholersterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of an HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. the nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistant with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain. 56 refs., 4 figs., 2 tabs.

  14. 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.

  15. 沙田柚无机焦磷酸酶基因的 cDNA 克隆及序列分析%Cloning and sequence analysis of inorganic pyrophosphatase gene from Citrus grandis var.shatianyu

    Institute of Scientific and Technical Information of China (English)

    秦新民; 万珊; 李惠敏; 覃屏生; 张渝

    2015-01-01

    related)and non-self(genetically unrelated)pollen.Inorganic pyrophosphatase(IPPase)play important roles in regu-lating the growth and development in plants.In order to better understand the mechanism of IPP gene in the self-in-compatibility of Citrus grandis var.shatianyu ,the inorganic pyrophosphatase gene of C .grandis var.shatianyu was cloned and physicochemical properties of pyrophosphatase were analyzed.The total RNA was isolated from style of C .grandis var.shatianyu used the total RNA Purification System(Invitrogen)and following the manufacture′s pro-tocols.According to the EST sequence(internal fragment of inorganic pyrophosphatase gene)in suppression subtrac-tive hybridization libraries of C .grandis var.shatianyu style,4 specific primers 5′-GSP1,5′-nGSP1,3′-GSP2 and 3′-nGSP2 were designed for amplifying 3-RACE and 5-RACE of the gene.The full-length sequences of cDNA of inor-ganic pyrophosphatase gene were obtained from suppression subtractive hybridization libraries of C .grandis var.shatianyu style by the SMART-RACE PCR method.A comparison of the similarity of the full-length cDNA se-quence of the inorganic pyrophosphatase gene was performed in the GenBank database used the BLAST program.DNAman software was used for mino acid sequence and homology analysis.Prediction of molecular weight, isoeletric point(pI)and hydrophobicity were performed by using on line software ExpASy and DNAman.A 150 bp band in 3-RACE,and a 900 bp band in 5-RACE were cloned by nested and non nested PCR method.For the full-length cDNA sequences,the middle sequence,3-RACE and 5-RACE sequence of the inorganic pyrophosphatase gene were spliced and formed the full-length cDNA sequences.As a result,the cDNA of inorganic pyrophosphatase was 1 136 bp in length containing an 654 bp open reading frame(ORF),which encoded a protein of 217 amino acids with a 170 bp 5 untranslated region(5 UTR)and a 321 bp 3 UTR.The sequence of the cloned cDNA of the inorganic pyro-phosphatase from C .grandis

  16. Cloning and sequence analysis of squalene synthase gene and cDNA in Glycyrrhiza uralensis%甘草鲨烯合酶基因及cDNA的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    荣齐仙; 刘春生; 黄璐琦; 张宁; 南博; 呙未

    2011-01-01

    Objective: To clone and sequence the open reading frame and genomic sequence of squalene synthase (SQS) from Glycyrrhiza uralensis. Method: The primers were designed according to cDNA sequence of SQS from G. glabra reported by Hiroaki HAYASHI, SQS cDNA was cloned with total RNA extracted from roots of G. uralensis. Specific fragments were amplified by RT-PCR and then were cloned and sequenced. SQS DNA was cloned with total DNA extracted from roots of G. uralensis. Specific fragments were amplified by PCR and then were cloned and sequenced. Result: GuSQS1 (GenBank accession number: GQ266154) was 1 242 bp in length encoding proteins with 412 amino acid. NCBI Blast x search results showed GuSQS1 had the highest amino acid similarity to the corresponding proteins from G. uralensis The identities of GuSQS1 with the two proteins were 98. 55% and 88. 62%. SQS ( GenBank accession number: GQ180932) gene with 4 484 bp containing 13 exons and 12 introns was then amplified by PCR with genomic DNA extracted from roots of G. uralensis. Conclusion: These findings of cloning and sequencing the open reading frame and genomic sequence of squalene synthase (SQS) from G. uralensis brought some new clues for the further exploration of SmSQS function in sterol and terpenes biosynthesis.%目的:对甘草鲨烯合酶(SQS)基因的cDNA及DNA进行克隆及序列分析.方法:根据已报道的光果甘草SQS1基因的cDNA序列设计引物,采用RT-PCR的方法,提取甘草根的RNA然后反转录成cDNA,以cDNA为模板,扩增出SQS基因的cDNA序列,以甘草总DNA为模板,扩增SQS的DNA序列.结果:序列分析表明,克隆获得的甘草SQS1的cDNA编码区为1242 bp,编码413个氨基酸残基,命名为GuSQS1,登录号为GQ266154,与卢虹玉等报道的甘草的2个SQS(SQS1和SQS2)的氨基酸序列一致性为98.55%,88.62%,对应DNA序列全长为4484 bp,含有13个外显子,12个内含子,登录号为GQ180932.结论:甘草SQS的cDNA及DNA序列的获得为进一步研究

  17. 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.

  18. 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.

  19. Expression of a cDNA encoding a member of the hexamerin storage proteins from the moth Sesamia nonagrioides (Lef.) during diapause.

    Science.gov (United States)

    Spyliotopoulos, Anastasios; Gkouvitsas, Theodoros; Fantinou, Argyro; Kourti, Anna

    2007-09-01

    We isolated and sequenced a cDNA clone corresponding to a storage protein (SnoSP1) from the corn stalk borer Sesamia nonagrioides (Lef.). The cDNA for SnoSP1 (2403 bp) codes for a 751 residue protein with predicted molecular mass of 88.3 kDa and calculated isoelectric point pI=8.72. A signal peptide of 16 amino acids is present at the N-terminus and the protein contained conserved insect larval storage protein signature sequence patterns. Multiple alignment analysis of the amino acid sequence revealed that SnoSP1 is most similar to the basic juvenile hormone-suppressible protein 2 precursor (TniSP2) from Trichoplusia ni (71% identity) and other moderately methionine-rich hexamers. According to both phylogenetic analyses and the criteria of amino acid composition, SnoSP1 belongs to the subfamily of moderately methionine-rich storage proteins (3.7% methionine, 11% aromatic amino acid). Treatment with the juvenile hormone analog, methroprene, after head ligation of larvae, is found to suppress the level of SnoSP1 gene, indicating hormonal effects at the transcriptional level. We also examined developmental profiles of SnoSP1 expression in fat body from diapausing and non-diapausing larvae by semi-quantitative and Real-Time PCR assays. In non diapause conditions the abundance of SnoSP1 was found in high levels during the last larval stage and decreased gradually during the pupal stage. Very low levels of this mRNA were detected in larvae that were preparing to enter diapause, but mRNA dramatically increased in those that were in diapause as well as in those that terminate diapause.

  20. A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause.

    Science.gov (United States)

    Liu, Ming; Zhang, Tian-Yi; Xu, Wei-Hua

    2005-06-01

    The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.

  1. Molecular Cloning of Spergen-4, Encoding a Spermatogenic Cell-Specific Protein Associated with Sperm Flagella and the Acrosome Region in Rat Spermatozoa.

    Science.gov (United States)

    Howida, Ali; Salaheldeen, Elsaid; Iida, Hiroshi

    2016-04-01

    We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

  2. Cloning, Characterization, and Distribution of an mRNA Encoding a H+-ATPase α Subunit in the Mantle of Pearl Oyster, Pinctada fucata

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Mitochondrial ATP synthase is responsible for the production of the majority of the cellular ATP,which is composed of two major units: F0 and F1. Although much is known about the active complex (5 subunits (αβγδε)), the role of the α subunit in the catalytic mechanism remains unclear, particularly in bivalve animals. This study first cloned and identified the full-length sequence of the mitochondrial H+-ATP synthase α subunit cDNA gene in Pinctada fucata using the reverse transcriptase polymerase chain reaction (RT-PCR)technique. The Pinctada fucata mitochondrial H+-ATP synthase α subunit contains 1991 nucleotides, with the translation start site at nt 48 (ATG) and the stop codon at nt 1660 (TAA), encoding a polypeptide 553 amino acids in length, which shares high similarity to that of other animals (81% identity to fruit fly, 82% to carp, and 83% to humans). Alignment analysis of the well-conserved amino acid domains in the ATPase α subunit, the α/β signal transduction domain, showed that two residues (Asp358 and Asn359) differ from any other ATP synthase α subunit. In situ hybridization analysis was used to reveal the wide-spread distribution of mitochondrial H+-ATP synthase in various tissues in Pinctada fucata. This work will help further research on pearl energy metabolism to increase the output and quality of pearls to more efficiently utilize our rich pearl oyster resources.

  3. Molecular cloning and characterization of duck interleukin-17

    Science.gov (United States)

    Interleukin-17 (IL-17) belonging to the Th17 family is a proinflammatory cytokine produced by activated T cells. A 1034-bp cDNA encoding duck IL-17 (duIL-17) was cloned from ConA-activated splenic lymphocytes of ducks. The encoded protein, predicted to consisted of 169 amino acids, displayed a molec...

  4. 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

  5. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

    Science.gov (United States)

    Baunsgaard, L; Dalbøge, H; Houen, G; Rasmussen, E M; Welinder, K G

    1993-04-01

    Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less

  6. 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.

  7. Cloning and characterization of 3-hydroxy-3-methylgIutary CoA reductase cDNA of Glycyrrhiza uralensis%乌拉尔甘草HMGR基因cDNA的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    荣齐仙; 许巧仙; 刘春生; 黄璐琦

    2011-01-01

    Objective: To clone and analysis the sequence of 3-hydroxy-3-methylglutary CoA reductase (HMGR) cDNA from Glycyrrhiza uralensis.Method: The primers were designed based on the conservative region of HMGR nucleic acids sequence from public database.The target gene was obtained from root of G.uralensis by use of homologous cDNA amplificati on and RACE technologies.The sequence alignment was performed using BLAST.The open reading frame was identified by use of the ORF Finder.The protein domains were defined by use of Prosite software.Clustal was used to conduct multiple amino acid sequence alignment and MEGA 5.0 was used to conduct the phylogenetic tree.Result: The GuHMGR cDNA sequence was obtained contains 1 842 bp contains a 1 722 bp ORF, encoding 573 amino acids with 3-hydroxy-3-methylglutary CoA reductases family profile.Deduced amino acid sequence had 84% and 76% homology to the amino acid sequence of Pisum sativum, Medicago truncatula.Conclusion: The cloning of 3-hydroxy-3-methylglutary CoA reductase (HMGR) cDNA will provide a foundation for exploring the function of HMGR in glycyrrhizin biosynthesis.%目的:对乌拉尔甘草3.羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3methylglutary CoA reductase,HMGR)的cDNA克隆并进行序列分析.方法:根据NCBI数据库中的豆科其他物种HMGR的cDNA保守区设计引物,利用同源扩增和cDNA末端快速扩增技术从甘草根中获得目的基因;利用BLAST进行序列比对,ORF Finder寻找开发阅读框,Prosite分析蛋白质的基本结构域,Clustal x比对已有HMGR的氨基酸序列,并构建进化树.结果:得到1个全长为1 842 bp的HMGR的cDNA序列,含有1 722 bp的开放阅读框(open reading frame,ORF),编码573个氨基酸,具有HMGR家族的特异序列,推测的氨基酸序列与豌豆、蒺藜苜蓿的氨基酸序列一致性分别为84%,76%.结论:对甘草HMGR基因的cDNA进行了克隆,为进一步研究3-羟基-3-甲基戊二酰辅酶A在甘草酸生物合成途径中的作用提供了理论依据.

  8. 诸氏鲻虾虎鱼卵黄蛋白原基因全长cDNA的克隆及表达%Full-length cDNA cloning and expression analysis of vitellogenin gene inMugilogobius chulae

    Institute of Scientific and Technical Information of China (English)

    余露军; 蔡磊; 李舸; 陈小曲; 陈琳; 李建军

    2016-01-01

    To explore the tissue distribution of the vitellogenin (Vg) gene inMugilogobius chulae and the effect of the pollutant 17β-estradiol on maleM. chulae, the full length of vitellogenin (Vg) cDNA in M. chulae was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) tech-niques using total RNA extracted from the liver of female fish. Meanwhile, the expression of Vg mRNA ofM. chu-lae was determined by real-time fluorescent quantitative PCR. The full length ofM. chulae’s Vg cDNA sequence contains 5 067 bp nucleotides, with a complete open reading frame (ORF) of 4 992 bp, which encodes 1 664 amino acid proteins with a deduced protein molecular weight of 186.2 ku. The ORF contains signal peptides and a polyserine domain. The real-time RT-PCR result showed thatM. chulae’s Vg mRNA was primarily detected in the liver. Furthermore, Vg mRNA expression levels at different time points of E2 treatment of male fish were deter-mined.M. chulae’s Vg mRNA expression levels reached the peak on the third day and then the expression levels declined on the seventh day. These results indicated that the full length of Vg cDNA in M. chulae was cloned suc-cessfully. This study suggests that Vg in maleM. chulae can be considered as a valid biomarker for offshore envi-ronmental monitoring of estrogenic substances.%为了探讨诸氏鲻虾虎鱼(Mugilogobius chulae)卵黄蛋白原组织分布及17β-雌二醇(E2)暴露对雄性诸氏鲻虾虎鱼Vg的影响,作者采用RT-PCR、RACE方法克隆并分析了诸氏鲻虾虎鱼卵黄蛋白原(Vg)基因的全长cDNA序列,并对Vg在诸氏鲻虾虎鱼体内的组织表达分布及E2诱导后不同时间表达规律进行了研究。结果表明:获得的Vg cDNA序列全长5067 bp,开放阅读框(ORF)含4992 bp,编码1663个氨基酸,含有信号肽、多丝氨酸区域,推测其编码氨基酸分子量为186.2 ku,等电点为9.31。荧光定量PCR结果显示, Vg在诸氏鲻虾虎鱼

  9. 羊驼垂体催乳素(PRL)基因全长cDNA的克隆及序列分析%Cloning and Sequence Analysis of the Full-length cDNA of PRL Gene from Alpaca Pituitary

    Institute of Scientific and Technical Information of China (English)

    薛霖莉; 董常生; 赫晓燕; 范瑞文; 王海东; 曹靖; 郝欢庆

    2011-01-01

    In order to provide theoretical basis for studying biological function and application of alpaca prolactin (PRL), the alpaca PRL cDNA sequence were cloned and analyzed.According to the known cDNA sequences from mammals, alpaca PRL primers was designed and the full-length cDNA of PRL from alpaca pituitary was cloned by RT-PCR and RACE techniques.The size of full-length cDNA of PRL from alpaca pituitary was 959 bp and it contained an open reading frame (ORF) of 687 bp which encoded PRL precursor protein with 229 AA.PRL precursor protein was a single-chain polypeptide composed of 30-AA signal peptide and 199-AA mature peptide.The spatial structure of alpaca PRL protein was similar to human GH.The result of the sequence alignment showed that the amino acids composition of alpaca PRL was similar to most mammals, but the methionine at 81-AA (51-AA for mature peptide) might lead to different spatial structure which might impact functions of alpaca PRL.A phylogenetic tree constructed basing on the amino acid sequences of alpaca PRL and other organisms showed that the relationships between alpaca PRL and camel PRL were closest and that the evolution speed of alpaca was very slow with no ' episodic' evolution pattern as most mammals such as primates, rodents and ruminant.%获得并分析羊驼PRL基因cDNA全序列结构,为研究羊驼催乳素(PRL)的各种生物学作用和生产应用提供理论依据.根据已知的不同哺乳动物的PRL基因cDNA序列,设计羊驼PRL引物,运用RT-PCR方法和cDNA末端快速扩增(RACE)技术获得羊驼PRL基因cDNA全序列.羊驼PRL基因cDNA序列全长959 bp,编码区为687bp,编码229个氨基酸的PRL碰前体蛋白.预测羊驼PRL蛋白质的空间结构类似人生长激素(GH),但在81位(成熟肽为51位)为蛋氨酸可能导致蛋白空间结构的不同而影响羊驼PRL的功能;序列比对结果表明,羊驼PRL的cDNA序列与大多数哺乳动物相似.构建的基因进化树分析结果显示,羊驼PRL与骆

  10. 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.

  11. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...

  12. Cloning, sequence analysis and detection of vitellogenin cDNA from Colisa fasciata%条纹密鲈卵黄蛋白原基因的克隆与检测

    Institute of Scientific and Technical Information of China (English)

    梁岳; 方展强

    2011-01-01

    Using RT-PCR,the partial length vitellogenin( VTG)cDNA sequence and (3-actin cDNA sequence of Colisa fasciata were cloned. VTG cDNA sequence contains 3 464 bp nucleotides and encodes 1 150 amino acids, p-actin cDNA sequence contains 1 253 bp nucleotides and encodes 375 amino acids. In vivo and in vitro methods were employed to investigate VTG mRNA expression under exposure to estradiol (E2), octylphenol (OP), cadmium ( Cd2 + ) and perfluorooctane sulfonates ( PFOS) and evaluate the estrogenic activity. The results showed that E2 and OP could induce VTG mRNA expression in dose-dependent way by in vivo and in vitro test. Cd2+ could induce VTG mRNA expression only in the low dose by in vivo test,but VTG mRNA expression was not observed in PFOS groups by in vivo and in vitro test. The results indicated that the strength of estrogenic effects was in the order E2 > OP > Cd2 +. Cd2 + estrogenic effects in vivo and in vitro results are inconsistent, suggesting that the mechanism of Cd2 + induced effects of estrogen and E2 may be different. The results also indicated that VTG cDNA of C. Fasciata is very sensitive to environmental hormone and very suitable to be a biomarker for monitoring the environmental hormones.%采用RT-PCR方法克隆并分析了条纹密鲈卵黄蛋白原(VTG)和β-肌动蛋白(β-actin)cDNA部分序列.获得的VTG cDNA序列片段长3464 bp,全部处于编码区,编码1150个氨基酸;β-actin cDNA序列片段长1253 bp,编码375个氨基酸.使用活体与离体的实验方法,检测了VTG mRNA转录情况,并以此评价雌二醇(E2)、辛基酚(OP)、镉(Cd2+)和全氟辛烷磺酸类化合物( PFOS)引起的雌激素效应.结果显示,E2和OP在活体和离体实验中均能剂量依赖性地诱导VTG mRNA表达.Cd2+仅在活体实验低剂量组诱导VTG mRNA表达,PFOS在活体和离体实验的各个浓度组均未见显著的VTG mRNA表达.结果表明,所诱导的雌激素效应强弱的排列顺序为E2>OP> Cd2.Cd2的雌激素效应

  13. Expression cloning of different bacterial phosphatase-encoding genes by histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green.

    Science.gov (United States)

    Riccio, M L; Rossolini, G M; Lombardi, G; Chiesurin, A; Satta, G

    1997-02-01

    A system for expression cloning of bacterial phosphatase-encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.

  14. 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.

  15. 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.

  16. 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.

  17. cDNA cloning and sequence analysis of genome segments S8 from rice black-streaked dwarf virus

    Institute of Scientific and Technical Information of China (English)

    张恒木; 陈剑平; 薛庆中; 雷娟利

    2002-01-01

    Genome segments S8 of two Chinese isolates of rice black-streaked dwarf virus (RBSDV), one from Zhejiang Province and another from Hebei Province, were amplified by RT-PCR and sequenced. Both segments consisted of 1936 nts in full length (EMBL accession numbers were AJ297431 and AJ297432, respectively) and contained only one big open reading frame which encoded a polypeptide with molecular weight of 68kD. The two Chinese isolates shared 94.0% and 96.5% identity at nucleotide and amino acid level, respectively. They shared 94.5-94.9% and 92.5-92.9% homology with S8 of RBSDV Japanese isolate at nucleotide and amino acid level, respectively; shared 85.1-87.6% and 91.7-91.9% homology with S7 of Italian MRDV (maize rough dwarf virus).

  18. Purification and cDNA cloning of a novel neurotoxic peptide (Acra3) from the scorpion Androctonus crassicauda.

    Science.gov (United States)

    Caliskan, Figen; García, Blanca I; Coronas, Fredy I V; Restano-Cassulini, Rita; Korkmaz, Ferhan; Sahin, Yalcin; Corzo, Gerardo; Possani, Lourival D

    2012-09-01

    Androctonus crassicauda is one of the Southeastern Anatolian scorpions of Turkey with ethno-medical and toxicological importance. Two toxic peptides (Acra1 and Acra2) were isolated and characterized from the venom of this scorpion. In this communication, the isolation of an additional toxin (Acra3) by chromatographic separations (HPLC and TSK-gel sulfopropyl) and its chemical and functional characterization is reported. Acra3 is a 7620Da molecular weight peptide, with 66 amino acid residues crosslinked by four disulfide bridges. The gene coding for this peptide was cloned and sequenced. Acra3 is anticipated to undergo post-translational modifications at the C-terminal region, having an amidated serine as last residue. Injection of Acra3 induces severe neurotoxic events in mice, such as: excitability and convulsions, leading to the death of the animals within a few minutes after injection. Electrophysiological assays conducted with pure Acra3, using cells that specifically expressed sodium channels (Nav1.1-Nav1.6) showed no clear effect. The exact molecular target of Acra3 remained undiscovered, similar to three other scorpion peptides that clustered very closely in the phylogenetic tree included here. The exact target of these four peptides is not very clear.

  19. 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.

  20. 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.

  1. Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

    Science.gov (United States)

    Legname, G; Bellosta, P; Gromo, G; Modena, D; Keen, J N; Roberts, L M; Lord, J M

    1991-08-27

    Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

  2. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

    Science.gov (United States)

    Tokunaga, T; Yano, O; Kuramoto, E; Kimura, Y; Yamamoto, T; Kataoka, T; Yamamoto, S

    1992-01-01

    Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

  3. Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH†

    Science.gov (United States)

    Russell, W. M.; Klaenhammer, T. R.

    2001-01-01

    The gusA gene, encoding a new β-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a β-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored β-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to β-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a β-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified β-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested. PMID:11229918

  4. 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

  5. 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

  6. Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A new MAPK gene, ZmSIMK1 (Zea mays L. salt-induced mitogen-activated protein kinase 1), is isolated from a maize cDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of encoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 cDNA into pET-42a(+), and transformed into E. coli strain BL21(DE3). A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1mmol/L IPTG.

  7. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

    Science.gov (United States)

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

  8. Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM 109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) fromβ-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.

  9. Nerve growth factor treatment of sensory neuron primary cultures causes elevated levels of the mRNA encoding the ATP synthase beta-subunit as detected by a novel PCR-based differential cloning method.

    Science.gov (United States)

    Kendall, G; Ensor, E; Crankson, H D; Latchman, D S

    1996-03-01

    The mRNA encoding the rat ATP synthase beta-subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase beta-subunit cDNA clones were isolated from a lambda library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA-derived probes from untreated and nerve-growth-factor-treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR-based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed.

  10. Molecular Cloning and Expression Analysis of Toll-like Receptor I cDNA in Japanese Flounder, Paralichthys olivaceus%牙鲆Toll样受体1基因全长cDNA的克隆及特征分析

    Institute of Scientific and Technical Information of China (English)

    吴恋; 孙金生; 耿绪云; 潘宝平; 魏俊利; 王雪惠; 高虹

    2012-01-01

    [Objective] The paper aimed to clone the full length gene of Toll-like recep- tors (TLRs) in Japanese flounder (Paralichthys olivaceus), and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1(TLR1) gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends (RACE). The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame (ORF), encoding 805 amino acid (aa) residues, including signal peptide, six leucine-rich repeat(LRR) motifs, two transmembrane zones and one TolI/IL 1 receptor (TIR) domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.%[目的]克隆牙鲆TLR1(Tolllikereceptors)全长基因,并对其结构特征和表达规律进行分析。[方法]采用同源克隆和快速扩增cDNA末端技术,从牙鲆头肾组织中克隆出TLRl基因cDNA全长序列,并对该基因进行生物信息学和表达模式分析。[结果]牙鲆TLR1基因cDNA全K2947bp,开放阅读框(ORF)2418bp,编码805个氨基酸,包括26个氨基酸组成的信号肽、2个跨膜区、6个富含亮氨酸重复结构域(LRR)和一个TIR结构域(Toll/interleukin(IL).1

  11. 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

  12. 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

  13. 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.

  14. 油茶CoUXS1基因全长cDNA克隆及生物信息学分析%Full-length cDNA Cloning and Bioinformatic Analysis of CoUXS1 Gene in Camellia oleifera

    Institute of Scientific and Technical Information of China (English)

    曾艳玲; 谭晓风; 王保明; 龙洪旭; 刘凯; 曾晓峰

    2012-01-01

    为了揭示油茶UDP-葡萄糖醛酸脱羧酶基因的序列特点和结构功能,为该基因的深入研究和开发应用奠定基础.根据油茶种子EST文库中调取的UDP-葡萄糖醛酸脱羧酶基因序列,设计特异引物,利用RT-PCR技术获得该基因全长cDNA克隆,并对其序列进行生物信息学分析.结果表明:该基因阅读框为1032bp,编码344个氨基酸,具有典型的UXS家族模体序列.与海岛棉GhUXS3基因亲缘关系最近,定位于细胞质,有较强的亲水性,将该基因命名为CoUXS1,GenBank登录号为JN017094.%In order to reveal sequence features and structural function of the UDP-glucuronate decarboxylase gene in Camellia oleifera, and based for the further research, the specific primers were designed according to the UDP-glucuronate decarboxylase gene sequence from Camellia oleifera seed EST library. Using RT-PCR technology, the gene full-length cDNA clones were obtained. Then the sequence was analyzed by bioinformatic software. The results showed that: the ORF box included 1032 bp and encoded 344 ami no acids. The sequence had typical UXS family motif sequence. The gene had closest phylogenetic relationship with GhUXS3 gene of Island cotton and localized in the cytoplasm with strong hydrophilicity. The gene was named CoUXSl and the GenBank accession number was JN017094.

  15. 乙醇脱氢酶Ⅰ类基因全长cDNA的克隆与表达%Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases

    Institute of Scientific and Technical Information of China (English)

    周文婷; 李景鹏; 崔羽; 张永红; 李世荣

    2007-01-01

    Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ~1.31 U/mg,0.09 ~0.15 U/mg and 0.76~1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.%目的:克隆编码人Ⅰ类乙醇脱氢酶基因,并探讨Ⅰ类乙醇脱氢酶(ADH)在乙醇的肝代谢中的作用.方法:从胎儿肝,肾提取的总RNA;经RT-PCR扩增得到cDNA并克隆至pGEM-T载体.cDNA序列用Kpn Ⅰ和Pst Ⅰ酶切鉴定,并检测其在大肠杆菌中表达活性.通过吸光法检测酶的活性.结果:成功克隆了人Ⅰ类乙

  16. Cloning, Sequencing and Characterization of 3-Hydroxybuty- rate Dehydrogenase Encoding Gene (bdh A) in Bradyrhizobium japonicum USDA110 Strain

    Institute of Scientific and Technical Information of China (English)

    DAI Mei-xue; WU Bo; BAI Xue-liang; ZHANG Cheng-gang; MA Qing-sheng; Charles Trevor C

    2002-01-01

    The current study describes the molecular characterization of a clone which can restore the ability of bdhA mutant strains NGRPA2 and Rm11107 to utilize 3-hydroxybutyrate as a sole carbon source (Hbu+). This clone was screened out by complementation experiment from Bradyrhizobium japonicum USDA110 genomic library, and the presence of bdhA gene in the clone was verified by Bdh assay and Southern blot analysis. Furthermore, the entire sequence of bdhA gene was sequenced and the sequence was deposited in GenBank database under the accession number AY077581. bdhA gene comprises 789 base pairs and encodes Bdh with 262 amino acid of MW 27.59 kDa. Interposon ΩKm was inserted into the bdh A ORF at EcoR I site and the bdhA mutant was constructed in B .japonicum by homologous recombination. Plant assay result did not show obvious effects of mutation of bdhA gene on nodulation and nitrogen-fixation.

  17. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  18. 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

  19. Molecular cloning and characterization of three genes encoding dihydroflavonol-4-reductase from Ginkgo biloba in anthocyanin biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Cheng Hua

    Full Text Available Dihydroflavonol-4-reductase (DFR, EC1.1.1.219 catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins, and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.

  20. Cloning and Characterization of an mRNA Encoding F1-ATPase Beta-Subunit Abundant in Epithelial Cells of Mantle and Gill of Pearl Oyster, Pinctadafucata

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process. All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1-ATPase beta-subunit (the F1-β-subunit, a major calalytic subunit of F-ATPase) from the pearl oyster (Pinctads fucata) was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals' F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals' F1-β-subunits, and had a unique phosphorylation site of PKC and CK Ⅱ on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder,and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.

  1. 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 biolog