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Sample records for cdna clones encoding

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

    OpenAIRE

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

    2015-01-01

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

  2. 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

  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, 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.

  5. 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.

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

    OpenAIRE

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

    1991-01-01

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

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

    OpenAIRE

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

    1995-01-01

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

  8. 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.

  9. 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.

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

    OpenAIRE

    1992-01-01

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

  12. 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

  13. 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.

  14. 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

  15. 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.

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

    Science.gov (United States)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

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

  19. Cloning 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.

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

    Directory of Open Access Journals (Sweden)

    S. S. Hasson

    2010-01-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

  2. 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.

  3. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-02-01

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

  5. 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.

  6. 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.

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

    Science.gov (United States)

    Shah; Cha

    2000-05-29

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

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

    DEFF Research Database (Denmark)

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

    1994-01-01

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

  9. 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

  10. 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.

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

    OpenAIRE

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

    1993-01-01

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

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

    Science.gov (United States)

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

    2010-02-28

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

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

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

    UTUT WIDYASTUTI; YASSIER ANWAR; SUHARSONO

    2009-01-01

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

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

    Science.gov (United States)

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

    1995-01-01

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

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

    DEFF Research Database (Denmark)

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

    1985-01-01

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

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUTI

    2009-07-01

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2002-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-03-20

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

  2. 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.

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

    OpenAIRE

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

    1989-01-01

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

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

    OpenAIRE

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

    1986-01-01

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

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    1993-11-01

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

  8. 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.

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

    International Nuclear Information System (INIS)

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

  10. 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.

  11. 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.

  12. Cloning and sequence of cDNA encoding 1-aminocyclo- propane-1-carboxylate oxidase in Vanda flowers

    Directory of Open Access Journals (Sweden)

    Pattana Srifah Huehne

    2013-08-01

    Full Text Available The 1-aminocyclopropane-1-carboxylate oxidase (ACO gene in the final step of ethylene biosynthesis was isolated from ethylene-sensitive Vanda Miss Joaquim flowers. This consists of 1,242 base pairs (bp encoding for 326 amino acid residues. To investigate the specific divergence in orchid ACO sequences, the deduced Vanda ACO was aligned with five other orchid ACOs. The results reveal that the ACO sequences within Doritaenopsis, Phalaenopsis and Vanda show highly conserved and almost 95% identical homology, while the ACOs isolated from Cymbidium, Dendrobium and Cattleya are 8788% identical to Vanda ACO. In addition, the 2-oxoglutarate- Fe(II_oxygenase (Oxy domain of orchid ACOs consists of a higher degree of amino acid conservation than that of the non-haem dioxygenase (DIOX_N domain. The overall homology regions of Vanda ACO are commonly folded into 12 α-helices and 12 β-sheets similar to the three dimensional template-structure of Petunia ACO. This Vanda ACO cloned gene is highly expressed in flower tissue compared with root and leaf tissues. In particular, there is an abundance of ACO transcript accumulation in the column followed by the lip and the perianth of Vanda Miss Joaquim flowers at the fully-open stage.

  13. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    OpenAIRE

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

    1993-01-01

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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  1. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    International Nuclear Information System (INIS)

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease

  2. 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.

  3. 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.

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

    Science.gov (United States)

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

    1997-07-15

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

  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 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

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

    International Nuclear Information System (INIS)

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

  12. Poliovirus type 3: molecular cloning of the genome and nucleotide sequence of the region encoding the protease and polymerase proteins.

    OpenAIRE

    1983-01-01

    Overlapping cDNA clones representing the entire genome of poliovirus type 3 have been prepared in E. coli by two separate methods. Cloning of RNA . cDNA hybrids produced a more comprehensive set of clones with generally larger cDNA inserts than cloning of double - stranded cDNA. A restriction map of the entire genome and the nucleotide sequence of 2003 bases from the 3' terminus, comprising the region encoding the protease and polymerase proteins, are presented.

  13. Partial purification of the chloroplast ATP synthase from Chlamydomonas reinhardtii and the cloning and sequencing of a cDNA encoding the gamma subunit

    International Nuclear Information System (INIS)

    The chloroplast ATP synthase was partially purified from the green alga Chlamydomonas reinhardtii by extracting membranes with deoxycholate and KCl, followed by centrifugation and ammonium sulfate fractionation of the supernatant. The enzyme assay involved the reconstitution of such fractions with bacteriorhodopsin and soybean phospholipids to form vesicles capable of light-dependent [32P]-phosphate esterification. A cDNA for the gamma subunit from Chlamydomonas was isolated, expressed in vitro and sequenced. It contains the entire coding region for the gamma subunit precursor. A 35 amino acid long transit peptide resides at the NH2-terminus of a 323 amino acid long mature peptide that is 77% similar to the spinach gamma subunit. Six cysteines were found; three were conserved in Chlamydomonas and spinach

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

    OpenAIRE

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

    1985-01-01

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

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

    OpenAIRE

    Bartels, D.; Thompson, R D

    1983-01-01

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

  16. 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...

  17. 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.

  18. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

  19. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1 and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Directory of Open Access Journals (Sweden)

    Oscar Rodríguez-Lima

    Full Text Available TATA-box binding protein (TBP is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1. Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5 and 2-Cys peroxiredoxin (Ts2-CysPrx gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  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. Isolation and Primary Expression Analysis of a Full-Length cDNA Clone Encoding Small GTP-Binding Protein Gene CaRab8 in Pepper%辣椒小G蛋白CaRab8基因全长cDNA的分离及表达特征的初步分析

    Institute of Scientific and Technical Information of China (English)

    赖燕; 肖翔; 林菁; 虞露; 陈桂信; 官德义; 何水林

    2011-01-01

    通过对辣椒均一化cDNA文库的筛选,分离获得了一个与烟草RAB8-2基因编码的蛋白有着高度同源性的cDNA阳性克隆,命名为CaR ab8.测序结果表明,该cDNA长度为961 bp,包含有651 bp的完整的开放阅读框,推测编码一个217个氨基酸的蛋白,该蛋白具有GTP/GDP结合活性必需的保守域和包括YYRGA在内的Rab家族成员特有的5个结构域.实时定量RT-PCR结果表明,CaRab8基因的表达水平在茉莉酸甲酯(MeJA)的诱导下在短时间内呈上调表达,在相应时间点受水杨酸(SA)诱导下调表达.%One 961bp full -length cDNA clone was isolated from pepper normalized cDNA library, which containing a complete 651 bp open reading frame and encoding a putative protein composed of 217 amino acids. Amino acid sequence deduced by this cDNA clone showed high similarity to NtRAB8-2 protein from tobacco. The full-length cDNA was named CaRab8. The protein encoded by this cDNA clone included conserved domains for guanine nucleotide binding and GTPase activities and five domains specific to Rab family including YYRGA domain. Real time quantitative PCR analysis showed the expression level of CaRab8 was induced by MeJA while was repressed by SA.

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

    OpenAIRE

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

    1984-01-01

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

  5. cDNA expression cloning in mammalian cells.

    Science.gov (United States)

    Hoffman, B J

    2001-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

  7. 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.

  8. 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).

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

    OpenAIRE

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

    1993-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

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

    OpenAIRE

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

    1988-01-01

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

  12. Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111

    DEFF Research Database (Denmark)

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

    1993-01-01

    transformation and because peptide sequences were available in the microsequence database. The cDNA was cloned from a fibroblast cDNA library using degenerate oligodeoxyribonucleotides and expressed in AMA cells using the vaccinia virus expression system. Database searches indicated that the predicted protein......Treatment of proliferating and quiescent primary human keratinocytes with interferon-gamma (IFN-gamma) (100 U/ml, 23.5 h) followed by two-dimensional gel analysis revealed three proteins, IGUP I-3421 (M(r) = 48,200, pI = 6.06); IGUP I-3524 (M(r) = 56,900, pI = 5.92), a protein homologous to peptide......-chain-release factor and tryptophanyl-tRNA synthetase; and IGUP I-5111 (M(r) = 30,400, pI = 5.76) recorded in the keratinocyte protein database (Celis et al., 1991, 1992) that are highly and specifically up-regulated by IFN-gamma among several agents tested including 14 other cytokines, second messengers [dibutyryl c...

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

    OpenAIRE

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

    1987-01-01

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

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

    International Nuclear Information System (INIS)

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

  15. 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.

  16. 5'-end sequences of budding yeast full-length cDNA clones - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

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

  17. 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.

  18. 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

  19. Cloning and characterization of a cDNA encoding thioredoxin peroxidase from Trichomonas vaginalis%阴道毛滴虫硫氧还原蛋白过氧化物酶的cDNA克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    章家新; 傅玉才; 刘红; 郑晓红

    2004-01-01

    目的:获取阴道毛滴虫硫氧还原蛋白过氧化物酶的cDNA克隆,研究其还原烷基氢过氧化物和氢过氧化物的抗氧化作用,以及调节由氢过氧化物介导的信号转换. 方法:提取阴道毛滴虫的总RNA,用λTripIEx2噬菌体载体构建cDNA表达文库,阳性质粒cDNA克隆进行测序,并用生物软件RPS-Blast,NCBI Blast和ClustalW等对6个读码框架进行序列分析. 结果:获得一株开放阅读框为588对碱基的cDNA克隆,推测肽链有196个氨基酸.序列分析表明,该肽链与衣滴虫过氧化物氧化还原酶(Prx1蛋白)及人的自然杀伤增强因子B分别有56%和61%的同源性;除了两个高度保守的半胱氨酸作用基序外,还有蛋白激酶C磷酸化和酪氨酸激酶磷酸化作用基序,N-豆蔻酰化作用位点,脂质运载蛋白信号位点等. 结论:该蛋白有>56%的可能性位于胞浆,和典型2-Cys Prx亚家族有很高(56%~62%)的同源性,很可能是其中的一个新成员.%Objective:To clone and characterize the thioredoxin peroxidase gene of Trichomonas vaginalis for studying its functions involving not only in the reduction of alkyl hydroperoxide and hydrogen peroxide as an antioxidant, but also in regulating hydrogen peroxide-mediated signal transduction. Methods: T. vaginalis mRNA was isolated to construct a cDNA library using λTripIEx2 phage vector. Sequencing of the positive plasmid cDNAs was performed and the resulted sequences were translated in all six reading frames and compared with the nonredundant databases using the NCBI BlastX, RPS-Blast and ClustalW programs. Results: A cDNA clone was isolated and the sequence analysis revealed that the cDNA clone had an open reading frame with 588 bp. The deduced amino acid sequence contained 196 residues and shared 56% identity with a recently identified Chlamydomonas peroxiredoxin 1 (Prx1) and 61% identity with Human natural killing enhance factor B (NKEFB). The conserved sequence elements including the motif of

  20. 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.

  1. 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.

  2. Molecular cloning and characterization of a cDNA encoding the prophenoloxidase gene in Bombyx mandarina%野桑蚕酚氧化酶原基因cDNA的分子克隆及其特征

    Institute of Scientific and Technical Information of China (English)

    张永亮; 黄重; 李志清; 张春冬; 朱勇

    2008-01-01

    Phenoloxidase plays an important role in defense mechanisms in insect immunity.The complemental deoxyribonucleic acid(cDNA)of Bombyx mandarins prophenoloxidase(PPO)gene was cloned by means of reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE).The cDNA was 2 134 bp in length and contained an open reading frame(ORF)of 2 082 bp which eneoded 693 amino acid residues.The deduced amino acid sequence showed a high identity to the reported sequence of PPO2 from other Lepidopterous insects and shared the typical structural features of PPO from other inseets.The results of RT-PCR showed that PPO mRNA was expressed in the integument,haemocyte,head,testis,ovary,fat body and midgut of 5th instar larvae and at different developmental stages of B.mandarina.These results provide molecular basis for further studying the function of ppo gene in B.mandarina.%酚氧化酶在昆虫的免疫防御机制中起着重要作用.利用RT-PCR和RACE方法,克隆了野桑蚕酚氧化酶原基因,获得了其cDNA序列.该序列长2 134 bp,含有一个2 082 bp的完整开放阅读框,编码一个由693个氨基酸残基组成的蛋白质.推导的氨基酸序列与其他鳞翅目昆虫PP02基因相应氨基酸序列有较高的同源性,该序列具有它们的PPO基因所共有的典型特征.组织特异性表达分析表明了该基因在野桑蚕5龄幼虫的血细胞、体壁、头部、精巢、卵巢、脂肪体和中肠等组织及其不同的发育阶段均有表达.这些结果为进一步研究野桑蚕酚氧化酶原基因的功能提供了分子基础.

  3. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

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

    Science.gov (United States)

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

    2007-06-01

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

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

    International Nuclear Information System (INIS)

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

  7. 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

  8. 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.

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

    International Nuclear Information System (INIS)

    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 106 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

  10. 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.

  11. 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...

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

    OpenAIRE

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

    2012-01-01

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

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

    Science.gov (United States)

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

    1993-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-05-01

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

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

    International Nuclear Information System (INIS)

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

  16. 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.

  17. 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.

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

    OpenAIRE

    Murphy, A.J.; Efstratiadis, A

    1987-01-01

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

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

    Science.gov (United States)

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

    2009-09-01

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

  1. 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. 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.

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

    Science.gov (United States)

    Ryabov, Eugene V

    2008-01-01

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

  5. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    OpenAIRE

    Myerowitz, R; Proia, R L

    1984-01-01

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

  8. 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

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

    International Nuclear Information System (INIS)

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

  10. 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)

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

    International Nuclear Information System (INIS)

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

  12. 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.

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

    Science.gov (United States)

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

    2014-02-01

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

  14. 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...

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

    Science.gov (United States)

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

    2016-04-01

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

  16. Systematic sequencing of cDNA clones using the transposon Tn5

    OpenAIRE

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

    2002-01-01

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

  17. 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.

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

    Science.gov (United States)

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

    1988-09-01

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

  19. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    Science.gov (United States)

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities. PMID:24381103

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

    International Nuclear Information System (INIS)

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

  1. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  3. 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

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

    Science.gov (United States)

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

    2006-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  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. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    OpenAIRE

    CLAUDIA TEREZIA SOCOL; A. VLAIC; VIORICA COSIER

    2013-01-01

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

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

    Science.gov (United States)

    Johnson, K N; Ball, L A

    2001-12-01

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

  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. 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......, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression...

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

    Science.gov (United States)

    San, Cha Thye; Shah, Farida Habib

    2005-12-01

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

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

    OpenAIRE

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

    1986-01-01

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

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

    OpenAIRE

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

    1984-01-01

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

  15. 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.

  16. cDNA Cloning and Expression Analysis of Mouse Gene Encoding The Protein Ercc61 Which Is a Novel Member of SNF2 Family%SNF2家族新成员Ercc61的cDNA克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    陈祥贵; 李勇; 臧明玺; 裴新荣; 许雅君; 高丽芳

    2004-01-01

    SNF2 family includes proteins essential to genome replication, repair, and expression. The cDNA cloning and initial characterization of a novel mouse member of this protein family was reported, and the new member was designated as Erccol ( excision repair cross-complementing rodent repair deficiency, complementation group 6-like) by MGNC. The cDNA of Erccol was 4 002 bp and firstly cloned in silico and then identified by RT-PCR in mouse embryo heart. This gene was composed of 2 extrons and mapped to mouse chromosome X. The longest ORF encoded a putative protein of 1240 amino acids. Eight highly conserved sequence regions of SNF2 protein were present in the deduced protein. Alignment against members of SNF2 family initially placed Ercc61 into ERCC6 subfamily of SNF2 family. EGFP-tagged Ercc61 protein was localized in cytoplasm of HeLa, NIH3T3 and B16 cells. BLAST search in EST database retrieved 69 homologous ESTs of Ercc6l, most of which were from mouse embryonic and tumorous tissues. RT-PCR performed on a panel of different mouse tissues demonstrated that Ercc6l strongly expressed in mouse embryo but significantly downregulated in neonate and adult normal tissue. These results suggest that Ercc61 is a novel member of SNF2 family and may be crucial for development of embryo and tumor.%SNF2家族蛋白在基因组复制、修复与表达中具有重要作用.报道了SNF2家族新成员Ercc61(excision repair crosscomplementing rodent repair deficiency,complementation group 6-like)的cDNA克隆、特性与表达分析.通过表达序列标签(EST)搜索和组装,获得了cDNA全长4002 bp的新基因Ercc6l(GenBank Acc.No AY172688),然后通过RT-PCR在小鼠胚胎心脏成功克隆了该基因.Ercc6l在小鼠基因组中由两个外显子和一个内含子组成,定位于X染色体,最大开放阅读框(ORF)编码一个含l 240个氨基酸的假定蛋白质.该假定蛋白质含有SNF2蛋白的8个保守基序(SNF2结构域).通过与SNF2家族各亚家族的成员

  17. 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.

  18. Molecular cloning of human interferon cDNA.

    OpenAIRE

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

    1980-01-01

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

  19. Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene.

    Science.gov (United States)

    Ramos-Martínez, Erick M; Herrera-Ramírez, Alejandra C; Badillo-Corona, Jesús Agustín; Garibay-Orijel, Claudio; González-Rábade, Nuria; Oliver-Salvador, María Del Carmen

    2012-07-01

    Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme. PMID:22543019

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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...

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

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

    OpenAIRE

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

    1980-01-01

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

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

    Science.gov (United States)

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2004-02-27

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

  8. 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.

  9. 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...

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

    Science.gov (United States)

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

    2011-06-01

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

  11. 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

  12. 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.

  13. 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.

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

    Science.gov (United States)

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

    1992-01-01

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

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

    Science.gov (United States)

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

    2012-06-01

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

  16. 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.

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

    Science.gov (United States)

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

    1995-04-01

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

  18. 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.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

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

  20. 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.

  1. 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.

  2. A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

    OpenAIRE

    Grafi, G; R.J. Burnett; Helentjaris, T; Larkins, B A; DeCaprio, J A; Sellers, W R; Kaelin, W G

    1996-01-01

    Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in ...

  3. 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

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

    International Nuclear Information System (INIS)

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

  5. 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.

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

  10. Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair.

    OpenAIRE

    Thomas, S M; Sedgwick, S G

    1989-01-01

    Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, ...

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

    Science.gov (United States)

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

    1998-07-01

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

  12. 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.

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

    Science.gov (United States)

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

    1988-07-01

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

  14. 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.

  15. 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.

  16. 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.

  17. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    International Nuclear Information System (INIS)

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    -terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-01-20

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

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

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-06-01

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

  2. 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.

  3. 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.

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

    OpenAIRE

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

    1989-01-01

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

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

    OpenAIRE

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

    1983-01-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

    Science.gov (United States)

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

    2015-04-15

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

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

    Science.gov (United States)

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

    1999-03-01

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

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

    Science.gov (United States)

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

    1999-01-01

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

  10. 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.

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

    International Nuclear Information System (INIS)

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

  12. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

    OpenAIRE

    Singh, M. B.; Hough, T.; Theerakulpisut, P; Avjioglu, A; S. Davies; Smith, P. M.; Taylor, P.; Simpson, R. J.; Ward, L. D.; McCluskey, J

    1991-01-01

    We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE...

  13. 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.

  14. 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.

  15. 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.

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

    Science.gov (United States)

    Lang, W H

    1988-09-20

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

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

    Science.gov (United States)

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

    2007-06-15

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

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

    OpenAIRE

    Crock, John; Wildung, Mark; Croteau, Rodney

    1997-01-01

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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

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

    Science.gov (United States)

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

    2016-06-01

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

  5. 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.

  6. Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

    International Nuclear Information System (INIS)

    Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking

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

    OpenAIRE

    Digweed, M; Günthert, U

    1989-01-01

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

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

    International Nuclear Information System (INIS)

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

  9. 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.

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

    OpenAIRE

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

    2002-01-01

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

  11. Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases

    Science.gov (United States)

    Biermann, B.; Johnson, E. M.; Feldman, L. J.

    1990-01-01

    Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

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

    Science.gov (United States)

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

    1996-01-01

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

  13. 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.

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

    OpenAIRE

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

    2005-01-01

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

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Nan-Nan Liu

    2016-07-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  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. Identification of a proglucagon cDNA from Rana tigrina rugulosa that encodes two GLP-1s and that is alternatively spliced in a tissue-specific manner.

    Science.gov (United States)

    Yeung, C M; Chow, B K

    2001-11-01

    Glucagon plays a pivotal role in the regulation of metabolism. A glucagon receptor has been previously characterized in the frog, Rana tigrina rugulosa, and the frog and human glucagon receptors have been shown to possess similar binding affinities toward human glucagon. To study the structural evolution of glucagon peptide and its receptor in vertebrates, in the current study, a proglucagon cDNA from the same frog species was cloned. Interestingly, in contrast to the mammalian proglucagons that contain only one GLP-1 peptide, the frog proglucagon cDNA encodes two GLP-1 peptides (GLP-1A and GLP-1B) in addition to a glucagon peptide and a glucagon-like peptide 2 (GLP-2). By reverse transcriptase-PCR (RT-PCR) analysis, the proglucagon gene expression was widely detected in the brain, colon, small intestine, liver, lung, and pancreas, suggesting that the proglucagon-derived peptides have diverse functions in frogs. Moreover, tissue-specific alternative mRNA splicing was observed in the brain, colon, and pancreas. In these tissues, proglucagon transcripts with a 135 bp in frame deletion encoding GLP-1A were found. This splicing event in R. tigrina rugulosa is novel because it deletes a GLP-1 encoding sequence instead of the GLP-2 observed in other vertebrates. These findings should enhance understanding of the proglucagon evolution, structure, and expression in vertebrates. PMID:11703080

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

    Science.gov (United States)

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

    2016-01-01

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

  2. 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.

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

    Institute of Scientific and Technical Information of China (English)

    SUN Feifei; HOU Xilin; LI Ying; CUI Xiumin

    2007-01-01

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

  4. 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.

  5. 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

  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. 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

  8. 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

  9. 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.

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

    OpenAIRE

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

    1985-01-01

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

  11. Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

    OpenAIRE

    Yan, Y C; Bai, Y.(Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China); Wang, L.F.; Miao, S Y; Koide, S S

    1990-01-01

    A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid r...

  12. 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.

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

    OpenAIRE

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

    1997-01-01

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

  14. Cloning 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. 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.

  16. Sequence of an intestinal cDNA encoding human gastric inhibitory polypeptide precursor

    International Nuclear Information System (INIS)

    Gastric inhibitory polypeptide (GIP) is a 42-amino acid hormone that stimulates insulin secretion in the presence of glucose. Complementary DNA clones encoding human GIP were isolated from a library prepared with RNA from duodenum. The predicted amino acid sequence indicates that GIP is derived by proteolytic processing of a 153-residue precursor, preproGIP. The GIP moiety is flanked by polypeptide segments of 51 and 60 amino acids at its NH2 and COOH termini, respectively. The former includes a signal peptide of about 21 residues and an NH2-terminal propeptide of 30 amino acids. GIP is released from the precursor by processing at single arginine residues. There is a region of nine amino acids in the COOH-terminal propeptide of the GIP precursor that has partial homology with a portion of chromogranin A as well as pancreastatin

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

    OpenAIRE

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

    2016-01-01

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

  18. 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.

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

    Science.gov (United States)

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

    2014-03-01

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

  20. 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].

  1. 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

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

    OpenAIRE

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

    1982-01-01

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

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

    OpenAIRE

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

    2013-01-01

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

  4. 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.

  5. 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.

  6. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-02-01

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

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

    International Nuclear Information System (INIS)

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

  9. Cloning and Sequencing of a Gene Encoding Aminopeptidase N in the Midgut of Helicoverpa armigera (Hübner)

    Institute of Scientific and Technical Information of China (English)

    WANG Gui-rong; LIANG Ge-mei; WU Kong-ming; GUO Yu-yuan

    2003-01-01

    A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACEtechnique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp inlength, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108.3kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features withaminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18 E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini ishydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accessionnumber is AY181026.

  10. 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

  11. 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.

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

    Science.gov (United States)

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

    1991-01-17

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

  14. 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.

  15. 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

  16. 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.

  17. Identification and characterization of the cDNA sequence encoding amelogenin in rabbit (Oryctolagus cuniculus).

    Science.gov (United States)

    Bai, Chunyan; Li, Yumei; Yan, Shouqing; Fang, Hengtong; Sun, Boxing; Zhang, Jiabao; Zhao, Zhihui

    2016-02-01

    Amelogenins, the most abundant proteins in tooth enamel extracellular matrix (ECM), are essential for tooth amelogenesis. The nucleotide sequence of amelogenin gene (AMEL) for rabbit, as an important member of mammals and good continuously growing incisor model, is important for comparative and evolutional study. Previous studies about rabbit amelogenin proteins got no consensus yet even as to their existence or size. In this study, with combined usage of in silico and molecular cloning technologies, we identified sequences of two transcripts of rabbit amelogenin, resulting from the alternative splicing of the 45-bp exon 4. The coding regions of the two transcripts are of 567- and 522-bp, encoding 188 and 173 amino acids including a 17-residue signal peptide, respectively. Sequence analysis revealed that rabbit amelogenin features in extremely high GC-content in nucleotide sequence and Alanine content in protein sequence. Detailed comparison of amino acid sequence with other mammals showed that the rabbit amelogenin protein is conserved in the sites and regions important for protein functions. Overall, our results uncovered the mysteries about rabbit amelogenin and revealed its sequence peculiarities. PMID:26551300

  18. 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).

  19. Cloning and Sequencing of a Gene Encoding GOBP2 in the Antenna of Spodoptera exigua

    Institute of Scientific and Technical Information of China (English)

    WANG Gui-rong; GUO Yu-yuan; XU Guang; WU Kong-ming

    2002-01-01

    A pair of degenerate primers was designed, based on the comparison of five insects' GOBP2 gene sequences reported previously. A specific band (about 400bp in length) was amplified from cDNA of Spodoptera exigua antenna and another specific band (about 2kb in length) was amplified from genomic DNA.The two segments were cloned into T-easy vector, respectively. Results of sequencing and structural analysis showed that the full-length of GOBP2Sexi ORF is 426bp, 141 amino acid residues were encoded. The predicted MW and pI are 16.07ku and 5.09, respectively. There are six conservative Cys locus in the sequence, which is the typical characteristic of OBPs. GOBP2Sexi gene was inserted by two introns between amino acid residue 22 and 23 and between 82 and 83. The length of two introns is 160bp and 1403bp. Results of Northern blot showed that GOBP2 gene expressed specifically in the antenna of Spodoptera exigua, and the expression level is nearly equal in the antenna of male and female moths. The sequence was deposited in GenBank/EMBL and the accession number is AJ294809.

  20. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  4. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-01-01

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

  6. 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.

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

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

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

    1985-01-01

    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, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-...

  10. 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.

  11. 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.

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

    Science.gov (United States)

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

    1990-08-01

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

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

    OpenAIRE

    Udo, Hiroshi

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-12-15

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

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

    Institute of Scientific and Technical Information of China (English)

    韩慧; 王希东; 姚正培; 夏木斯亚·卡坎

    2014-01-01

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

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

    OpenAIRE

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

    1983-01-01

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

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

    OpenAIRE

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

    1996-01-01

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

  18. 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.

  19. 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.

  20. 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.

  1. Cloning of the LamA3 gene encoding the alpha 3 chain of the adhesive ligand epiligrin. Expression in wound repair.

    Science.gov (United States)

    Ryan, M C; Tizard, R; VanDevanter, D R; Carter, W G

    1994-09-01

    We have isolated cDNA clones encoding the entire 170-kDa chain of epiligrin (alpha 3Ep) and a genomic clone encoding the alpha 3Ep gene (LamA3). Analysis of multiple cDNA clones revealed two distinct transcripts (alpha 3EpA and alpha 3EpB). Sequencing of the alpha 3EpA transcript indicated sequence and structural homology to laminin alpha 1 and alpha 2 chains that extend from domain IIIa through the carboxyl-terminal G domain. The alpha 3EpB transcript encodes a larger amino-terminal domain and contains additional epidermal growth factor repeats and sequences corresponding to domain IV of alpha 1 laminin. Fluorescence in situ hybridization indicated that the LamA3 gene is located on chromosome 18q11.2, a locus distinct from the LamA1 gene (18p11.3). The G domain of the epiligrin alpha 3 chain contains five subdomains that are individually related to the G subdomains reported for Drosophila and vertebrate laminin alpha chains. Sequence divergence within the G domain of alpha 3 epiligrin suggests that it is functionally distinct from laminin, consistent with our previous report showing that epiligrin interacts with different integrin adhesion receptors. Analysis of RNA from human foreskin keratinocytes (HFKs) identified multiple epiligrin transcripts that were down-regulated by viral transformation and differentiation. In contrast, epiligrin expression was up-regulated in wound sites of human skin. PMID:8077230

  2. 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.

  3. Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

    OpenAIRE

    Hendy, G N; Kronenberg, H M; Potts, J T; Rich, A

    1981-01-01

    We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' ...

  4. 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.

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    2012-12-01

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

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

    OpenAIRE

    Lang, W H; van Holde, K E

    1991-01-01

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

  9. Nucleotide sequence of the cDNA encoding the precursor of the beta subunit of rat lutropin.

    OpenAIRE

    Chin, W W; Godine, J E; Klein, D. R.; Chang, A S; Tan, L K; Habener, J F

    1983-01-01

    We have determined the nucleotide sequences of cDNAs encoding the precursor of the beta subunit of rat lutropin, a polypeptide hormone that regulates gonadal function, including the development of gametes and the production of steroid sex hormones. The cDNAs were prepared from poly(A)+ RNA derived from the pituitary glands of rats 4 weeks after ovariectomy and were cloned in bacterial plasmids. Bacterial colonies containing transfected plasmids were screened by hybridization with a 32P-labele...

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

    Science.gov (United States)

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

    2015-08-01

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

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

    Science.gov (United States)

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

    2001-05-01

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

  12. 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.

  13. 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.

  14. 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.

  15. cDNA cloning, genomic structure, and chromosome mapping of the human epithelial membrane protein CL-20 gene (EMP1), a member of the PMP22 family.

    Science.gov (United States)

    Chen, Y; Medvedev, A; Ruzanov, P; Marvin, K W; Jetten, A M

    1997-04-01

    CL-20 is a novel gene encoding a protein that is structurally related to but distinct from the peripheral myelin protein PMP22. Like PMP22, CL-20 is likely to play important roles in the regulation of cell proliferation, differentiation, and cell death. In this study, we describe the cloning and sequencing of a cDNA encoding the human homologue of CL-20 and characterize the genomic structure of this gene. The hCL-20 gene (HGMW-approved symbol EMP1) encodes a protein of 157 amino acids that exhibits 76% identity to the rabbit CL-20 and to the rat EMP-1, which have been described recently, and 39% identity to human PMP22. CL-20 contains four hydrophobic domains, suggesting that it is an integral membrane protein. In particular the second hydrophobic domain encoded within the fourth exon is highly conserved among CL-20, EMP-1, and PMP22, suggesting a functional role for this region. CL-20 mRNA is abundant in squamous-differentiated bronchial epithelial cells; however, low levels of CL-20 mRNA can be detected in several human tissues by Northern analysis. Retinoic acid, which inhibits squamous differentiation, represses CL-20 expression in normal human bronchial epithelial cells. The genomic structure of the hCL-20 gene was analyzed using a P1 vector containing this gene. The hCL-20 gene contains five exons about 0.2, 0.12, 0.1, 0.14, and 2.2 kb and four introns about 15, 1.9, 0.1, and 0.7 kb. We have mapped the hCL-20 gene to chromosome 12p12 by fluorescence in situ hybridization. PMID:9126480

  16. 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...

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

    Directory of Open Access Journals (Sweden)

    Abolhassani Mohsen

    2009-06-01

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

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

    OpenAIRE

    Mehdy, Mona C.; Lamb, Christopher J.

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-10-01

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

  20. 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).

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

    Science.gov (United States)

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

    1999-07-30

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

  2. Cloning and Sequence Analysis of Tocopherol Cyclase cDNA from Wheat (T. Aestivum L.)%小麦生育酚环化酶基因全长cDNA的克隆与分析

    Institute of Scientific and Technical Information of China (English)

    邹礼平; 高和平

    2009-01-01

    生育酚环化酶(TC)是维生素E生物合成途径中的%Tocopherol cyclase (TC) is a key enzyme for plant vitamin E biosynthesis. The cDNA of wheat TC was cloned by RACE-PCR with primers designed based on a EST (BQ161011) sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1404 bp, which encoding 467 amino acid residues. Homology analysis of the deduced amino acid showed 91%, 88%, 71%, 68% and 67% identity with TC from rice, maize, sesame, potato and Arabidopsis thaliana, respectively. The result of phylogenetic analysis indicated that various TC sequences were clustered into three groups.

  3. 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.

  4. [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.

  5. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    Science.gov (United States)

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

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

    Science.gov (United States)

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

    1989-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Yan-xia Bai; Qing-yong Ma; Guang-xiao Yang

    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) apoptotic peptide was amplified by using self-complementation polymerase chain reaction (PCR) method and cloned into the pGEM-T Easy vector. The constructed plasmid was confirmed by endonuclease analysis and sequencing. Results The insertion of objective DNA fragment was confirmed by plasmid DNA enzyme spectrum analysis, p53 (N37) gene was successfully synthesized chemically in vitro. The sequencing result of positive clone was completely identical to the human p53(N37) sequence in GenBank using BLAST software (http://www. ncbi. him. nih. gov/cgi-bin /BLASTn). Conclusion The cloning of DNA fragment encoding p53(N37) apoptotic peptide was constructed by using DNA synthesis and pGEM-T Easy cloning methods. With the constructed plasmid, we could further investigate the function of N37 peptide.

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

    Directory of Open Access Journals (Sweden)

    Mohammad Hadi Sekhavati

    2013-01-01

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

  17. 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.

  18. 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.

  19. Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

    Science.gov (United States)

    Uchida, K; Tsuchida, J; Tanaka, H; Koga, M; Nishina, Y; Nozaki, M; Yoshinaga, K; Toshimori, K; Matsumiya, K; Okuyama, A; Nishimune, Y

    2000-10-01

    We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation. PMID:10993819

  20. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone

    International Nuclear Information System (INIS)

    We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions

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

    Directory of Open Access Journals (Sweden)

    Hiroshi Udo

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

  2. 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.

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

    International Nuclear Information System (INIS)

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

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

    DEFF Research Database (Denmark)

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

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

  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

    [ 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.

  6. 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.

  7. 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.

  8. 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.

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

    Science.gov (United States)

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

    1991-02-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    International Nuclear Information System (INIS)

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

  12. Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes.

    OpenAIRE

    Nishimura, Y; Rosenfeld, M G; Kreibich, G; Gubler, U; Sabatini, D. D.; Adesnik, M; Andy, R

    1986-01-01

    We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome. The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries. The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues. The polypepti...

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

    Science.gov (United States)

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

    2009-07-01

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

  14. 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.

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

    Science.gov (United States)

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

    1995-03-01

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

  16. 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.

  17. 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.

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1985-01-01

    The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage λD69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed...... by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent...... deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells...

  4. 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.

  5. 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.

  6. 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

  7. 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

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

    OpenAIRE

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

    1983-01-01

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

  9. Molecular Cloning, Expression Analysis, and Preliminarily Functional Characterization of the Gene Encoding Protein Disulfide Isomerase from Jatropha curcas.

    Science.gov (United States)

    Wang, Haibo; Zou, Zhurong; Gong, Ming

    2015-05-01

    Reactive oxygen species (ROS) in plants, arising from various environmental stresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formation of disulfide bonds, which might be alleviated by a relatively specific modifier called protein disulfide isomerase (PDI). From our previous data of the transcriptome and digital gene expression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant. In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing the entire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 amino acids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminal signal peptide (-MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoring sequence motif (-KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxin domains (-CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, the expression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, but weakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeast could notably enhance the cold resistance of host cells. Conclusively, these results explicitly suggested a considerable association of JcPDI to cold response and a putative application potential for its correlated genetic engineering. PMID:25825250

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

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

    Science.gov (United States)

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

    2010-09-01

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

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

    Science.gov (United States)

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

    2004-02-01

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

  17. 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.

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

    Science.gov (United States)

    Lang, W H; van Holde, K E

    1991-01-01

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

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

    Science.gov (United States)

    Kiriake, Aya; Shiomi, Kazuo

    2011-11-01

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

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

    International Nuclear Information System (INIS)

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

  1. cDNA cloning, purification, properties, and function of a beta-1,3-glucan recognition protein from a pyralid moth, Plodia interpunctella.

    Science.gov (United States)

    Fabrick, J A; Baker, J E; Kanost, M R

    2003-06-01

    Microorganisms possess distinctive biochemical or molecular patterns on their cell surfaces, such as those formed by the lipopolysaccharides, lipoteichoic acids, and/or peptidoglycans of bacteria and the beta-1,3-glucans of fungi. Pattern recognition proteins that bind to these surface moieties have been implicated in the activation of the innate immune response in insects and other invertebrates. We report the purification and cloning of a cDNA for a 53-kDa beta-1,3-glucan recognition protein (betaGRP) from the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). BetaGRP cDNA contains an open reading frame that encodes 488 amino acids, of which the first 17 residues comprise the secretion signal peptide. The calculated molecular mass of the 471-residue mature protein is 53,311 Da. The protein consists of a carboxyl-terminal domain that is similar to other recognition proteins from invertebrates, beta-1,3-glucanases from bacteria, and a beta-1,3-glucanase from the sea urchin, Strongylocentrotus purpuratus. The amino-terminus of betaGRP shares sequence similarity with other invertebrate recognition molecules and the beta-1,3-glucanase from S. purpuratus. Affinity purification of a 53-kDa protein and subsequent sequencing of a peptide produced by tryptic cleavage confirmed the presence of the betaGRP in P. interpunctella larval hemolymph. RT-PCR analysis indicates that betaGRP is constitutively expressed in all life-stages, with no detectable induction following exposure of wandering larvae to microbial elicitors. Northern blot analysis indicates that the 1.8-kb betaGRP transcript is transcribed within the fat body. Recombinant betaGRP retains beta-1,3-glucan-binding activity, binds to lipopolysaccharide and lipoteichoic acid in vitro, causes aggregation of microorganisms, and activates the prophenoloxidase cascade in the presence of soluble beta-1,3-glucan. These data support the hypothesis that the 53-kDa betaGRP functions to recognize

  2. cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.

    Institute of Scientific and Technical Information of China (English)

    HU Zhen-di; FENG Xia; LIN Qing-sheng; CHEN Huan-yu; LI Zhen-yu; YIN Fei; LIANG Pei; GAO Xi-wu

    2016-01-01

    Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a ful-length cDNA of carboxylesterasepxCCE016b was cloned and exogenously expressed inEscherichia coliat the ifrst time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of thepxCCE016b was signiifcantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore,pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfuly with prokaryotic cel. Western blot analysis showed that it was coded by pxCCE016b. Al the above ifndings provide important information for further functional study, although we are uncertainty

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  5. 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.

  6. 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.

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

    OpenAIRE

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

    1992-01-01

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

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

    Science.gov (United States)

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

    2009-05-01

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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Isolation and sequence of a cDNA encoding the precursor of a bombesinlike peptide from brain and early embryos of Xenopus laevis.

    OpenAIRE

    Wechselberger, C; Kreil, G; Richter, K.

    1992-01-01

    A cDNA encoding the precursor of a bombesinlike peptide was isolated from brain of Xenopus laevis. The predicted end product resembles neuromedin B, which was originally isolated from mammalian spinal cord. The mRNA for this precursor was also present in gastrointestinal tract and in ovaries. Moreover, it could be detected in early embryos (stage 2 and stage 10) of X. laevis. These findings suggest novel roles for peptides of the bombesin family in oocyte maturation and early amphibian develo...

  14. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    International Nuclear Information System (INIS)

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in λ phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (∼ 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia

  15. 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.

  16. Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.

    OpenAIRE

    Latimer, M T; Ferry, J G

    1993-01-01

    The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of th...

  17. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    OpenAIRE

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein se...

  18. 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...

  19. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems

    OpenAIRE

    Kagale Sateesh; Yang Wen; Rozwadowski Kevin

    2008-01-01

    Abstract Background Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular res...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Cloning of a down-regulated gene encoding small GTP binding protein in hybrid wheat

    Institute of Scientific and Technical Information of China (English)

    YAO Yingyin; NI Zhongfu; CHEN Rongmin; WU Limin; SUN Qixin

    2005-01-01

    Previous studies showed that differential gene expression between wheat hybrids and their parents was responsible for the heterosis. To provide an insight into the molecular basis of wheat heterosis, one cDNA, designated TaRab, was identified from the cDNA library of wheat seedling leaves. The sequence comparison in GenBank revealed that TaRab is homologous to a group of genes encoding Rab-GTP binding protein. Semi-quantitative RT-PCR analysis indicated that TaRab was expressed in all plant tissues examined,but at slightly higher level in leaves. Further analysis exhibited that TaRab displayed lower expression in hybrid than in its patents in both roots and leaves, which was in agreement with the original results of suppression subtractive hybridization. TaRab was located on chromosome 7B and C-7DS5-0.36 by in silico mapping. The relationship between differential expression of TaRab and the molecular basis of wheat heterosis was also discussed.

  5. 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.

  6. 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.

  7. 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. PMID:22288192

  8. (+)-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.

  9. 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.

  10. 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.

  11. Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth

    International Nuclear Information System (INIS)

    Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a λgt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggest that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution

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

    International Nuclear Information System (INIS)

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

  13. 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...

  14. Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.

    Science.gov (United States)

    Lieb, B; Altenhein, B; Lehnert, R; Gebauer, W; Markl, J

    1999-10-01

    We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octopus dofleini hemocyanin. HtH2-e is the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequences are compared to their counterparts in Octopus, Helix pomatia and HtH1 (from the latter, the sequences of FU-f, FU-g and FU-h have recently been determined) and discussed in relation to the recent 2.3 A X-ray structure of FU-g from Octopus hemocyanin and the 15 A three-dimensional reconstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamers in vivo, the additional N-glycosylation sites in HtH1 compared to HtH2 are discussed. PMID:10491167

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin.

    OpenAIRE

    Tweten, R K; Collier, R J

    1983-01-01

    Two restriction fragments from corynebacteriophage beta vir tox+ that encode peptides similar to diphtheria toxin fragment A and the chain termination fragment, CRM45, have been cloned into Escherichia coli in plasmid pBR322. Clones containing the recombinant plasmids produced gene products that were active in catalyzing the ADP ribosylation of elongation factor 2 and were reactive with diphtheria toxin antiserum. Toxin-related peptides were found primarily in the periplasmic compartment and ...

  3. 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...

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

    OpenAIRE

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

    1999-01-01

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

  5. 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.

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

    International Nuclear Information System (INIS)

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

  7. 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.

  8. cDNA cloning, characterization and expression analysis of a novel antimicrobial peptide gene penaeidin-3 (Fi-Pen3) from the haemocytes of Indian white shrimp Fenneropenaeus indicus.

    Science.gov (United States)

    Shanthi, S; Vaseeharan, B

    2012-03-20

    A new member of antimicrobial peptide genes of the penaeidin family, penaeidin 3, was cloned from the haemocytes of Indian white shrimp Fenneropeneaus indicus (F. indicus), by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE-PCR) methods. The complete nucleotide sequence of cDNA clone of Indian white shrimp F. indicus Penaeidin 3 (Fi-Pen3) was 243bp long and has an open reading frame which encodes 80 amino acid peptide. The homology analysis of Fi-Pen3 sequence with other Penaeidins 3 shows higher similarity with Penaeus monodon (92%). The theoretical 3D structure generated through ab initio modelling indicated the presence of two-disulphide bridges in the alpha-helix. The signal peptide sequence of Fi-Pen3 is almost entirely homologous to that of other Penaeidin 3 of crustaceans, while differing relatively in the N-terminal domain of the mature peptide. The mature peptide has a predicted molecular weight of 84.9kDa, and a theoretical pI of 9.38. Phylogenetic analysis of Fi-Pen3 shows high resemblance with other Pen-3 from P. monodon, Litopenaeus stylirostris, Litopenaeus vannamei and Litopenaeus setiferus. Fi-Pen3 found to be expressed in haemocytes, heart, hepatopancreas, muscles, gills, intestine, and eyestalk with higher expression in haemocytes. Microbial challenge resulted in mRNA up-regulation, up to 6h post injection of Vibrio parahemolyticus. The Fi-Pen3 mRNA expression of F. indicus in the premolt stage (D(01) and D(02)) was significantly up-regulated than the postmolt (A and B) and intermolt stages (C). The findings of the present paper underline the involvement of Fi-Pen3 in innate immune system of F. indicus. PMID:21885268

  9. 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.

  10. Molecular Cloning and Sequence Analysis of the cDNAs Encoding Toxin-Like Peptides from the Venom Glands of Tarantula Grammostola rosea

    OpenAIRE

    Tadashi Kimura; Seigo Ono; Tai Kubo

    2012-01-01

    Tarantula venom glands produce a large variety of bioactive peptides. Here we present the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared from the venom glands of the Chilean common tarantula, Grammostola rosea. The cDNA sequences of about 1500 clones out of 4000 clones were analyzed after selection using several criteria. Forty-eight novel toxin-like peptides (GTx1 to GTx7, and GTx-TCTP and GTx-CRISP) were predicted from the nucleotide s...

  11. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    OpenAIRE

    Dong, G.; Vieille, C; Zeikus, J G

    1997-01-01

    The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three protei...

  12. 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.

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

    International Nuclear Information System (INIS)

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

  14. 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.

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

    Science.gov (United States)

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

    2007-01-01

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

  16. 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.

  17. 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.

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

    Science.gov (United States)

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

    2009-11-01

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

  19. Cloning and Expression Analysis of an XET cDNA in the Peel and Pulp of Banana Fruit Ripening and Softening

    Institute of Scientific and Technical Information of China (English)

    LUWang-Jin; RyoheiNAKANO; YasutakaKUBO; AkitsuguINABAt; JIANGYue-Ming

    2004-01-01

    Xyloglucan endotransglycosylase (XET) is thought to be involved in fruit softening throughdisassembly of xyloglucan, which is the predominant hemicellulose of cell wall. To study the relationshipbetween fruit softening and XET during banana (Musa acuminata Colla cv. Grand Nain) fruit ripening, a fulllength cDNA (1 095 bp) encoding an XET, MA-XET1, was isolated from ripening banana fruit using RT-PCRand RACE-PCR (rapid amplification of cDNA ends) methods. Sequence analysis showed that the cDNAcontains 5' untranslated region of 66 bp, 3' untranslated region of 189 bp and ORF of 840 bp, encoding apredicted polypeptide of 280 amino acids, including DE|DFEFL motif, which is a presumptive catalyticdomain conserved in XETs. DNA gel blot analysis demonstrated that MA-XET1 is encoded by a multi-copyfamily in the banana genome. RNA gel blot analysis revealed that the level of MA-XET1 transcript in thepulp was undetectable, increased and decreased slightly at the preclimacteric, climacteric and postclimactericstages, respectively. In the peel, accumulation of MA-XET1 transcript was low, increased dramatically andthen decreased rapidly, at preclimacteric, climacteric and postclimacteric stages, respectively. Treatmentof fruit with propylene, an analog of ethylene, decreased the firmness and enhanced the accumulation ofMA-XET1 transcript in the peel and pulp. These results suggest that MA-XET1 is involved in softening ofthe peel and pulp during banana fruit ripening and its expression is regulated by ethylene at transcriptionallevel.

  20. Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Goldgaber, D.; Lerman, M.I.; McBride, O.W.; Saffiotti, U.; Gajdusek, D.C.

    1987-02-20

    Four clones were isolated from an adult human brain complementary DNA library with an oligonucleotide probe corresponding to the first 20 amino acids of the ..beta.. peptide of brain amyloid from Alzheimer's disease. The open reading frame of the sequenced clone coded for 97 amino acids, including the known amino acid sequence of this polypeptide. The 3.5-kilobase messenger RNA was detected in mammalian brains and human thymus. The gene is highly conserved in evolution and has been mapped to human chromosome 21.

  1. 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

  2. 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.

  3. Cloning and Analysis of microRNAs Encoded by the Primate γ-Herpesvirus Rhesus Monkey Rhadinovirus

    OpenAIRE

    Schäfer, Alexandra; Cai, Xuezhong; Bilello, John P.; Desrosiers, Ronald C.; Cullen, Bryan R.

    2007-01-01

    Several pathogenic human herpesviruses have recently been shown to express virally encoded microRNAs in infected cells. Although the function of these microRNAs is largely unknown, they are hypothesized to play a role in mediating viral replication by down-regulating cellular mRNAs encoding antiviral factors. Here, we report the cloning and analysis of microRNAs encoded by Rhesus Monkey Rhadinovirus (RRV), an animal virus model for the pathogenic human γ-herpesvirus Kaposi’s Sarcoma-Associate...

  4. 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

  5. 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 a...

  6. 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.

  7. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

    Science.gov (United States)

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-01-18

    Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN. PMID:9931470

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

    OpenAIRE

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

    2002-01-01

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

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

    OpenAIRE

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

    2004-01-01

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

  10. Molecular characterization of cDNA encoding oxygen evolving enhancer protein 1 increased by salt treatment in the mangrove Bruguiera gymnorrhiza.

    Science.gov (United States)

    Sugihara, K; Hanagata, N; Dubinsky, Z; Baba, S; Karube, I

    2000-11-01

    Young plants of the common Okinawa mangrove species Bruguiera gymnorrhiza were transferred from freshwater to a medium with seawater salt level (500 mM NaCl). Two-dimensional gel electrophoresis revealed in the leaf extract of the plant a 33 kDa protein with pI 5.2, whose quantity increased as a result of NaCl treatment. The N-terminal amino acids sequence of this protein had a significant homology with mature region of oxygen evolving enhancer protein 1 (OEE1) precursor. The cloning of OEE1 precursor cDNA fragment was carried out by means of reverse transcription-PCR (RT-PCR) using degenerated primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence consisted of 322 amino acids and was 87% identical to that of Nicotiana tabacum. In B. gymnorrhiza, the predicted amino acid sequence of the mature protein starts at the residue number 85 of the open reading frame. The first 84-amino acid residues correspond to a typical transit sequence for the signal directing OEE1 to its appropriate compartment of chloroplast. The expression of OEE1 was analyzed together with other OEE subunits and D1 protein of photosystem II. The transcript levels of all the three OEEs were enhanced by NaCl treatment, but the significant increase of D1 protein was not observed. PMID:11092914

  11. 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

  12. 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.

  13. 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.

  14. Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

    OpenAIRE

    Wallace, B; Yang, Y. J.; Hong, J S; Lum, D

    1990-01-01

    A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter co...

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

    OpenAIRE

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

    1985-01-01

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

  16. Identification and characterization of a cDNA encoding a crustin-like, putative antibacterial protein from the American lobster Homarus americanus.

    Science.gov (United States)

    Christie, Andrew E; Rus, Szymon; Goiney, Christopher C; Smith, Christine M; Towle, David W; Dickinson, Patsy S

    2007-07-01

    Pathogenic challenges in decapod crustaceans are combated by innate immune responses, including the production and secretion of soluble antibacterial proteins into the hemolymph. Among the antibacterials that have been identified in decapod species are the crustins, a group of four-disulfide core/whey-acidic-protein (WAP) domain-containing proteins, which target marine/salt tolerant Gram-positive bacteria. To begin to assess the possible role of crustins in combating bacterial invasion in the American lobster Homarus americanus, we identified and sequenced a 744 base pair cDNA that encodes a novel 96 amino acid crustin-like protein. Comparison of H. americanus crustin (Hoa-crustin) with crustins from other decapod species showed that it is most similar to an isoform predicted from the European lobster Homarus gammarus ( approximately 86% identity). With our identification of the Hoa-crustin cDNA, we are positioned to begin molecular and physiological investigations of the regulation and function of this putative antibacterial protein in H. americanus. PMID:17418897

  17. 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.

  18. 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.

  19. 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.

  20. From Plant Extract to a cDNA Encoding a Glucosyltransferase Candidate: Proteomics and Transcriptomics as Tools to Help Elucidate Saponin Biosynthesis in Centella asiatica.

    Science.gov (United States)

    de Costa, Fernanda; Barber, Carla J S; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant. PMID:26843164

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

    Science.gov (United States)

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

    2002-02-01

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

  2. 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.

  3. Cloning and Characterization of a Gene (mspA) Encoding the Major Sheath Protein of Treponema maltophilum ATCC 51939T

    OpenAIRE

    Heuner, Klaus; Choi, Bong-Kyu; Schade, Rüdiger; Moter, Annette; Otto, Albrecht; Göbel, Ulf B.

    1999-01-01

    The major sheath protein-encoding gene (mspA) of the oral spirochete Treponema maltophilum ATCC 51939T was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The mspA gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an mspA-s...

  4. 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...

  5. tef1, a Phytophthora infestans gene encoding translation elongation factor 1-alpha

    NARCIS (Netherlands)

    Klooster, van 't J.W.; Berg-Velthuis, van den G.; West, van P.; Govers, F.

    2000-01-01

    From a set of Phytophthora infestans cDNA clones randomly selected from a potato-P. infestans interaction cDNA library, three out of 22 appeared to correspond to a gene encoding translation elongation factor 1α. The gene, called tef1, is a single copy gene in P. infestans. During the life cycle of P

  6. Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus.

    OpenAIRE

    Colombo, S.; G. Toietta; Zecca, L.; Vanoni, M; Tortora, P.

    1995-01-01

    Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic resi...

  7. Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor.

    OpenAIRE

    Zhang, Y. X.; Tang, L.; Hutchinson, C R

    1996-01-01

    A homolog of the mmsA gene of Pseudomonas aeruginosa, which encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is involved in valine catabolism in pseudomonads and mammals, was cloned and sequenced from Streptomyces coelicolor. Of the two open reading frames (ORFs) found, which are convergently transcribed and separated by a 62-nucleotide noncoding region, the deduced amino acid sequence of the msdA ORF (homologous to mmsA) is similar to a variety of prokaryotic and eukaryotic a...

  8. Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus.

    OpenAIRE

    Theisen, M.; Rioux, C R; Potter, A A

    1993-01-01

    Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an...

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

    Science.gov (United States)

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

    1998-06-01

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

  10. 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.

  11. CDNA cloning and mRNA expression of the six mouse insulin-like growth factor binding proteins

    NARCIS (Netherlands)

    A.G.P. Schuller (Alwin); C. Groffen (Cora); J.W. van Neck (Han); E.C. Zwarthoff (Ellen); S.L.S. Drop (Stenvert)

    1994-01-01

    textabstractThe insulin-like growth factor binding proteins (IGFBPs) comprise a family of six distinct proteins which modulate insulin-like growth factor action. We have isolated cDNAs encoding the six mouse IGFBPs (mIGFBPs). In addition, we studied the mRNA expression of the six mIGFBPs during deve

  12. 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

  13. 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.

  14. 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.

  15. Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA.

    Science.gov (United States)

    Richman, A M; Bulet, P; Hetru, C; Barillas-Mury, C; Hoffmann, J A; Kafalos, F C

    1996-08-01

    Larvae of the mosquito vector of human malaria, Anopheles gambiae, were inoculated with bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phylogenetically conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is induced in response to bacterial infection, in both adult and larval stages. In contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance. PMID:8799739

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

    Science.gov (United States)

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

    1981-02-01

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

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

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

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

  18. 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

  19. 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...

  20. 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.

  1. 番茄GDP-D-甘露糖焦磷酸化酶cDNA的克隆、表达及定位%Cloning, Expression, and Mapping of GDP-D-mannose Pyrophosphorylase cDNA from Tomato (Lycopersicon esculentum)

    Institute of Scientific and Technical Information of China (English)

    邹礼平; 李汉霞; 欧阳波; 张俊红; 叶志彪

    2006-01-01

    GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled.The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1 086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato,tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.%GDP-D-甘露糖焦磷酸化酶催化GDP-D-甘露糖的合成,是植物抗坏血酸生物合成途径中上游的关键酶.以马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列为信息探针,在GenBank dbEST数据库中找到65条高度同源的番茄EST序列,通过序列拼接及RACE-PCR得到了番茄该基因的全长cDNA序列,命名为LeGMP.LeGMP与马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列一致率为96%,推导的氨基酸序列与马铃薯、烟草、紫苜蓿、拟南芥的GDP-D-甘露糖焦磷酸化酶基因的一致率分别为99%、97%、91%、89%.经Northern杂交分析,LeGMP在番茄根、茎、叶、花、果实中都有表达,但表达水平有差异.利用75个番茄远缘杂交重组系(IL系)将LeGMP定位在番茄第3

  2. 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.

  3. cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes).

    Science.gov (United States)

    Xia, Xiaohua; Zhao, Jie; Du, Qiyan; Chang, Zhongjie

    2010-08-01

    The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads. PMID:20861569

  4. cDNA cloning and expression analysis of two distinct Sox8 genes in Paramisgurnus dabryanus (Cypriniformes).

    Science.gov (United States)

    Xia, Xiaohua; Zhao, Jie; Du, Qiyan; Chang, Zhongjie

    2010-08-01

    The Sox9 gene attracts a lot of attention because of its connection with gonadal development and differentiation. However, Sox8, belonging to the same subgroup SoxE, has rarely been studied. To investigate the function as well as the evolutionary origin of SOXE subgroup, we amplified the genomic DNA of Paramisgurnus dabryanu using a pair of degenerate primers. Using rapid amplification of the cDNA ends (RACE), it was discovered that P. dabryanu has two duplicates: Sox8a and Sox8b. Each has an intron of different length in the conserved HMG-box region. The overall sequence similarity of the deduced amino acid of PdSox8a and PdSox8b was 46.26%, and only two amino acids changed in the HMG-box. This is the first evidence showing that there are two distinct duplications of Sox8 genes in Cypriniformes. Southern blot analysis showed only one hybrid band, with lengths 7.4 or 9.2 kb. Both semi-quantitative RT-PCR and real-time quantitative PCR assay displayed that both PdSox8a and PdSox8b are downregulated during early embryonic development. In adult tissues, the two Sox8 genes expressed ubiquitously, and expression levels are particularly high in the gonads and brain. In gonads, both PdSox8a and PdSox8b are expressed at a higher level in the tesis than in the ovary. PdSox8a and PdSox8b may have functional overlaps and are essential for the neuronal development and differentiation of gonads.

  5. 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.

  6. 菊花花瓣XTH基因cDNA序列的克隆与分析%Cloning and Analysis of the cDNA Sequence of XTH Gene in Chrysanthemum × morifolium Petal

    Institute of Scientific and Technical Information of China (English)

    孙悦; 苏媚; 王蕾; 姬筱雅; 郑必平; 谈建中

    2012-01-01

    [Objective] The aim was to study the structural characteristics of xyloglucan endotransglycosylase/ hydrolase ( XTH) gene in eth-ylene-insensitive feverfew. [ Method] The total RNA was extracted from Chrysanthemum x morifolium petal using Trizol reagent, and the cD-NA fragment of XTH gene was cloned by RT-PCR and T/A cloning, and then analyzed by agarose gel eleetrophresis and sequencing. [ Result] The cloned cDNA sequence was 911 bp. It was predicted to encode a polypeptide of 293 amino acids and had seven active sites of XTH family, and then named as CmXTH (gene accession number HM752243). In addition, the BLAST analysis showed that the deduced amino acid sequence of CmXTH showed high homology with other 19 chosen plant XTHs. Among these, CmXTH had closer genetic relationship with Ger-bera hybrid cuhivar XET, Solatium lycopersicum XTH7, whereas had relatively distant relationship with Populus euparatica XET, Fragaria ananassa XET1, Actinidia deliciosa XET, etc. [Conclusion] The cloned fragment was certainly cUNA sequence of XTH gene, which was associated with the petal, growth and senescence in Chrysanthemum x morifolium. The study lays foundation for further studying the structure and biological functions of XTH genes as well as the growth of Chrysanthemum X morifolium petals.%[目的]研究非乙烯敏感型菊科植物的木葡聚糖内糖基转移酶/水解酶(xyloglucan endo-transglycosylase/hydrolase,XTH)基因的结构特征.[方法]采用Trizol方法从菊花花瓣中提取总RNA,利用RT-PCR技术和T/A载体克隆XTH基因的cDNA序列,并对目的序列进行琼脂糖凝胶电泳和测序分析.[结果]克隆的cDNA片段大小为911 bp,编码293个氨基酸残基,具有XTH蛋白家族的7个活性位点,命名该基因序列为CmXTH(基因登录号为HM752243);其推导的氨基酸序列与其他19种植物的XTH具有较高的同源性,其中与非洲菊、蕃茄的亲缘关系最近,而与猕猴桃、草莓、胡杨等的亲缘关系较远.[结论

  7. Molecular Cloning of a Lectin cDNA from Alocasia macrorrhiza and Prediction of Its Characteristics%海芋凝集素cDNA的分子克隆及其性质预测

    Institute of Scientific and Technical Information of China (English)

    朱亚然; 王捷; 黄炳球; 侯学文

    2006-01-01

    用cDNA末端快速扩增-聚合酶链式反应(RACE-PCR)方法克隆了海芋(Alocasia macrorrhiza)凝集素的全长cDNA(GenBank检索号DQ340864),并用多种生物信息学工具对其性质进行了预测.根据来源于天南星科其他植物的凝集素和类似蛋白的保守区的DNA序列,设计了几个海芋凝集素基因aml特异引物(GSP).用RNeasy试剂盒从海芋块茎中提取出总RNA,并以此为模板,用SMARTTM RACE cDNA扩增试剂盒提供的经特殊设计的通用引物以及不同的基因特异引物,分别获得海芋凝集素5'-和3'-RACE-PCR扩增片段.这些PCR产物经0.8%琼脂糖凝胶纯化后,分别与T克隆载体pMD 18-T相连,筛选获得阳性克隆并提取质粒,经双酶切和特异引物的PCR验证无误后,进行序列分析.从5'-和3'-RACE-PCR测序结果拼接出全长海芋凝集素cDNA序列,并用新设计自5'-RACE-PCR 5'末端的引物GSP7进行全长3'-RACE-PCR反应,获得全长海芋凝集素cDNA克隆并再次测序验证.这一新克隆的海芋凝集素cDNA的长度为1124核苷酸,分析表明它是一个编码270个氨基酸残基的蛋白质,其等电点为pH 5.7,相对分子量为29.7 kD.同源性分析结果表明,海芋凝集素与其他来源于天南星科的甘露糖凝集素以及相似蛋白具有高度同源性.在海芋凝集素序列中发现了2个B型凝集素功能区域和3个甘露糖的结合位点.综合上述信息,认为这一新克隆的海芋凝集素cDNA是一个编码甘露糖识别凝集素的基因序列.%The cDNA of Alocasia macrorrhiza lectin(aml, GenBank accession number: DQ340864) was cloned by RACE-PCR and its characteristics were predicted by various bioinformatics tools. GSPs (Gene Specific Primers) were designed according to the conserved regions of the genes encoded for lectins and similar proteins from the same family Araceae. Total RNAs were extracted from the tubers of A. macrorrhiza by Qiagen RNeasy mini kit. The 3'- and 5'-RACE-PCRs were performed with the

  8. 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.

  9. Characterization and construction of functional cDNA clones of Pariacoto virus, the first Alphanodavirus isolated outside Australasia.

    Science.gov (United States)

    Johnson, K N; Zeddam, J L; Ball, L A

    2000-06-01

    Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as a Nodavirus. As such, PaV is the first Alphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3' end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor alpha. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins beta and gamma, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5' and 3' termini. Transcription of these plasmids in cells

  10. 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).

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

    Institute of Scientific and Technical Information of China (English)

    Weijun Mai; Chaoqun Hu

    2009-01-01

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

  12. 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.

  13. Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone.

    Science.gov (United States)

    Nagashima, Shigeo; Kobayashi, Tominari; Tanaka, Toshinori; Tanggis; Jirintai, Suljid; Takahashi, Masaharu; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-09-01

    To characterize the genomic mutations of hepatitis E virus (HEV) during consecutive passages associated with adaptation to growth in cell culture, a cloned genotype 3 HEV [pJE03-1760F/wt, starting virus (SV)] was passaged 10 times in A549 cells, and the entire genomic sequence of the passage 10 (P10) progeny was determined. Compared to SV, P10 virus possessed two non-synonymous (T2808C and A5054G) and four synonymous mutations (C1213T, T2557C, C3118T and C4435T) in the ORF1. Full-length infectious cDNA clones with a single, double (T2808C and A5054G), or all six mutations, identical to P10, were constructed, and their replication capacity was compared. Four (C1213T, T2557C, T2808C and A5054G) of the six viruses with a single mutation grew more efficiently than SV. The P10 virus propagated more rapidly and grew more efficiently than SV and T2808C+A5054G and reached a higher viral load (95.1- and 8.5-fold, respectively) at 20days post-inoculation. An immunofluorescence analysis revealed that a high percentage (>80%) of cells inoculated with the P10 virus expressed ORF2 proteins, while relatively low percentages (nearly 30% or 5%) inoculated with T2808C+A5054G or SV, respectively, expressed ORF2 proteins. We found that not only non-synonymous but also synonymous HEV mutations are independently associated with increased virus production. PMID:27485920

  14. 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

  15. 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

  16. 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

  17. A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

    OpenAIRE

    Valéria Pereira Ferrer; Thiago Lopes de Mari; Luiza Helena Gremski; Dilza Trevisan Silva; Rafael Bertoni da Silveira; Waldemiro Gremski; Olga Meiri Chaim; Andrea Senff-Ribeiro; Helena Bonciani Nader; Silvio Sanches Veiga

    2013-01-01

    Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide...

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

    Science.gov (United States)

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

    2006-05-01

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

  19. Cloning and Expression of Glucoamylase Genes from Aspergilus niger cDNA Library in Pichia pastrois%黑曲霉糖化酶基因在毕赤酵母中的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    汤斌; 钱鹏

    2012-01-01

    从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。%An expression cDNA library was constructed from high-yielding glucoamylase strains of A.niger and the glucoamylase gene was isolated,then the expression of the gene in Pichia pastoris was studied.The cDNA sequence of glucoamylase from A.niger was obtained by RT-PCR.The cDNA fragment was cloned into the expression vector pPIC9K and the linearized recombinant vector was transformed to Pichia pastrois GS115 by electroporation.The positive clones were analyzed subsequently.The recombined Pichia pastrois were cultured in the MM medium,using 1% methanol to induce the expression of recombinant gene.The results showed that the maximum activity of glucoamylase was 15.6 U/mL after it was fermented for 72 h.Sequence analysis revealed that glucoamylase had 1908 bp,which encodes a putative polypeptide of 636 amino acids.The expressed protein was purified from the fermented supernatant using DEAE column and determined by SDS-PAGE.The result of SDS-PAGE also showed that the molecular weights of the enzyme was 80 kDa.The expression vector of the glucoamylase gene was constructed successfully,and it could express glucoamylase in Pichia pastrois.

  20. Cloning of the Streptococcus gordonii PK488 gene, encoding an adhesin which mediates coaggregation with Actinomyces naeslundii PK606.

    OpenAIRE

    Andersen, R N; Ganeshkumar, N.; Kolenbrander, P. E.

    1993-01-01

    Coaggregation between Streptococcus gordonii PK488 and Actinomyces naeslundii PK606 is mediated by a 38-kDa streptococcal protein, designated ScaA. The gene, scaA, which encodes this protein has been cloned into Escherichia coli. A genomic S. gordonii PK488 library (in Lambda ZAP II) was screened with anti-S. gordonii immunoglobulin G absorbed with S. gordonii PK1804, an isogenic coaggregation-defective mutant of strain PK488. A positive recombinant phage was isolated, and a phagemid designat...