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Sample records for cdna cloning chromosomal

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-01-15

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

  2. Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

    Science.gov (United States)

    Kuroiwa, A; Matsubara, K; Nagase, T; Nomura, N; Seong, J K; Ishikawa, A; Anunciado, R V; Tanaka, K; Yamagata, T; Masangkay, J S; Dang, V B; Namikawa, T; Matsuda, Y

    2001-01-01

    The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.

  3. Cloning chromosome specific genes by reciprocal probing of arrayed cDNA and cosmid libraries

    Energy Technology Data Exchange (ETDEWEB)

    Yazdani, A.; Lee, C.C.; Wehnert, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    A human gene map will greatly facilitate the association of genes to single locus diseases and provide candidates for genes involved in complex genetic traits. Given the estimated 100,000 human genes an integrated strategy with a high throughput approach for isolation and mapping of expressed sequences is needed to create such a gene map. We have developed an approach that allows high throughput gene isolation and mapping using arrayed genomic and cDNA lambda libraries. Reciprocal probing of the arrayed genomic and cDNA cosmic libraries can rapidly establish cDNA-cosmid associations. Fluorescence in situ hybridization (FISH) chromosomal mapping and expressed sequence tag/sequence tag site (EST/STS) primers generated from DNA sequence of PCR-based mapping using somatic hybrid cell line mapping panels were utilized to characterize further the hybridization-based cDNA cosmid association. We have applied this approach to chromosome 17 using a placental cDNA library and have identified a total of 30 genes out of which 11 are novel. Furthermore seven cDNAs were mapped to 17q21 in this study, providing novel candidate genes for BRCA-1 gene for early onset breast cancer. The results of our study clearly show that an integration of an expression map into physical and genetic maps can provide candidate genes for human diseases that have been mapped to specific regions. This approach combined with the current physical mapping efforts could efficiently provide a detailed human gene map.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-05-01

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

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    1994-11-11

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-07-03

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

  9. 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

  10. Isolation and characterization of a steroid sulfatase cDNA clone: genomic deletions in patients with X-chromosome-linked ichthyosis

    Energy Technology Data Exchange (ETDEWEB)

    Ballabio, A.; Parenti, G.; Carrozzo, R.; Sebastio, G.; Andria, G.; Buckle, V.; Fraser, N.; Craig, I.; Rocchi, M.; Romeo, G.; Jobsis, A.C.; Persico, M.G.

    1987-07-01

    The authors have isolated several cDNA clones from a lambdagt11 expression library by screening with antibodies prepared against the microsomal enzyme steroid sulfatase, which is deficient in classical X-chromosome-linked ichthyosis patients. One of these clones (p422) has been assigned by mapping with a somatic cell hybrid panel and by in situ hybridization to Xp22.3. Clone p422 therefore has a coincident localization with the previously identified locus for steroid sulfatase expression in the region of the X chromosome escaping from inactivation. Twelve steroid sulfatase-deficient patients, including eight cases of classical ichthyosis, were found to be deleted for genomic sequences detected by the clone.

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

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    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.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  13. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-06-01

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

  15. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-06-01

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

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

    Science.gov (United States)

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

    1997-11-01

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

  18. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-06-10

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

  20. Sequential cloning of chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  1. Sequential cloning of chromosomes

    Science.gov (United States)

    Lacks, S.A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

  2. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    Energy Technology Data Exchange (ETDEWEB)

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. (McGill Univ. and Royal Victoria Hospital, Montreal Quebec (Canada)); Mattei, M.G. (INSERM, Marseille (France)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Riviere, M.; Szpirer, J. (Universite Libre de Bruxelles, Rhode-St-Genese (Belgium)) (and others)

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acelpeptide hydrolase gene (APEH). These results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes. 34 refs., 7 figs. 1 tab.

  3. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes.

    Science.gov (United States)

    Pausova, Z; Bourdon, J; Clayton, D; Mattei, M G; Seldin, M F; Janicic, N; Rivière, M; Szpirer, J; Levan, G; Szpirer, C

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acylpeptide hydrolase gene (APEH). Our results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes.

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

    Science.gov (United States)

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

    1992-11-01

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

  5. 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.

  6. 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.

  7. Infectious Maize rayado fino virus from cloned cDNA

    Science.gov (United States)

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  8. Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis

    Energy Technology Data Exchange (ETDEWEB)

    Bonifas, J.M.; Morley, B.J.; Oakey, R.E.; Kan, Y.W.; Epstein, E.J. Jr.

    1987-12-01

    A human steroid sulfatase cDNA 2.4 kilobases long was isolated from a human placental lambda gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-08-10

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-02-01

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

  12. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-10-01

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

  14. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    Energy Technology Data Exchange (ETDEWEB)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-10-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11..beta..-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11..beta..-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

  15. 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.

  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. 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...

  18. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    Energy Technology Data Exchange (ETDEWEB)

    Gafvels, M.E.; Strauss, J.F. III (Univ. of Pennyslvania, Philadelphia, PA (United States)); Caird, M.; Patterson, D. (Eleanor Roosevelt Institute, Denver, CO (United States)); Britt, D.; Jackson, C.L. (Brown Univ., Providence, RI (United States))

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  19. 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.

  20. 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.

  1. Human estrogen sulfotransferase gene (STE): Cloning, structure, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Aksoy, I.A.; Weinshilboum, M. [Mayo Foundation, Rochester, MI (United States)] [and others

    1995-09-01

    Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DBEA) ST gene, STD. The 5{prime} flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5{prime}-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues. 50 refs., 6 figs., 1 tab.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1992-02-15

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

  3. Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences

    Indian Academy of Sciences (India)

    Chuan-Liang Deng; Wei-Li Zhang; Ying Cao; Shao-Jing Wang; Shu-Fen Li; Wu-Jun Gao; Long-Dou Lu

    2015-12-01

    The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related , m and . Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However, there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

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

    Science.gov (United States)

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

    2002-01-01

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

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

    OpenAIRE

    1997-01-01

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

  12. Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Vianey-Saban, C

    1996-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE...

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

    Directory of Open Access Journals (Sweden)

    Joana M.S. CARDOSO

    2012-09-01

    Full Text Available 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 of these clones was found to mimic the wild-type OMMV, making it useful in future studies on protein structure and function by site directed mutagenesis of individual genes. This is the first report on constructing full-length cDNA clones of OMMV from which infectious RNAs can be transcribed in vitro.

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

    OpenAIRE

    1991-01-01

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

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

    NARCIS (Netherlands)

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

    1985-01-01

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

  16. 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.

  17. 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).

  18. Cloning and analysis of the mouse Fanconi anemia group a cDNA and an overlapping penta zinc finger cDNA

    NARCIS (Netherlands)

    Wong, JCY; Alon, N; Norga, K; Kruyt, FAE; Youssoufian, H; Buchwald, M

    2000-01-01

    Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a mode I system, we cloned and characterized the mouse homolog of the human FANCA cDNA, The mouse cDNA

  19. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, S.; Weinshilboum, R.M. [Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, MN (United States); Brandriff, B.F. [Lawrence Livermore National Lab., CA (United States); Ward, A.; Little, P.F.R. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1995-10-10

    Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5{prime}-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5{prime}-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

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

    OpenAIRE

    1994-01-01

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

  1. 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.

  2. Characterization of the soybean early nodulin cDNA clone GmENOD55.

    NARCIS (Netherlands)

    Blank, de C.; Mylona, P.; Katinakis, P.; Bisseling, T.; Franssen, H.; Yang, W.C.

    1993-01-01

    Two cDNA clones of the soybean early nodulin GmENOD55 were characterized. These clones may represent two members of the soybean early nodulin gene family GmENOD55. GmENOD55 has an N-terminal signal peptide and it contains an internal domain consisting of proline and serine residues. Analyses of

  3. Cloning, structure, and chromosome localization of the mouse glutaryl-CoA dehydrogenase gene

    Energy Technology Data Exchange (ETDEWEB)

    Koeller, D.M.; DiGiulio, A.; Frerman, F.E. [Univ. of Colorado Health Sciences Center, Denver, CO (United States)] [and others

    1995-08-10

    Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, and inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains and open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped. 14 refs., 3 figs.

  4. Molecular Cloning of the Bovine Liver ADPRT cDNA

    Science.gov (United States)

    1988-12-13

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

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

    Science.gov (United States)

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

    2004-07-01

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

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    1994-01-01

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

  9. Cloning and expression of murine immune interferon cDNA.

    OpenAIRE

    1983-01-01

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

  10. 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

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

    OpenAIRE

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

    1987-01-01

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

  12. 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...

  13. 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...

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

    Indian Academy of Sciences (India)

    T Venugopal; S Mathavan; T J Pandian

    2002-06-01

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

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

    Science.gov (United States)

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

    2002-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Michel Guenter

    2007-10-01

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

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

    Science.gov (United States)

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

    1990-09-25

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

  18. 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.

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

    Science.gov (United States)

    Schaefer, B C

    1995-05-20

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

  20. 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.

  1. 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.

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

    OpenAIRE

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

    1993-01-01

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

  3. Cloning and expression of a novel human HCUTA cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Copper is one of the most important trace elements to life. Human HCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 of E. coli. The whole opening reading frame of HCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed in E. coli BL21 (DE3).

  4. 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.

  5. 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.

  6. 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

  7. Localization of the human fibromodulin gene (FMOD) to chromosome 1q32 and completion of the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Sztrolovics, R.; Grover, J.; Roughley, P.J. [McGill Univ., Montreal (Canada)] [and others

    1994-10-01

    This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig.

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

    Science.gov (United States)

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

    1995-04-03

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

  9. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-05-01

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

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  12. 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.

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

    Science.gov (United States)

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

    2008-03-10

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

  14. The preparation of an infectious full-length cDNA clone of Saffold virus

    OpenAIRE

    Okuwa Takako; Shimizu Hiroyuki; Asif Naeem; Hosomi Takushi; Himeda Toshiki; Muraki Yasushi; Ohara Yoshiro

    2011-01-01

    Abstract The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV...

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

    Science.gov (United States)

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

    2013-04-01

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

  16. 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.

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

    Science.gov (United States)

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

    1993-04-15

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

  18. 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.

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

    Science.gov (United States)

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

    1987-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2001-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    Science.gov (United States)

    Bujaki, Erika

    2016-01-01

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

  3. 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

    obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication......We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA...... clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence...

  4. 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.

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

    Science.gov (United States)

    Hedrick, S A; Bell, J N; Boller, T; Lamb, C J

    1988-01-01

    Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

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

    Science.gov (United States)

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

    1989-03-01

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

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

    Science.gov (United States)

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

    1997-12-19

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

  8. Strategies for cloning and manipulating natural and synthetic chromosomes.

    Science.gov (United States)

    Karas, Bogumil J; Suzuki, Yo; Weyman, Philip D

    2015-02-01

    Advances in synthetic biology methods to assemble and edit DNA are enabling genome engineering at a previously impracticable scale and scope. The synthesis of the Mycoplasma mycoides genome followed by its transplantation to convert a related cell into M. mycoides has transformed strain engineering. This approach exemplifies the combination of newly emerging chromosome-scale genome editing strategies that can be defined in three main steps: (1) chromosome acquisition into a microbial engineering platform, (2) alteration and improvement of the acquired chromosome, and (3) installation of the modified chromosome into the original or alternative organism. In this review, we outline recent progress in methods for acquiring chromosomes and chromosome-scale DNA molecules in the workhorse organisms Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. We present overviews of important genetic strategies and tools for each of the three organisms, point out their respective strengths and weaknesses, and highlight how the host systems can be used in combination to facilitate chromosome assembly or engineering. Finally, we highlight efforts for the installation of the cloned/altered chromosomes or fragments into the target organism and present remaining challenges in expanding this powerful experimental approach to a wider range of target organisms.

  9. 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 List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality score...s Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality score...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  10. 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.

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

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

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

  12. 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.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  14. 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.

  15. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  16. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-01-15

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

  18. 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.

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

    OpenAIRE

    Bordat, Amandine; Houvenaghel, Marie-Christine

    2015-01-01

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

  20. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Assembly, Verification, and Initial Annotation of the NIA Mouse 7.4K cDNA Clone Set

    Science.gov (United States)

    VanBuren, Vincent; Piao, Yulan; Dudekula, Dawood B.; Qian, Yong; Carter, Mark G.; Martin, Patrick R.; Stagg, Carole A.; Bassey, Uwem C.; Aiba, Kazuhiro; Hamatani, Toshio; Kargul, George J.; Luo, Amber G.; Kelso, Janet; Hide, Winston; Ko, Minoru S.H.

    2002-01-01

    A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from embryonic and newborn organ libraries consisting of ∼120,000 cDNA clones, which were initially re-arrayed into a set of ∼11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers. [The sequence data from this study have been submitted to GenBank under accession nos. BQ550036–BQ563104.] PMID:12466305

  6. Molecular cloning and chromosome assignment of murine N-ras.

    OpenAIRE

    Ryan, J.; Hart, C P; Ruddle, F H

    1984-01-01

    The murine N-ras gene was cloned by screening an EMBL-3 recombinant phage library with a human N-ras specific probe. Hybridization of two separate unique sequence N-ras probes, isolated from the 5' and 3' flanking sequences of the murine gene, to a mouse-Chinese hamster hybrid mapping panel assigns the N-ras locus to mouse chromosome three.

  7. An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

    Science.gov (United States)

    Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

    2012-07-01

    cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-04-01

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

  14. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik

    2013-01-01

    . This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable......Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described...... recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses....

  15. cDNA cloning and life-cycle stage-specific expression of coronin from Physarum polycephalum.

    Science.gov (United States)

    Minami, Yoshiko; Ishihara, Masaaki; Hayase, Masato; Sakaguchi, Tomohisa; Yubisui, Toshitsugu

    2009-03-23

    Coronin cDNA was cloned from the plasmodia of Physarum polycephalum. The amino acid sequence deduced from the cDNA was comprised of 449 residues and showed 60% identity to that of Dictyostelium discoideum coronin. Southern blot analysis suggested that the coronin gene present in the P. polycephalum genome might be a single copy. Coronin was expressed in diploid plasmodia, while it was not detected in haploid amoebae or spores.

  16. 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.

  17. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-01-15

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

  19. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, C.; Raftogianis, R.; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States)

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  20. 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.

  1. The preparation of an infectious full-length cDNA clone of Saffold virus.

    Science.gov (United States)

    Himeda, Toshiki; Hosomi, Takushi; Asif, Naeem; Shimizu, Hiroyuki; Okuwa, Takako; Muraki, Yasushi; Ohara, Yoshiro

    2011-03-09

    The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

  2. The preparation of an infectious full-length cDNA clone of Saffold virus

    Directory of Open Access Journals (Sweden)

    Okuwa Takako

    2011-03-01

    Full Text Available Abstract The pathogenicity of Saffold virus (SAFV among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3. The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

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

    Science.gov (United States)

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

    2014-10-27

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

  4. Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E.

    1991-09-01

    The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

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

    Science.gov (United States)

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

    2004-06-01

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

  6. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2015-12-01

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

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

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

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

  11. 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

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUT

    2009-01-01

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

  14. A cDNA Clone-Launched Platform for High-Yield Production of Inactivated Zika Vaccine

    Directory of Open Access Journals (Sweden)

    Yujiao Yang

    2017-03-01

    Full Text Available A purified inactivated vaccine (PIV using the Zika virus (ZIKV Puerto Rico strain PRVABC59 showed efficacy in monkeys, and is currently in a phase I clinical trial. High-yield manufacture of this PIV is essential for its development and vaccine access. Here we report an infectious cDNA clone-launched platform to maximize its yield. A single NS1 protein substitution (K265E was identified to increase ZIKV replication on Vero cells (a cell line approved for vaccine production for both Cambodian FSS13025 and Puerto Rico PRVABC59 strains. The NS1 mutation did not affect viral RNA synthesis, but significantly increased virion assembly through an increased interaction between NS1 and NS2A (a known regulator of flavivirus assembly. The NS1 mutant virus retained wild-type virulence in the A129 mouse model, but decreased its competence to infect Aedes aegypti mosquitoes. To further increase virus yield, we constructed an infectious cDNA clone of the clinical trial PIV strain PRVABC59 containing three viral replication-enhancing mutations (NS1 K265E, prM H83R, and NS3 S356F. The mutant cDNA clone produced >25-fold more ZIKV than the wild-type parent on Vero cells. This cDNA clone-launched manufacture platform has the advantages of higher virus yield, shortened manufacture time, and minimized chance of contamination.

  15. 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.

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

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1994-01-01

    We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

  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

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

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

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

  20. 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.

  1. 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

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...

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

    OpenAIRE

    1988-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-06-01

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

  4. 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.

  5. Positional cloning of disease genes on chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Doggett, N. [Los Alamos National Lab., NM (United States); Bruening, M. [Leiden Univ. (Netherlands); Callen, D. [Adelaide Women`s and Children`s Hospital, North Adelaide, South Australia (Australia); Gardiner, M. [University Coll., London (United Kingdom); Lerner, T. [Massachusetts General Hospital, Boston, MA (United States)

    1996-04-01

    The project seeks to elucidate the molecular basis of an important genetic disease (Batten`s disease) by molecular cloning of the affected gene by utilizing an overlapping clone map of chromosome 16. Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomoter disturbances. The Batten disease gene was genetically mapped to the chromosome region 16p 12.1 in close linkage with the genetic markers D16S299 and D16S298. Exon amplification of a cosmid containing D16S298 yielded a candidate gene that was disrupted by a 1 kb genomic deletion in all patients containing the most common haplotype for the disease. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the gene as the Batten disease gene. The disease gene encodes a novel 438 amino acid membrane binding protein of unknown function.

  6. 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.

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

    Institute of Scientific and Technical Information of China (English)

    周兆斓; 朱祯; 刘春明; 张海涛; 肖桂芳; 李向辉

    1996-01-01

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

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

    Science.gov (United States)

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

    1998-08-01

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

  9. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-10-01

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

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

    Science.gov (United States)

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

    2005-09-01

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

  12. 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.

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

    OpenAIRE

    Anderberg, R J; Walker-Simmons, M K

    1992-01-01

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

  14. Cloning, structural organization, and chromosomal mapping of the human phenol sulfotransferase STP2 gene

    Energy Technology Data Exchange (ETDEWEB)

    Gaedigk, A.; Beatty, B.G.; Grant, D.M. [Hospital for Sick Children, Toronto, Ontario (Canada)

    1997-03-01

    Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p1.2) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family. 27 refs., 3 figs.

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

    Science.gov (United States)

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

    2014-11-07

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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

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

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

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

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

    Science.gov (United States)

    Shen

    1999-01-01

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

  3. A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

    Science.gov (United States)

    Cui, Y H; Zhu, G Q; Chen, Q J; Wang, Y F; Yang, M M; Song, Y X; Wang, J G; Cao, B Y

    2011-11-25

    Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

  4. 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.

  5. 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

  6. 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

  7. Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

    Institute of Scientific and Technical Information of China (English)

    徐军望; 冯德江; 李旭刚; 常团结; 朱祯

    2002-01-01

    The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-05-01

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

  9. 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).

  10. 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.

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

    Science.gov (United States)

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

    1987-10-01

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

  12. 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.

  13. 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.

  14. 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...

  15. 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

  16. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

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

    Science.gov (United States)

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

    1997-05-16

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

  18. 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......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

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

    Institute of Scientific and Technical Information of China (English)

    Rong Long Jiang; Qiao Sheng Lu; Kang Xian Luo

    2000-01-01

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

  20. 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.

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

    Science.gov (United States)

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

    2002-01-01

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

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

    Science.gov (United States)

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

    2004-05-01

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

  3. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  4. 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

  5. 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.

  6. cDNA sequence, gene structure, and cholinesterase-like domains of an esterase from Caenorhabditis elegans mapped to chromosome V.

    Science.gov (United States)

    Fedon, Y; Cousin, X; Toutant, J P; Thierry-Mieg, D; Arpagaus, M

    1993-01-01

    The structure of an esterase gene from Caenorhabditis elegans has been determined by comparison of the sequences in genomic and cDNA clones. The gene was mapped close to the center of chromosome V (1.7 centimorgans to the left of dpy-11) and is therefore distinct from the gut esterase gene ges-1. It possessed 7 short introns. The 5' splice site of intron 3 presented the sequence GC instead of the usual GT that was found in the other six introns. The cDNA was trans-spliced with the short leader SL1. The open reading frame indicated that a protein of 557 aminoacids was encoded. The deduced aminoacid sequence did not present a signal peptide at the N-terminal but a potential N-myristoylation site (GXXXS) provided that the initiator methionine was removed. This protein should therefore remain intracellular. Comparison of this C. elegans sequence to other protein sequences in databases, as well as the analysis of the secondary structure in the protein showed that it belongs to the subgroup of esterases in the alpha/beta hydrolase fold family.

  7. 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.

  8. Cloning, Structural Organization and Chromosomal Mapping of Rat Costimulatory Molecule 4-1BBL

    Institute of Scientific and Technical Information of China (English)

    Qiu-Ming DONG; Xue-Guang ZHANG; Li-Jie MA; Guang-Bo ZHANG; Ya-Fang WU; Jia-Yao SHEN; Ying CHEN; Yong-Jing CHEN; Xiang-Ke PU; Sai-Yu HANG

    2005-01-01

    4-1BBL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL (GenBank accession No. AY259541). The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type Ⅱ transmembrane glycoprotein, the same as human and murine 4-1BBL. "SDAA" in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I ("SXXS" motif), which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned (GenBank accession No.AY332409). Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues: brain, lung, colon, liver,thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 (GenBank accession UniGene No. Rn.46783). Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological function of this molecule.

  9. 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.

  10. Molecular cloning and chromosomal assignment of the human brain-type phosphodiesterase I/nucleotide pyrophosphatase gene (PDNP2)

    Energy Technology Data Exchange (ETDEWEB)

    Kawagoe, Hiroyuki; Soma, Osamu; Goji, Junko [Kobe Univ. School of Medicine (Japan)] [and others

    1995-11-20

    Phosphodiesterase I/nucleotide pyrophosphatase is a widely expressed membrane-bound enzyme that cleaves diester bonds of a variety of substrates. We have cloned brain-type cDNA for this enzyme from rat brain and designated it PD-I{alpha}. In this study we have isolated cDNA and genomic DNA encoding human PD-I{alpha}. Human PD-I{alpha} cDNA, designated PDNP2 in HGMW nomenclature, has a 2589-nucleotide open reading frame encoding a polypeptide of 863 amino acids with a calculated M{sub r} of 99,034. Northern blot analysis revealed that human PD-I{alpha} transcript was present in brain, lung, placenta, and kidney. The database analysis showed that human PD-I{alpha} was identical with human autotaxin (ATX), a novel tumor motility-stimulating factor, except that human PD-I{alpha} lacks 156 nucleotides and 52 amino acids of human ATX. Human PD-I{alpha} and human ATX are likely to be alternative splicing products from the same gene. The 5{prime} region of the human PDNP2 gene contains four putative binding sites of transcription factor Sp1 without typical TATA or CAAT boxes, and there is a potential octamer binding motif in intron 2. From the results of fluorescence in situ hybridization, the human PDNP2 gene is located at chromosome 8q24.1. 17 refs., 3 figs.

  11. Molecular cloning, genomic structure, chromosomal localization, and alternative splice forms of the platelet collagen receptor glycoprotein VI.

    Science.gov (United States)

    Ezumi, Y; Uchiyama, T; Takayama, H

    2000-10-14

    Glycoprotein VI (GPVI) is the major collagen receptor underlying platelet activation. We cloned the full-length cDNA for GPVI (GPVI-1) and its two isoforms (GPVI-2 and -3) from phorbol-ester-stimulated CMK cells. The GPVI-1 cDNA was identical in the coding region with the cDNA that has recently been reported to belong to the immunoglobulin superfamily. The GPVI gene consisted of 8 exons spanning over 23 kbp and was mapped on the chromosome 19q13. 4. The promoter of GPVI gene lacked TATA and CAAT boxes and had multiple transcription start sites like other megakaryocytic genes. When COS-7 cells were cotransfected with the GPVI isoforms and Fc receptor gamma chain, Fc receptor gamma chain was associated with GPVI-1 and -2 but did not affect the GPVI expression levels. GPVI-1 and -2 could bind the collagen-related peptide, which exhibits triple-helical and polymeric structure of collagen to activate platelets via GPVI. Copyright 2000 Academic Press.

  12. Aquifer parameter identification by the best chromosome clone plus younger generation chromosome prepotency genetic algorithm

    Institute of Scientific and Technical Information of China (English)

    LI Jing-sheng; LI Kai; YAO Lei-hua

    2005-01-01

    This paper developed an improved combinatorial method called the best chromosome clone plus younger generation chromosome prepotency genetic algorithm (BCC-YGCP-GA) to evaluate aquifer parameters. This method is based on a decimal simple genetic algorithm (SGA). A synthetic example for unsteady-state flow in a two-dimensional, inhomogeneous, confined aquifer containing three hydraulically distinct zones, is used to develop data to test the model. The simulation utilizes SGA and BCC-YGCP-GA coupled to the finite element method to identify the mean zonal hydraulic conductivities, and storage coefficients of the three-compartment model. For this geometrically simple model, used as a prototype of more complex systems, the SGA does not reach convergence within 100 generations. Conversely, the convergence rate of the BCC-YGCD-GA model is very fast. The objective function value calculated by BCC-YGCD-GA is reduced to 1/1 000th of the starting value within 100 generations,and the hydraulic conductivity and storage of three zones are within a few percent of the "true" values of the ideal model, highlighting the power of the method for aquifer parameterization.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  14. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  15. Mapping and ordered cloning of the human X chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  16. [Cloning and sequencing of KIR2DL1 framework gene cDNA and identification of a novel allele].

    Science.gov (United States)

    Sun, Ge; Wang, Chang; Zhen, Jianxin; Zhang, Guobin; Xu, Yunping; Deng, Zhihui

    2016-10-01

    To develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China. Total RNA was isolated from peripheral blood sample, and complementary DNA (cDNA) transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DL1 framework gene was amplified with a pair of KIR2DL1-specific PCR primers. The PCR products with a length of approximately 1.2 kb were then subjected to cloning and haplotype sequencing. A specific target fragment of the KIR2DL1 framework gene was obtained. Following allele separation, a wild-type KIR2DL1*00302 allele and a novel variant allele, KIR2DL1*031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD-KIR Database showed that the novel allele KIR2DL1*031 has differed from the closest allele KIR2DL1*00302 by a non-synonymous mutation at CDS nt 188A>G (codon 42 GAG>GGG) in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1*031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1*031 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee. An assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.

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

    Science.gov (United States)

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

    2015-11-01

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

  18. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  19. 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.

  20. cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

    Energy Technology Data Exchange (ETDEWEB)

    Rennex, D.; Hemmings, B.A.; Hofsteenge, J.; Stone, S.R. (Friedrich Miescher-Institut, Basel (Switzerland))

    1991-02-26

    Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. ({sup 3}H)Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

  1. Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

    Science.gov (United States)

    Baldini, A; Archidiacono, N; Carbone, R; Bolino, A; Shridhar, V; Miller, O J; Miller, D A; Ward, D C; Rocchi, M

    1992-01-01

    We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

  2. Isolation, cDNA, and genomic structure of a conserved gene (NOF) at chromosome 11q13 next to FAU and oriented in the opposite transcriptional orientation.

    Science.gov (United States)

    Kas, K; Lemahieu, V; Meyen, E; Van de Ven, W J; Merregaert, J

    1996-06-15

    In our effort to characterize a gene at chromosome 11q13 involved in a t(11;17)(q13;q21) translocation in B-non-Hodgkin lymphoma, we have identified a novel human gene, NOF (Neighbour of FAU). It maps right next to FAU in a head to head configuration separated by a maximum of 146 nucleotides. cDNA clones representing NOF hybridized to a 2. 2-kb mRNA present in all tissues tested. The largest open reading frame appeared to contain 166 amino acids and is proline rich, and the sequence shows no homology with any known gene in the public databases. The NOF gene consists of 4 exons and 3 introns spanning approximately 5 kb, and the boundaries between exons and introns follow the GT/AG rule. The NOF locus is conserved during evolution, with the predicted protein having over 80% identity to three translated mouse and rat ESTs of unknown function. Moreover, the mouse ESTs map in the same organization, closely linked to the FAU gene, in the mouse genome. NOF, however, is not affected by the t(11;17)(q13;q21) chromosomal translocation.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  4. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fra...

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

    Science.gov (United States)

    Huang, C F; Chuang, N N

    1998-12-11

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

  6. cDNA cloning and mRNA expression of neuropeptide Y in orange spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Chen, Rong; Li, Wensheng; Lin, Haoran

    2005-09-01

    A full-length cDNA encoding the neuropeptide Y (NPY) was cloned from the hypothalamus of orange spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends approaches. The NPY cDNA sequence is 688 bp long and has an open reading frame of 300 bp encoding prepro-NPY with 99 amino acids. The deduced amino acid sequences contain a 28-amino-acids signal peptide followed by a 36-amino-acids mature NPY peptide. mRNA expression of NPY was determined using semi-quantitative RT-PCR followed by Southern blot analysis. NPY mRNA was expressed in olfactory bulb, telencephalon, pituitary, hypothalamus, optic tectum-thalamus, medulla oblongata, cerebellum and spinal cord. Low levels of NPY mRNA expression were found in retina, ovary and stomach, while much lower levels of expression were detected in liver, heart, gill, skin, anterior intestine, thymus and blood. No NPY mRNA expression was observed in unfertilized eggs, newly fertilized eggs, 16-cells stage and morula stage of the embryo and lower levels of expression were detected in the blastula, gastrula and neurula stages. It was highly expressed from lens formation stage to 52-day-old larval stage. NPY might be involved in the late embryonic and larval development of the orange spotted grouper.

  7. 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.

  8. 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.

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  11. 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.

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

    Science.gov (United States)

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

  13. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR ... RNA species and approximately 80 structurally distinct ... a reverse transcription kit with Oligo dT as the primers according to ... 2). On the basis of the high identity, we concluded that the cDNA isolated is ...

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

    Science.gov (United States)

    Mieszczanek, J; Kofta, W; Wedrychowicz, H

    2000-12-01

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

  15. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence.

    Science.gov (United States)

    D'Aiuto, L; Antonacci, R; Marzella, R; Archidiacono, N; Rocchi, M

    1993-11-01

    We have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed.

  16. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))

    1993-11-01

    The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

  17. Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

    Directory of Open Access Journals (Sweden)

    Quan Zhang

    Full Text Available Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17 that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining

  18. Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

    Science.gov (United States)

    Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

    2014-01-01

    Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    Science.gov (United States)

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

    1996-02-15

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

  1. 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

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

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

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

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

    Science.gov (United States)

    Arenhart, Sandra; Silva, José Valter Joaquim; Flores, Eduardo Furtado; Weiblen, Rudi; Gil, Laura Helena Vega Gonzales

    The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  4. 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.

  5. 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.

  6. Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence

    Science.gov (United States)

    Li, Jie; Jin, Zhonghui; Gao, Yueyi; Zhou, Lei; Ge, Xinna; Guo, Xin; Yang, Hanchun

    2017-01-01

    The recently emerged highly virulent variants of porcine epidemic and diarrhea virus (PEDV) remain a huge threat to the worldwide swine industry. Here, we describe the development of a bacterial artificial chromosome (BAC) reverse genetics system for PEDV based on two recent Chinese field isolates, namely CHM2013 and BJ2011C. Phylogenetically, CHM2013 is closely related to the vaccine strain SM98 whereas the isolate BJ2011C belongs to the GIIb group, a cluster that contains many recent pandemic strains. The full-length cDNA clones of the two isolates were constructed into BAC under the control of CMV promoter. The rescued viruses rBJ2011C and rCHM2013 were found to replicate at the kinetics similar to their respective parental viruses in cell culture. When tested in the 2-day-old pig model, rBJ2011C caused severe diarrhea of piglets with extensive damages to the intestinal epithelium, leading to 100% fatality within 48 hours. In contrast, the rCHM2013-inoculated piglets all survived with only very minor tissue damage observed. Thus, we have successfully established a convenient platform for PEDV genome manipulation. This study also represents the first description of a DNA-launched reverse genetics system for the highly virulent PEDV. PMID:28301551

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

    OpenAIRE

    1993-01-01

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

  8. Cloning and gene expression of a cDNA for the chicken follicle-stimulating hormone (FSH)-beta-subunit.

    Science.gov (United States)

    Shen, San-Tai; Yu, John Yuh-Lin

    2002-02-15

    Follicle-stimulating hormone (FSH) is a member of pituitary glycoprotein hormones that are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSH-beta in avian species. For better understanding of the phylogenic diversity and evolution of FSH molecule, we have isolated and sequenced the complete complementary DNA (cDNA) encoding chicken FSH-beta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned chicken FSH-beta cDNA consists of 2457-bp nucleotides, including 44-bp nucleotides of the 5'-untranslated region (UTR), 396 bp of the open reading frame, and an extraordinarily long 3'-UTR of 2001-bp nucleotides followed by a poly(A)((16)) tail. It encodes a 131-amino-acid precursor molecule of FSH-beta-subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the chicken FSH-beta-subunit. Four proline residues, presumably responsible for changing the backbone direction of protein structure, are conserved in chicken FSH-beta-subunit as well. The nucleotide sequence of chicken FSH-beta cDNA shows high homology with quail FSH-beta cDNA, 97% homology in the open reading frame, and 85% homology in the 3'-UTR. The deduced amino acid sequence of chicken FSH-beta-subunit shows a remarkable similarity to other avian FSH-beta-subunits, 98% homology with quail, and 93% homology with ostrich, whereas a lower similarity (66 to 70%) is noted when compared with mammalian FSH-beta-subunits. By contrast, when comparing with the beta-subunits of chicken luteinizing hormone and thyroid-stimulating hormone, the homologies are as low as 37 and 40%, respectively. FSH-beta mRNA was only expressed in pituitary gland out of various

  9. 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.

  10. 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.

  11. 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.

  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. 454 sequencing of pooled BAC clones on chromosome 3H of barley

    Directory of Open Access Journals (Sweden)

    Yamaji Nami

    2011-05-01

    Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

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

    Science.gov (United States)

    Ju, B G; Kim, W S

    2000-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  16. 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.

  17. 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.

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

    Science.gov (United States)

    Jia, Airong; Zhang, Xiao-Hua

    2009-09-01

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

  19. Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense).

    Science.gov (United States)

    Inamine, Saki; Onaga, Shoko; Ohnuma, Takayuki; Fukamizo, Tamo; Taira, Toki

    2015-01-01

    Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.

  20. Cloning and functional expression of a chitinase cDNA from the apple leaf miner moth Lithocolletis ringoniella.

    Science.gov (United States)

    Fan, Xiao-Jun; Mi, Yan-Xia; Ren, Hui; Zhang, Chang; Li, Yao; Xian, Xiao-Xiao

    2015-02-01

    Insect chitinase plays essential roles in chitin catabolism involved in digestion and molting during insect development. In the current work, we cloned a chitinase cDNA, LrCht5, from the apple leaf miner moth Lithocolletis ringoniella and characterized its amino acid sequence and protein properties. The L. ringoniella chitinase cDNA was 2136 bp in length with an open reading frame of 1737 bp that encodes a polypeptide of 579 amino acid residues with a predicted molecular mass of 64.4 kDa and pI of 5.49. The catalytic domain has several phosphorylation and glycosylation sites. The recombinant LrCht5 was expressed in Escherichia coli and the Spodoptera frugiperda cell line Sf9, and the LrCht5 expressed in insect cells exhibited chitinolytic activity. LrCht5 was most stable at pH 6.0 and 45°C. This work has potential application in the development of novel and more specific synthetic chitinase inhibitors for use as bioinsecticides.

  1. Functional cDNA expression cloning: Pushing it to the limit

    Science.gov (United States)

    OKAYAMA, Hiroto

    2012-01-01

    The 1970s and the following decade are the era of the birth and early development of recombinant DNA technologies, which have entirely revolutionized the modern life science by providing tools that enable us to know the structures of genes and genomes and to dissect their components and understand their functions at the molecular and submolecular levels. One major objective of the life sciences is to achieve molecular and chemical understandings of the functions of genes and their encoded proteins, which are responsible for the manifestation of all biological phenomena in organisms. In the early 1980s, I developed, together with Paul Berg, a new technique that enables the cloning of full-length complementary DNAs (cDNAs) on the basis of their functional expression in a given cell of interest. I review the development, application and future implications in the life sciences of this gene-cloning technique. PMID:22450538

  2. 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.

  3. cDNA Clones with Rare and Recurrent Mutations Found in Cancers | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at UT- MD Anderson Cancer Center has developed High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB)1,2 pipeline to construct mutant alleles open reading frame expression clones that are either recurrent or rare in cancers. These barcoded genes can be used for context-specific functional validation, detection of novel biomarkers (pathway activation) and targets (drug sensitivity).

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

    DEFF Research Database (Denmark)

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

    1985-01-01

    DNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha......'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human...

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

    Science.gov (United States)

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

    2009-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

  8. 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.

  9. 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 ...

  10. 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...

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

  13. 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.

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

    Science.gov (United States)

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

    2010-11-01

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

  15. Cloning and characterization of a novel gene (C17orf25) from the deletion region on chromosome 17p13.3 in hepatocelular carcinoma

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open reading frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17orf25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma.

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

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

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

  17. 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.

  18. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-01-15

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

  20. 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

  1. 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.

  2. Cloning and Expression Analysis of a Giant Gourami Vasa-Like cDNA

    Directory of Open Access Journals (Sweden)

    ALIMUDDIN

    2011-09-01

    Full Text Available Molecular marker is useful in the development of testicular cells transplantation for detecting donor-derived germ cells in the recipient gonad. In this study, a giant gourami (Osphronemus goramy vasa-like gene (GgVLG was cloned and characterized for use as a molecular marker for germ cells in this species. Nucleotide sequence analysis revealed that GgVLG comprises 2,340 bps with an open reading frame of 1,962 bps encoding 653 amino acids. The deduced amino acid sequence contained 17 arginine-glycine or arginine-glycine-glycine motifs and eight conserved motifs belonging to the DEAD-box protein family. The GgVLG sequence showed high similarity to Drosophila vasa, common carp vasa homolog and tilapia vasa homolog for 66.2, 85.9, and 90.7%, respectively. In adult tissues, the GgVLG transcripts were specifically detected in ovary and testis. In situ hybridization analysis showed that GgVLG mRNA was detected in oocytes of the ovary and spermatogonia of the testis. There was no signal detected in the spermatocytes, spermatids and other gonadal somatic cells. Thus, consensus sequences, specific localization of GgVLG mRNA in the germ cells, amino acid sequence similarity and phylogenic analysis all suggest that GgVLG is the giant gourami vasa-like gene. Further, GgVLG can be used as a molecular marker for giant gourami germ cells.

  3. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

    Science.gov (United States)

    Tobian, J A; Macrina, F L

    1982-10-01

    A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

  4. 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

  5. Sequence determination of cDNA clones of transcripts from the tumor-associated region of the Marek's disease virus genome.

    Science.gov (United States)

    Iwata, A; Ueda, S; Ishihama, A; Hirai, K

    1992-04-01

    The number of 132-bp tandem direct repeats within the long inverted repeat region of the Marek's disease virus type 1 (MDV1) genome increases concomitantly with the loss of oncogenicity during serial passages in cultured cells. Twelve clones carrying the 132-bp sequence were isolated from a cDNA library constructed from chicken embryo fibroblasts infected with the MDV1 Md5 strain. Through sequence analysis of a cDNA clone and primer extension analysis, the corresponding mRNA was found to be a linear transcript which included the two 132-bp tandem direct repeats. Two open reading frames were found in this transcript. One had a week homology with v-fms. The other should increase its size concomitantly with expansion of the 132-bp tandem direct repeat. PCR analysis of both cDNA clones and RNA gave amplified products which were as large as that produced from the genomic clone, indicating that a majority of mRNA from this region is composed of unspliced transcripts.

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

    Science.gov (United States)

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

    2005-12-15

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

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

    Science.gov (United States)

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

    2003-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

    BAI Yong; GONG Wei; LIU Tianyun; ZHU Yuxian

    2003-01-01

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

  9. 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.

  10. 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

  11. 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.

  12. Comparative mapping of a gorilla-derived alpha satellite DNA clone on great ape and human chromosomes.

    Science.gov (United States)

    Baldini, A; Miller, D A; Shridhar, V; Rocchi, M; Miller, O J; Ward, D C

    1991-11-01

    We have isolated an alpha satellite DNA clone, pG3.9, from gorilla DNA. Fluorescence in situ hybridization on banded chromosomes under high stringency conditions revealed that pG3.9 identifies homologous sequences at the centromeric region of ten gorilla chromosomes, and, with few exceptions, also recognizes the homologous chromosomes in human. A pG3.9-like alphoid DNA is present on a larger number of orangutan chromosomes, but, in contrast, is present on only two chromosomes in the chimpanzee. These results show that the chromosomal subsets of related alpha satellite DNA sequences may undergo different patterns of evolution.

  13. 45,X/46,XY chromosome mosaicism detected by midtrimester amniocentesis in amniocyte clones.

    Science.gov (United States)

    Hecht, F; Hecht, B K

    1982-07-01

    Amniocyte clones from a mild-trimester pregnancy disclosed 45,X/46,XY sex chromosome mosaicism. Because of the uncertainty concerning the phenotype of the fetus, the parents elected to terminate the pregnancy. Mixed (asymmetrical) gonadal dysgenesis was not found. The fetus appeared to have a normal male uro-genital system. No malformations of any type were detected, although as expected, the fetus did have 45,X/46,XY mosaicism.

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2011-01-01

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

  17. 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.

  18. 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.

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

    Science.gov (United States)

    Elgart, G W; Stanley, J R

    1993-08-01

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

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

    Science.gov (United States)

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

    1991-03-28

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

  1. Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone.

    Science.gov (United States)

    Liu, R; Zhang, H H; Chen, Z X; Shahid, M Q; Fu, X L; Liu, X D

    2015-10-29

    Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

  2. 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.

  3. 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...

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

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

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

  5. An infectious full-length cDNA clone of duck Tembusu virus, a newly emerging flavivirus causing duck egg drop syndrome in China.

    Science.gov (United States)

    Li, Shuang; Zhang, Lijiao; Wang, Yongyue; Wang, Shuxia; Sun, Haigang; Su, Wenliang; He, Weiyong; Han, Bo; Su, Jingliang

    2013-01-01

    Duck Tembusu virus (TMUV) is a recently identified pathogenic flavivirus that causes severe egg drop and encephalitis in Chinese ducks and geese. It has been found to be most closely related to the mosquito-origin Tembusu virus and chicken Sitiawan virus reported in Malaysia. However, the ecological characteristics and the pathogenesis of duck TMUV are largely unknown. We report the construction of full-length cDNA clone of duck TMUV strain JXSP. The virus genome was reverse transcribed, amplified as seven overlapping fragments and successively ligated into the low copy number vector pWSK29 under the control of a T7 promoter. Transfection of BHK-21 cells with the transcribed RNA from the full-length cDNA clone resulted in production of highly infectious progeny virus. In vitro growth characteristics in BHK-21 cells and virulence in ducklings and BALB/c mice were similar for the rescued and parental viruses. This stable infectious cDNA clone will be a valuable tool for studying the genetic determinants of duck TMUV. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Identification and cloning of the CHL4 gene controlling chromosome segregation in yeast.

    Science.gov (United States)

    Kouprina, N; Kirillov, A; Kroll, E; Koryabin, M; Shestopalov, B; Bannikov, V; Zakharyev, V; Larionov, V

    1993-10-01

    A collection of chl mutants characterized by decreased fidelity of chromosome transmission and by minichromosome nondisjunction in mitosis was examined for the ability to maintain nonessential dicentric plasmids. In one of the seven mutants analyzed, chl4, dicentric plasmids did not depress cell division. Moreover, nonessential dicentric plasmids were maintained stably without any rearrangements during many generations in the chl4 mutant. The rate of mitotic heteroallelic recombination in the chl4 mutant was not increased compared to that in an isogenic wild-type strain. Analysis of the segregation of a marked chromosome indicated that sister chromatid nondisjunction and sister chromatid loss contributed equally to chromosome malsegregation in the chl4 mutant. A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation and was physically mapped to the right arm of chromosome IV near the SUP2 gene. Nucleotide sequence analysis of CHL4 clone revealed a 1.4-kb open reading frame coding for a 53-kD predicted protein which does not have homology to published proteins. A strain containing a null allele of CHL4 is viable under standard growth conditions but has a temperature-sensitive phenotype (conditional lethality at 36 degrees). We suggest that the CHL4 gene is required for kinetochore function in the yeast Saccharomyces cerevisiae.

  7. Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

    Science.gov (United States)

    Hizume, M; Shibata, F; Maruyama, Y; Kondo, T

    2001-09-01

    Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Murphy Brian R

    2004-10-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-04-01

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

  11. 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.

  12. 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.

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

    Science.gov (United States)

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

    1991-06-01

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

  14. Pathogenicity of a Very Virulent Strain of Marek's Disease Herpesvirus Cloned as Infectious Bacterial Artificial Chromosomes

    Directory of Open Access Journals (Sweden)

    Lorraine P. Smith

    2011-01-01

    Full Text Available Bacterial artificial chromosome (BAC vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130 of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

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

    NARCIS (Netherlands)

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

    1991-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  17. Next Generation Sequencing of Classical Swine Fever Virus and Border Disease virus cloned in Bacterial Artificial Chromosomes

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Höper, Dirk; Beer, martin

    2012-01-01

    Next generation sequencing is a powerful tool for complete sequencing of large amounts of DNA. We have recently cloned full genome cDNA copies (obtained by long-range RT-PCR) of entire genomes of classical swine fever virus (CSFV) strain Koslov and Border disease virus strain Gifhorn into bacterial...

  18. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  19. 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.

  20. 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.

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Science.gov (United States)

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

    2012-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-08-01

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

  4. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Directory of Open Access Journals (Sweden)

    Kalpana Dulal

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV. The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  5. Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse;

    Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stabl...... single-copy bacterial artificial chromosome (BAC) generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived. Our strategy allows construction of stable infectious BAC DNAs from a single full-length PCR product....

  6. Cloning, chromosome localization and features of a novel human gene, MATH2

    Indian Academy of Sciences (India)

    Lingchen Guo; Min Jiang; Yushu Ma; Haipeng Cheng; Xiaohua Ni; Yangsheng Jin; Yi Xie; Yumin Mao

    2002-04-01

    We report cloning and some features of a novel human gene, MATH2, which encodes a protein of 337 amino acid residues with a basic helix–loop–helix domain and exhibits 98% similarity to mouse Math2. Results of Northern blot analysis revealed two transcripts of the MATH2 gene of 1.7 kb and 2.4 kb in human brain. We localized MATH2 to chromosome 7 at 7p14–15 by matching with the Human Genome Sequence Database. Human MATH2 and mouse Math2 may have the same functions in the nervous system.

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Carolyn A. Owen

    2016-06-01

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

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

    Science.gov (United States)

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

    2016-06-14

    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.

  10. 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.

  11. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

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

    Science.gov (United States)

    Shigemori, Yasushi

    2005-06-01

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

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

    Science.gov (United States)

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

    2012-08-01

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

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

    Science.gov (United States)

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

    1983-04-25

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

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

    Science.gov (United States)

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

    2004-01-01

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

  17. 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.

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

    OpenAIRE

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

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surf...

  19. Molecular cloning and analysis of breakpoints on ring chromosome 17 in a patient with autism.

    Science.gov (United States)

    Vazna, Alzbeta; Havlovicova, Marketa; Sedlacek, Zdenek

    2008-01-15

    The breakpoint junction on a ring chromosome 17 in a girl with autism, mental retardation, mild dysmorphism and neurofibromatosis was identified and analysed at the nucleotide level. The extent of the deleted segments was about 1.9 Mb on 17p and about 1.0 Mb on 17q. The structure of the junction between the 17p and 17q arms, especially the lack of significant homology between the juxtaposed genomic regions and the presence of short microhomology at the junction site, indicated non-homologous end joining as the most likely mechanism leading to the rearrangement. In addition to the 17p-17q junction itself, a de novo 1 kb deletion in a distance of 400 bp from the junction was identified, which arose most likely as a part of the rearrangement. The defect directly inactivated 3 genes, and the deleted terminal chromosome segments harboured 27 and 14 protein-coding genes from 17p and 17q, respectively. Several of the genes affected by the rearrangement are candidates for the symptoms observed in the patient. Additional rearrangements similar to the 1 kb deletion observed in our patient may remain undetected but can participate in the phenotype of patients with chromosomal aberrations. They can also be the reason for repeated failures to clone breakpoint junctions in other patients described in the literature.

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

    Science.gov (United States)

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

    1992-07-01

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

  1. Cloning, sequencing, and analysis of inv8 chromosome breakpoints associated with recombinant 8 syndrome.

    Science.gov (United States)

    Graw, S L; Sample, T; Bleskan, J; Sujansky, E; Patterson, D

    2000-03-01

    Rec8 syndrome (also known as "recombinant 8 syndrome" and "San Luis Valley syndrome") is a chromosomal disorder found in individuals of Hispanic descent with ancestry from the San Luis Valley of southern Colorado and northern New Mexico. Affected individuals typically have mental retardation, congenital heart defects, seizures, a characteristic facial appearance, and other manifestations. The recombinant chromosome is rec(8)dup(8q)inv(8)(p23.1q22.1), and is derived from a parental pericentric inversion, inv(8)(p23.1q22.1). Here we report on the cloning, sequencing, and characterization of the 8p23.1 and 8q22 breakpoints from the inversion 8 chromosome associated with Rec8 syndrome. Analysis of the breakpoint regions indicates that they are highly repetitive. Of 6 kb surrounding the 8p23.1 breakpoint, 75% consists of repetitive gene family members-including Alu, LINE, and LTR elements-and the inversion took place in a small single-copy region flanked by repetitive elements. Analysis of 3.7 kb surrounding the 8q22 breakpoint region reveals that it is 99% repetitive and contains multiple LTR elements, and that the 8q inversion site is within one of the LTR elements.

  2. 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.

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

    Science.gov (United States)

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

    1996-07-15

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

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

    Science.gov (United States)

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

    2011-01-01

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

  5. 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.

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    2003-02-01

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

  9. 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.

  10. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  12. 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.

  13. Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

    Institute of Scientific and Technical Information of China (English)

    HE Cong-fen; MA You-zhi; XIN Zhi-yong; XU Qiong-fang; LI Lian-cheng

    2004-01-01

    The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) was identified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes were microisolated and collected. After two rounds of PCR amplification, the PCR products were ranged from 150 - 3 000 bp,with predominant fragments at about 200 - 2 000 bp. Using Ag.intermediumgenomic DNA as a probe, Southern blotting analysis confirmed the products originated from Ag. intermediumgenome. The products were purified, ligated to pUC18 and then transformed into competence E.coli DH5α to produce a 2Ai-2 chromosome DNA library. The microcloning experiments produced approximately 5×105 clones, the size range of the cloned inserts was 200- 1 500 bp, with an average of 580bp. Using Ag. intermediumgenomic DNA as a probe, dot blotting results showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences in the library. Four Ag. intermedium clones were screened from the library by RFLP, and three clones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitive sequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitive DNA sequence.

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

    OpenAIRE

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

    2015-01-01

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

  15. cDNA cloning, structural, and functional analyses of venom phospholipases A₂ and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi.

    Science.gov (United States)

    Tsai, Inn-Ho; Wang, Ying-Ming; Cheng, An Chun; Starkov, Vladislav; Osipov, Alexey; Nikitin, Ilya; Makarova, Yana; Ziganshin, Rustam; Utkin, Yuri

    2011-02-01

    Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. The mapping of novel genes to human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Buenaventura, J.M. [Sarah Lawrence College, Bronxville, NY (United States)

    1994-12-01

    The principle goal of our laboratory is the discovery of new genes on human chromosome 19. One of the strategies to achieve this goal is through the use of cDNA clones known as {open_quotes}expressed sequence tags{close_quotes} (ESTs). ESTs, short segments of sequence from a cDNA clone that correspond to the mRNA, occur as unique regions in the genome and, therefore, can be used as markers for specific positions. In collaboration with researchers from Genethon in France, fifteen cDNA clones from a normalized human infant brain cDNA library were tested and determined to map to chromosome 19. A verification procedure is then followed to confirm assignment to chromosome 19. First, primers for each cDNA clone are developed and then amplified by polymerase chain reaction from genomic DNA. Next, a {sup 32}P-radiolabeled probe is made by polymerase chain reaction for each clone and then hybridized against filters containing an LLNL chromosome 19-specific cosmid library to find putative locations on the chromosome. The location is then verified by running a polymerase chain reactions from the positive cosmids. With the Browser database at LLNL, additional information about the positive cosmids can be found. Through use of the BLAST database at the National Library of Medicine, homologous sequences to the clones can be found. Among the fifteen cDNA clones received from Genethon, all have been amplified by polymerase chain reaction. Three have turned out as repetitive elements in the genome. Ten have been mapped to specific locations on chromosome 19. Putative locations have been found for the remaining two clones and thus verification testing will proceed.

  17. 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.

  18. Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (Citrus grandis) as a model. II. Cloning of resistance gene analogs from single chromosomes.

    Science.gov (United States)

    Huang, D; Wu, W; Lu, L

    2004-05-01

    Amplification of resistance gene analogs (RGAs) is both a useful method for acquiring DNA markers closely linked to disease resistance (R) genes and a potential approach for the rapid cloning of R genes in plants. However, the screening of target sequences from among the numerous amplified RGAs can be very laborious. The amplification of RGAs from specific chromosomes could greatly reduce the number of RGAs to be screened and, consequently, speed up the identification of target RGAs. We have developed two methods for amplifying RGAs from single chromosomes. Method 1 uses products of Sau3A linker adaptor-mediated PCR (LAM-PCR) from a single chromosome as the templates for RGA amplification, while Method 2 directly uses a single chromosomal DNA molecule as the template. Using a pair of degenerate primers designed on the basis of the conserved nucleotide-binding-site motifs in many R genes, RGAs were successfully amplified from single chromosomes of pomelo using both these methods. Sequencing and cluster analysis of RGA clones obtained from single chromosomes revealed the number, type and organization of R-gene clusters on the chromosomes. We suggest that Method 1 is suitable for analyzing chromosomes that are unidentifiable under a microscope, while Method 2 is more appropriate when chromosomes can be clearly identified.

  19. 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.

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

    Science.gov (United States)

    Santhanam, R; Naz, R K

    2001-09-01

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

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

    Science.gov (United States)

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

    1990-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Yunwi Heo

    2016-07-01

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

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

    Science.gov (United States)

    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 constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

  4. Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes

    Directory of Open Access Journals (Sweden)

    Ackermann Mathias

    2009-10-01

    Full Text Available Abstract Background Bovine herpesviruses type 1 (BoHV1 and type 5 (BoHV5 are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves. Findings The viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes (BACs. First, recombinant viruses carrying the genetic elements for propagation in bacteria were generated. Second, DNA from these recombinant viruses were transferred into prokaryotic cells. Third, DNA from these bacteria were transferred into eukaryotic cells. Progeny viruses from BAC transfections showed similar kinetics as their corresponding wild types. Conclusion The two viral genomes of BoHV1 and BoHV5 cloned as BACs are accessible to the tools of bacterial genetics. The ability to easily manipulate the viral genomes on a molecular level in future experiments will lead to a better understanding of the difference in pathogenesis induced by these two closely related bovine herpesviruses.

  5. Molecular cloning, chromosomal mapping, and characterization of the mouse UDP-galactose: Ceramide galactosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Coetzee, T.; Fujita, N.; Marcus, J. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1996-07-01

    UDP-galactose:ceramide galactosyltransferase (CGT) (EC 2.11.62) catalyzes the final step in the synthesis of galactocerebroside, a glycosphingolipid characteristically abundant in myelin. In this report, we describe the isolation of genomic clones spanning the mouse CGT gene. The mouse CGT gene consists of six exons that span a minimum of 70 kb of DNA and that encode a 541 amino acid translation product with extensive sequence similarity to the rat CGT enzyme and to UDP-glucuronosyltransferases (UGT). The 5{prime}-untranslated region of the mouse CGT gene is encoded by a separate exon located approximately 25 kb upstream of the first protein-encoding exon. Furthermore, the genomic organization of the five encoding region exons of the mouse CGT gene resembles that of the human UGT1 and rat UGT2B1 genes. Finally, analysis of somatic cell hybrids by PCR and fluorescence in situ hybridization to metaphase chromosomes has localized the mouse CGT gene to chromosome 3, bands E3-F1. 26 refs., 5 figs., 1 tab.

  6. Cloning and deletion mapping of the recF dnaN region of the Escherichia coli chromosome.

    Science.gov (United States)

    Ream, L W; Clark, A J

    1983-09-01

    By cloning a 3.6-kb EcoRI fragment of the Escherichia coli chromosome with pBR322 we located more precisely recF relative to dnaN. By deletion mapping we localized functional recF to a 1.65-kb region of the cloned fragment and allowed rough mapping of the C terminus of dnaN. Cloned recF+, separated from functional flanking genes dnaN and gyrB, complemented chromosomal recF mutations presumably by coding for a cytodiffusible product. The protein encoded by dnaN was observed as a band on a polyacrylamide gel from minicells. Identification of a recF protein was not made.

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

    Science.gov (United States)

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

    1992-05-18

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

  8. Screening of YAC clones and building a map of the chromosome 13 region often deleted during chronic B-cell lymphocytic leucosis

    NARCIS (Netherlands)

    Brodyanskii, VM; Sulimova, GE; Udina, IG; Aitova, SS; Shaikhaev, GO; Sharikova, OA; Zakharev, VM; Fedorova, LI; Zelenin, AV; Eikhorn, S; Baush, C; Laland, M; Ross, M; Yankovskii, NK

    1995-01-01

    Pools of YAC clones from the ICRF library were analyzed by PCR using PBKpt, MGG15, and D13S25 markers that flank the chromosome 13 region often deleted during chronic lymphocytic leukemia. Ten clones were found and described. Nine mega-YAC clones from the CEPH library flanking the region of interest

  9. Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

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

    Science.gov (United States)

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

    2006-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  12. 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.

  13. 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.

  14. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

    Science.gov (United States)

    Lima, Fabio Mitsuo; Souza, Renata Torres; Santori, Fábio Rinaldo; Santos, Michele Fernandes; Cortez, Danielle Rodrigues; Barros, Roberto Moraes; Cano, Maria Isabel; Valadares, Helder Magno Silva; Macedo, Andréa Mara; Mortara, Renato Arruda; da Silveira, José Franco

    2013-01-01

    Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

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

    Science.gov (United States)

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

    2004-01-01

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

  16. Molecular cloning of the cDNA encoding follicle-stimulating hormone beta subunit of the Chinese soft-shell turtle Pelodiscus sinensis, and its gene expression.

    Science.gov (United States)

    Chien, Jung-Tsun; Shen, San-Tai; Lin, Yao-Sung; Yu, John Yuh-Lin

    2005-04-01

    Follicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family. These hormones are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSHbeta in reptilian species. For better understanding of the phylogenetic diversity and evolution of FSH molecule, we have isolated and sequenced the complementary DNA (cDNA) encoding the Chinese soft-shell turtle (Pelodiscus sinensis, Family of Trionychidae) FSHbeta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned Chinese soft-shell turtle FSHbeta cDNA consists of 602-bp nucleotides, including 34-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 206-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHbeta subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the Chinese soft-shell turtle FSHbeta subunit. The deduced amino acid sequence of the Chinese soft-shell turtle FSHbeta shares identities of 97% with Reeves's turtle (Family of Bataguridae), 83-89% with birds, 61-70% with mammals, 63-66% with amphibians and 40-58% with fish. By contrast, when comparing the FSHbeta with the beta-subunits of the Chinese soft-shell turtle luteinizing hormone and thyroid stimulating hormone, the homologies are as low as 38 and 39%, respectively. A phylogenetic tree including reptilian species of FSHbeta subunits, is presented for the first time. Out of various tissues examined, FSHbeta mRNA was only expressed in the pituitary gland and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated by fluorescence real-time PCR analysis.

  17. 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.

  18. 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.

  19. 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.

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

    Science.gov (United States)

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

    2011-01-01

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

  1. 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.

  2. Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni.

    Science.gov (United States)

    Dores, Robert M; Cameron, Erin; Lecaude, Stephanie; Danielson, Phillip B

    2003-08-01

    Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for beta-endorphin, ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH. Port Jackson shark delta-MSH has 83% primary sequence identity with dogfish and stingray delta-MSH, and it appears that the delta-MSH sequence may have been the result of an internal domain duplication and reinsertion of the beta-MSH sequence. The presence of the delta-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the delta-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.

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

    Science.gov (United States)

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

    2004-06-01

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

  4. 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.

  5. 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.

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

    Science.gov (United States)

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王家庆; 边佳; 李代宗; 马爽; 王亮; 那广宁

    2013-01-01

    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

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

    Science.gov (United States)

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

    2012-10-01

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

  9. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

  11. The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, C.A.; Holmgren, A. [Karolinska Inst., Stockholm (Sweden); Bajalica, S.; Lagercrantz, J. [Karolinska Hospital, Stockholm (Sweden)

    1996-03-05

    Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe. 13 refs., 2 figs.

  12. 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.

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Science.gov (United States)

    Mehdy, M C; Lamb, C J

    1987-06-01

    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 lambdagt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 10 recombinants with an antiserum to purified bean CHI. The identity of the cloned sequences was confirmed by hybrid-select translation and the production of antigenic polypeptides from transcripts synthesized in vitro. Addition of elicitor to cell cultures resulted in the rapid accumulation of CHI mRNA, with maximum levels achieved 3-4 h after elicitation. CHI mRNA also accumulated during the natural infection of hypocotyls with the fungal pathogen Colletotrichum lindemuthianum, and in mechanically wounded hypocotyls. The kinetics of accumulation of CHI mRNA in response to these environmental signals were strikingly similar to those of mRNAs encoding two other phenylpropanoid pathway enzymes, phenylalanine ammonialyase and chalcone synthase. In contrast to the multi-gene families encoding these two enzymes, chalcone isomerase is encoded by a single gene which is regulated by several environmental stimuli.

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

    Science.gov (United States)

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

    1992-04-13

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

  16. Evidence for different origin of sex chromosomes in snakes, birds, and mammals and step-wise differentiation of snake sex chromosomes.

    Science.gov (United States)

    Matsubara, Kazumi; Tarui, Hiroshi; Toriba, Michihisa; Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Agata, Kiyokazu; Matsuda, Yoichi

    2006-11-28

    All snake species exhibit genetic sex determination with the ZZ/ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python (Python molurus bivittatus) and the habu (Trimeresurus flavoviridis), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata, respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis. Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus, whereas they are fully differentiated in T. flavoviridis, and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids.

  17. 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.

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

    Science.gov (United States)

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

    2006-09-01

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

  19. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

    DEFF Research Database (Denmark)

    Gottwein, Judith; Scheel, Troels; Callendret, Benoit

    2010-01-01

    Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic...... analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees...

  20. cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica.

    Science.gov (United States)

    Kaneko, Kensuke; Washio, Kenji; Umezawa, Taiki; Matsuda, Fuyuhiko; Morikawa, Masaaki; Okino, Tatsufumi

    2014-01-01

    The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their Km values for Br(-) were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.

  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. 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.

  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.

  4. 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.

  5. 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.

  6. 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.

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

    Science.gov (United States)

    Mino, Masako; Sawada, Hitoshi

    2016-04-01

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

  8. 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.

  9. cDNA Cloning, Overexpression, Purification and Pharmacologic Evaluation for Anticancer Activity of Ribosomal Protein L23A Gene (RPL23A from the Giant Panda

    Directory of Open Access Journals (Sweden)

    Si-Nan Zhang

    2012-02-01

    Full Text Available RPL23A gene encodes a ribosomal protein that is a component of the 60S subunit. The protein belongs to the L23P family of ribosomal proteins, which is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of ribosomal protein L23A (RPL23A gene of the Giant Panda (Ailuropoda melanoleuca. The cDNA of RPL23A was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL23A cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. Recombinant protein of RPL23A obtained from the experiment acted on Hep-2 cells and human HepG-2 cells, then the growth inhibitory effect of these cells was observed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide assay. The result indicated that the length of the fragment cloned is 506 bp, and it contains an open-reading frame (ORF of 471 bp encoding 156 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL23A protein is 17.719 kDa with a theoretical pI 11.16. The molecular weight of the recombinant protein RPL23A is 21.265 kDa with a theoretical pI 10.57. The RPL23A gene can be really expressed in E. coli and the RPL23A protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 22 KDa polypeptide. The data showed that the recombinant protein RPL23A had a time- and dose-dependency on the cell growth inhibition rate. The data also indicated that the effect at low concentrations was better than at high concentrations on Hep-2 cells, and that the concentration of 0.185 μg/mL had the best rate of growth inhibition of 36.31%. All results of the experiment revealed that the recombinant protein RPL23A exhibited anti-cancer function on the Hep-2 cells. The study provides a scientific basis and aids

  10. 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.

  11. 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.

  12. Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

    Energy Technology Data Exchange (ETDEWEB)

    Hamann, J. [Univ. of Amsterdam (Netherlands)]|[Max Planck Society, Berlin-Buch (Germany); Hamann, D.; Lier, R.A.W. [Univ. of Amsterdam (Netherlands)] [and others

    1995-08-15

    CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).

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

    Science.gov (United States)

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

    2008-02-01

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

  14. 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.

  15. (+)-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.

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

    Science.gov (United States)

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

    2011-12-01

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

  17. 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.

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

    Science.gov (United States)

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

    2012-09-01

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

  19. 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.

  20. 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.

  1. Mapping and ordered cloning of the human X chromosome. Progress report, September 1991--November 1992

    Energy Technology Data Exchange (ETDEWEB)

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  2. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  3. 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

    Science.gov (United States)

    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 deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg2+ ≈ 150 μM; Km for Mn2+ ≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-β-farnesene (85%), (Z)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (Km ≈ 0.6 μM; Vrel = 100) and Mg2+ as cofactor, and (E)-β-farnesene (98%) and (Z)-β-farnesene (2%) with Mn2+ as cofactor (Vrel = 80). With the C10 analog, GDP, as substrate (Km = 1.5 μM; Vrel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced. PMID:9371761

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

    Science.gov (United States)

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

    2015-11-01

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

  5. cDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India

    Indian Academy of Sciences (India)

    THOMAS NAICY; THIRUPATHY VENKATACHALAPATHY; THAZHATHUVEETTIL ARAVINDAKSHAN; KUNNIYOOR CHEEMANI RAGHAVAN; MANGATTUMURUPPEL MINI; KULANGARA SHYAMA

    2017-06-01

    Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the full-length coding sequence of the caprine IGF1 gene from Attappady Black and Malabari breeds, two indigenous goat breeds of south India, to analyse its structure, and to ascertainthe relative abundance of IGF1 mRNA in different tissues. The caprine IGF1 cDNA (GenBank accession nos: KJ549851 and KJ549852) contained a 465-bp open reading frame encoding IGF1 protein with 154 amino acid residues. A novel SNP was detected in the 3' UTR region, g.931A>G. Genotyping was performed in 277 goats from the two genetic groups using the PCR-single strand conformational polymorphism (SSCP) and two genotypes, AA and AG were observed at this locus. IGF1 is a secretary pathway protein with 49 amino acid-long signal peptide with 19 phosphorylation sites. Caprine IGF1 amino acid sequence was 83–99% identical to other species with highest identity with the ruminants. Relative expression of IGF1 was highest in uterus and liver (P< 0.05), followed by oviduct and muscle. This work provided an important experimental basis for further research on the functions of IGF1 in goats.

  6. Expression at the cell surface of native fusion protein of the Newcastle disease virus (NDV) strain Italien from cloned cDNA.

    Science.gov (United States)

    Espion, D; de Henau, S; Letellier, C; Wemers, C D; Brasseur, R; Young, J F; Gross, M; Rosenberg, M; Meulemans, G; Burny, A

    1987-01-01

    A cDNA library was constructed with poly(A)+-mRNAs from NDV-Italien infected BHK-21 cells. A clone, that hybridized to the F gene mRNA, was sequenced. A long open reading frame encodes for a protein of 553 amino acids, with a calculated molecular weight of 59,153, consisting of twelve cysteine residues and six potential glycosylation sites. The protein sequence contains a hydrophobic region at the N-terminus of F1 and a presumptive long transmembrane fragment near the C-terminus. Comparison of the F proteins from NDV strains Italien and Australia-Victoria shows that the sequences are very similar, with conservation of most cysteine residues and of the potential glycosylation sites. The F coding sequence was inserted into the genome of vaccinia virus under the control of vaccinia P7.5 transcriptional regulatory sequences. Expression of F protein was demonstrated by indirect immunofluorescence with five anti-F monoclonal antibodies known to react with conformational epitopes.

  7. A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

    Science.gov (United States)

    Tomkins, J P; Mahalingam, R; Smith, H; Goicoechea, J L; Knap, H T; Wing, R A

    1999-09-01

    We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

  8. [CHL15--a new gene controlling the replication of chromosomes in saccharomycetes yeast: cloning, physical mapping, sequencing, and sequence analysis].

    Science.gov (United States)

    Kuprina, N Iu; Krol', E S; Koriabin, M Iu; Shestopalov, B V; Bliskovskiĭ, V V; Bannikov, V M; Gizatullin, R Z; Kirillov, A V; Kravtsov, V Iu; Zakhar'ev, V M

    1993-01-01

    We have analyzed the CHL15 gene, earlier identified in a screen for yeast mutants with increased loss of chromosome III and artificial circular and linear chromosomes in mitosis. Mutations in the CHL15 gene lead to a 100-fold increase in the rate of chromosome III loss per cell division and a 200-fold increase in the rate of marker homozygosis on this chromosome by mitotic recombination. Analysis of segregation of artificial circular minichromosome and artificially generated nonessential marker chromosome fragment indicated that sister chromatid loss (1:0 segregation) is a main reason of chromosome destabilization in the chl15-1 mutant. A genomic clone of CHL15 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL15 revealed a 2.8-kb open reading frame with a 105-kD predicted protein sequence. At the N-terminal region of the protein sequences potentially able to form DNA-binding domains defined as zinc-fingers were found. The C-terminal region of the predicted protein displayed a similarity to sequence of regulatory proteins known as the helix-loop-helix (HLH) proteins. Data on partial deletion analysis suggest that the HLH domain is essential for the function of the CHL15 gene product. Analysis of the upstream untranslated region of CHL15 revealed the presence of the hexamer element, ACGCGT (an MluI restriction site) controlling both the periodic expression and coordinate regulation of the DNA synthesis genes in budding yeast. Deletion in the RAD52 gene, the product of which is involved in double-strand break/recombination repair and replication, leads to a considerable decrease in the growth rate of the chl15 mutant. We suggest that CHL15 is a new DNA synthesis gene in the yeast Saccharomyces cerevisiae.

  9. Cloning

    Science.gov (United States)

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

  10. Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha.

    Science.gov (United States)

    Okada, S; Fujisawa, M; Sone, T; Nakayama, S; Nishiyama, R; Takenaka, M; Yamaoka, S; Sakaida, M; Kono, K; Takahama, M; Yamato, K T; Fukuzawa, H; Brennicke, A; Ohyama, K

    2000-11-01

    Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

  11. 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

  12. Chromosome

    Science.gov (United States)

    Chromosomes are structures found in the center (nucleus) of cells that carry long pieces of DNA. DNA ... is the building block of the human body. Chromosomes also contain proteins that help DNA exist in ...

  13. Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning.

    Science.gov (United States)

    Liu, Yao-Guang; Liu, Hongmei; Chen, Letian; Qiu, Weihua; Zhang, Qunyu; Wu, Hao; Yang, Chunyi; Su, Jing; Wang, Zhonghua; Tian, Dongsheng; Mei, Mantong

    2002-01-09

    The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.

  14. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

    DEFF Research Database (Denmark)

    Cottingham, Matthew G; Andersen, Rikke F; Spencer, Alexandra J

    2008-01-01

    The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC) in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20). The genomic BAC clone was 'rescued' back...... to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full......-2006). In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+) T cells against an epitope from Plasmodium berghei was created using Gal...

  15. Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA

    DEFF Research Database (Denmark)

    Ali, Ahmd; Thomsen, Preben Dybdahl; Babar, M.E.

    2009-01-01

    An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG....../CGG(n) trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human...... showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X....

  16. Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

    Science.gov (United States)

    Reddy, Sanjay M; Sun, Aijun; Khan, Owais A; Lee, Lucy F; Lupiani, Blanca

    2013-06-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

  17. Identification of a provirally activated c-Ha-ras oncogene in an avian nephroblastoma via a novel procedure: cDNA cloning of a chimaeric viral-host transcript.

    Science.gov (United States)

    Westaway, D; Papkoff, J; Moscovici, C; Varmus, H E

    1986-01-01

    Retrovirus without oncogenes often exert their neoplastic potential as insertional mutagens of cellular proto-oncogenes. This may be associated with the production of chimaeric viral-host transcripts; in these cases; activated cellular genes can be identified by obtaining cDNA clones of bipartite RNAs. This approach was used in the analysis of chicken nephroblastomas induced by myeloblastosis-associated virus (MAV). One tumor contained a novel mRNA species initiated within a MAV LTR. cDNA cloning revealed that this mRNA encodes a protein of 189 amino acids, identical to that of normal human Ha-ras-1 at 185 positions, including positions implicated in oncogenic activation of ras proto-oncogenes; there are no differences between the coding sequences of presumably normal Ha-ras cDNA clones from chicken lymphoma RNA and the tumor-derived cDNAs. The chimaeric mRNA in the nephroblastoma is at least 25-fold more abundant than c-Ha-ras mRNA in normal kidney tissue, and a 21-kd ras-related protein is present in relatively large amounts in the tumor. We conclude that a quantitative change in c-Ha-ras gene expression results from an upstream insertion mutation and presumably contributes to tumorigenesis in this single case. Little or no increase in c-Ha-ras RNA or protein was observed in other nephroblastomas. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 10. PMID:3011401

  18. Chromosomal assignment of chicken clone contigs by extending the consensus linkage map

    NARCIS (Netherlands)

    Aerts, J.; Veenendaal, T.; Poel, van der J.J.; Crooijmans, R.P.M.A.; Groenen, M.A.M.

    2005-01-01

    The bacterial artificial clone-based physical map for chicken plays an important role in the integration of the consensus linkage map and the whole-genome shotgun sequence. It also provides a valuable resource for clone selection within applications such as fluorescent in situ hybridization and posi

  19. 蒙古绵羊Ghrelin基因的克隆和原核表达%The cDNA Cloning and Prokaryotic Expression of Mongolia Sheep Ghrelin Gene

    Institute of Scientific and Technical Information of China (English)

    梁建荣; 曹贵方; 赵鹏伟; 温世勇; 程兰玲; 凃勇

    2011-01-01

    To construct Mongolia sheep Ghrelin prokaryotic expression vector, and observe its expression in host Escherichia coli strain BL2KDE3), the cDNA of Ghrelin gene was amplified from abomasum fundic gland mRNA of Mongolia sheep by RT-PCR. PCR product was cloned into the T vector pMD-19T to construct pMD-19T-Ghrelin for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET-32a( + ) and transformed into host Escherichia coli strain BL21(DE3)for expression. The clone was induced by IPTG and was identified by SDS-PAGE and Western-blot. Comparing with the Ghrelin sequence of sheep reported in GenBank, there were two nucleotide differences, but it did not affect the amino acid sequence; the expression product was observed with soluble protein; The result of Western-blot showed that the recombinant protein was recognized by His-antibody specifically. The nucleotide sequence isolated from the recombinant plasmid pET-32a-Ghrelin was the same as expected; the Ghrelin protein was expressed successfully in Escherichia coli, which provided a basis for further investigation of Ghrelin function.%为了克隆蒙古绵羊Ghrelin基因,构建其原核表达载体,并检测其在大肠杆菌中的表达情况.本研究用RT-PCR方法从蒙古绵羊胃组织mRNA扩增获得Ghrelin基因的cDNA序列,克隆到pMD-19T载体后进行序列分析.将测序正确的cDNA序列与原核表达载体pET-32a(+)连接,并转化BL21( DE3)大肠杆菌.经IPTG诱导后进行SDS-PAGE和Western-blot分析.结果表明克隆的蒙古绵羊Ghrelin基因序列与已发表基因序列有2个碱基的差异,该碱基的变化并不影响氨基酸序列,目的蛋白主要以可溶性形式存在,Western-blot初步证实了所获得的融合蛋白是特异的.本研究成功构建了蒙古绵羊Ghrelin基因的原核表达载体,并在大肠杆菌中获得高效表达,为进一步研究蒙古绵羊Ghrelin基因的功能提供参考.

  20. 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

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

    Science.gov (United States)

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

    2014-12-10

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

  2. Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.

  3. Engineering infectious foot-and-mouth disease virus in vivo from a full-lensth genomic cDNA clone of the A/AKT/58 strain

    Institute of Scientific and Technical Information of China (English)

    BAI XingWen; LI PingHua; CAO YiMei; LI Dong; LU ZengJun; GUO JianHong; SUN DeHui; ZHENG HaiXue; SUN Pu; LIU XiangTao; LUO JianXun; LIU ZaiXin

    2009-01-01

    Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10-7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10-6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran-scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.

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

    Institute of Scientific and Technical Information of China (English)

    石瑞; 曹诣斌; 陈文荣; 郭卫东

    2011-01-01

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

  5. Molecular cloning, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice ( Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    李子银; 张劲松; 陈受宜

    1999-01-01

    By using differential display PCR (DD-PCR) technique, two salt-inducible and one salt-repressed cDNA fragments were isolated from rice. The three eDNA fragments were characterized respectively as partial sequence of rice S-adenosylmethionine deearboxylase (SAMDC) gene, a new member of translation elongation factor 1A gene (named REF1A ), and a novel gene whose function is unknown (named SRG1). The full-length cDNA of SAMDC gene (named SAMDC1) was further isolated by RT-PCR approach and the deduced polypeptide was found to be homologous to SAMDC proteins of other plants, yeast and human. Northern hybridization revealed that expression of SAMDC1 and REF1A was induced, while SRG1 was dramatically repressed, by salinity stress. Southern blot analysis demonstrated that SAMDC1 and SRG1 were present as a single copy gene in rice genome, whereas rice REF1A gene was organized as a gene family. The REF1A, SAMDC1, and SRG1 genes were located on chromosome 3, 4, and 6 respectively by RFLP mapping approach using ZYQ

  6. Molecular cloning and sequence analysis of a cDNA encoding pituitary thyroid stimulating hormone beta-subunit of the Chinese soft-shell turtle Pelodiscus sinensis and regulation of its gene expression.

    Science.gov (United States)

    Chien, Jung-Tsun; Chowdhury, Indrajit; Lin, Yao-Sung; Liao, Ching-Fong; Shen, San-Tai; Yu, John Yuh-Lin

    2006-04-01

    A cDNA encoding thyroid stimulating hormone beta-subunit (TSHbeta) was cloned from pituitary of the Chinese soft-shell turtle, Pelodiscus sinensis, and its regulation of mRNA expression was investigated for the first time in reptile. The Chinese soft-shell turtle TSHbeta cDNA was cloned from pituitary RNA by reverse transcription and polymerase chain reaction (RT-PCR), and rapid amplification cDNA end (RACE) methods. The Chinese soft-shell turtle TSHbeta cDNA consists of 580-bp nucleotides, including 67-bp nucleotides of 5'-untranslated region (UTR), 402-bp of the open reading frame, and 97-bp of 3'-UTR followed by a 14 poly (A) trait. It encodes a precursor protein molecule of 133 amino acids with a putative signal peptide of 19 amino acids and a putative mature protein of 114 amino acids. The number and position of 12 cysteine residues, presumably forming six disulfide bonds, one putative asparagine-linked glycosylation site, and six proline residues that are found at positions for changing the backbone direction of the protein have been conserved in the turtle as in other vertebrate groups. The deduced amino acid sequence of the Chinese soft-shell turtle TSHbeta mature protein shares identities of 82-83% with birds, 71-72% with mammals, 49-57% with amphibians, and 44-61% with fish. The Chinese soft-shell turtle pituitaries were incubated in vitro with synthetic TRH (TSH-releasing hormone), thyroxine and triiodothyronine at doses of 10(-10) and 10(-8)M. TRH stimulated, while thyroid hormones suppressed, TSHbeta mRNA levels in dose-related manner. The sequences of cDNA and its deduced peptide of TSHbeta as well as the regulation of its mRNA level were reported for the first time in reptile.

  7. Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC%以绵羊MHC 区段BAC 克隆酶切片段为探针杂交筛选绵羊混合组织cDNA 文库

    Institute of Scientific and Technical Information of China (English)

    杨小亮; 白大章; 邱巍; 董慧芹; 李大全; 陈芳; 马润林; Hugh T Blair; 高剑峰

    2012-01-01

    Under the premise what we have known bacterial artificial chromosome(BAC) clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.%在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC 文库MHC 区段的6 个BAC 克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA 文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC 克隆比对以及在NCBI Blastn 数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析.实验中,经过两轮杂交共筛选出27 个cDNA 阳性克隆(序

  8. Cloning of the VASP (Vasodilator-Stimulated Phosphoprotein) genes in human and mouse: Structure, sequence, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Zimmer, M.; Fischer, L.; Hauser, W. [Medizinische Universitaetsklinik, Wuerzburg (Germany)] [and others

    1996-09-01

    The genes encoding the vasodilator-stimulated phosphoprotein (VASP) in human and mouse were isolated, and major parts were sequenced. In both species the gene is composed of 13 exons with conserved exon-intron positions. The mouse VASP cDNA sequence was deduced from the genomic sequence. The predicted amino acid sequence is 89% identical to the human protein. The high nucleotide sequence homology extends not only over the coding regions but also into the 3{prime}-UTRs, indicating a possible function in mRNA targeting or regulation of translation. Prominent 5{prime} CpG islands including multiple SP1 sites indicate a housekeeping function of VASP. Using cosmid DNA as a probe for fluorescence in situ hybridization, the human VASP gene was assigned to chromosome 19q13.2-q13.3, an extended region with homology to mouse chromosome 7. A sequence overlap of the VASP 5{prime}-region with the telomeric end of a cosmid contig physically links the VASP gene with ERCC1. VASP is located about 92 kb distal to ERCC1 and about 300 kb proximal to the myotonic dystrophy protein kinase gene. 43 refs., 6 figs.

  9. Identification of 2nd chromosome region translocated onto the W chromosome by RFLP with EST-cDNA clones in the Gensei-kouken strains of the mulberry silkworm, Bombyx mori L

    Directory of Open Access Journals (Sweden)

    Sivaramakurup Sreekumar

    2010-01-01

    Full Text Available In silkworms, sex-limited strains are either obtained spontaneously or induced by X-rays or gamma rays. When a fragment of an autosome carrying a dominant allele of those genes responsible for certain characters is translocated onto a W chromosome, the female of the successive generations will express these phenotypic characters and sex discrimination can be facilitated. Gensei-kouken strains are sex-limited strains of silkworms developed by irradiating the pupae with gamma rays, by which a portion of the second chromosome is translocated onto the W chromosome. In these improved strains, the females are yellow-blooded and spin yellow cocoons. By using the EST-cDNA clones mapped on the Z chromosome, we identified the sex according to the polymorphic banding pattern or intensity of the signals. Furthermore, by using the clones on the second chromosome, the region of the second chromosome translocated onto the W chromosome was also defined. In both the A95 and A 96 strains selected for the present study, only the mid-portion of the second chromosome was translocated. The differences in length of the fragments translocated in these strains are discussed.

  10. 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.

  11. Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa).

    Science.gov (United States)

    Feng, Bi-Hong; Wu, Bei; Zhang, Chun-Rong; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

    2012-01-15

    Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv. Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin (KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be involved in the modulation of TDZ-induced morphogenesis responses. However, very little is known about the genes involved in ethylene formation during somatic embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved in the effect of TDZ on inhibition of embryogenic competence of the alfalfa callus. In this study we cloned full-length ACO cDNA from the alfalfa callus, named MsACO, and observed changes in this gene expression during callus formation and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis showed that during the EC subcultural period, the expression level of MsACO in EC was significantly increased on the 2nd day, rose to the highest level on the 8th day and remained at this high level until the 21st day. However, the ACO expression in the TDZ (0.93 μM)-treated callus was higher than in the EC especially on the 8th day. Moreover the ACO expression level increased with increasing TDZ concentration during the subcultural/maintenance period of the callus. It is worth noting that comparing the treatment with TDZ alone, the treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene expressions and ACO activity in the treated callus. These results indicate that the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic competence of alfalfa callus could be mediated by ACO gene expression. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.

  12. 醛糖还原酶cDNA的克隆和表达%Cloning and Expression cDNA of Aldose Reductase

    Institute of Scientific and Technical Information of China (English)

    项军强; 蒋红亮; 张虹; 丁明; 赵辅昆

    2011-01-01

    Aldose reductase(AR), the rate-limiting enzyme of the polyol pathway of sugar metabolism, has been implicated in the pathogenesis of diabetic complications, in particular D-glucose reduction to sorbitol. Aldose reductase cDNA is amplified by reverse transcription-PCR (RT-PCR) from the isolated SMMC7721 total RNA. The PCR product is cloned and inserted into E. coli expression vector pET22b(+) to create a recombinant plasmid with 6× His Tag, which is named pET22b(+)-AR. The AR-(His)6 fusion protein is expressed in E. coli BL21(DE3) by IPTG. And AR-(His)6 is purified by Ni-NTA affinity chromotagraphy. Finally the purified AR-(His)6 activity enzyme is measured by AR assay with ultraviolet spectrophotometry. The results of this study may represent a basis for development of potential drug in preventing diabetic complications.%醛糖还原酶能促使葡萄糖转化成山梨醇,是多元醇代谢通路的限速酶,与糖尿病并发症的发生和发展有密切关系.用RT-PCR扩增醛糖还原酶基因,将扩增产物克隆到大肠杆菌表达载体pET22b(+),构建重组表达载体pET22b(+)-AR.经PCR、双酶切和序列测定鉴定后,转化E.coli BL21(DE3),经IPTG诱导表达,SDS-PAGE和Western blotting对重组蛋白进行分析和鉴定后,利用Ni-NTA琼脂糖亲和层析纯化获得重组蛋白AR-(His)6.紫外分光光度法对AR-(His)6进行酶活检测,其比活力为0.45 U/mg,为下一步筛选具有抑制醛糖还原酶活性的化合物及开发有临床应用价值的醛糖还原酶抑制剂提供参考.

  13. Transfer of stem cells carrying engineered chromosomes with XY clone laser system.

    Science.gov (United States)

    Sinko, Ildiko; Katona, Robert L

    2011-01-01

    Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.

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

    Institute of Scientific and Technical Information of China (English)

    曹运长; 李文笙; 叶卫; 林浩然

    2004-01-01

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

  15. Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Yu, Feng; Lei, Yunting; Li, Yuan; Dou, Quanwen; Wang, Haiqing; Chen, Zhiguo

    2013-07-01

    Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70-80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.

  16. Cloning and characterization of dispersed repetitive DNA derived from microdissected sex chromosomes of Rumex acetosa.

    Science.gov (United States)

    Mariotti, Beatrice; Navajas-Pérez, Rafael; Lozano, Rafael; Parker, John S; de la Herrán, Roberto; Rejón, Carmelo Ruiz; Rejón, Manuel Ruiz; Garrido-Ramos, Manuel; Jamilena, Manuel

    2006-02-01

    Rumex acetosa is characterized by a multiple chromosome system (2n = 12 + XX for females, and 2n = 12 + XY1Y2 for males), in which sex is determined by the ratio between the number of X chromosomes and autosome sets. For a better understanding of the molecular structure and evolution of plant sex chromosomes, we have generated a sex chromosome specific library of R. acetosa by microdissection. The screening of this library has allowed us to identify 5 repetitive DNA families that have been characterized in detail. One of these families, DOP-20, has shown no homology with other sequences in databases. Nevertheless, the putative proteins encoded by the other 4 families, DOP-8, DOP-47, DOP-60, and DOP-61, show homology with proteins from different plant retroelements, including poly proteins from Ty3-gypsy- and Ty1-copia-like long terminal repeat (LTR) retroelements, and reverse transcriptase from non-LTR retro elements. Results indicate that sequences from these 5 families are dispersed throughout the genome of both males and females, but no appreciable accumulation or differentiation of these types of sequences have been found in the Y chromosomes. These repetitive DNA sequences are more conserved in the genome of other dioecious species such as Rumex papillaris, Rumex intermedius, Rumex thyrsoides, Rumex hastatulus, and Rumex suffruticosus, than in the polygamous, gynodioecious, or hermaphrodite species Rumex induratus, Rumex lunaria, Rumex con glom er atus, Rumex crispus, and Rumex bucephalo phorus, which supports a single origin of dioecious species in this genus. The implication of these transposable elements in the origin and evolution of the heteromorphic sex chromosomes of R. acetosa is discussed.

  17. Sequence and chromosomal localization of the mouse brevican gene

    DEFF Research Database (Denmark)

    Rauch, U; Meyer, H; Brakebusch, C

    1997-01-01

    Brevican is a brain-specific proteoglycan belonging to the aggrecan family. Phage clones containing the complete mouse brevican open reading frame of 2649 bp and the complete 3'-untranslated region of 341 bp were isolated from a mouse brain cDNA library, and cosmid clones containing the mouse bre...... to an alternative brevican cDNA, coding for a GPI-linked isoform. Single strand conformation polymorphism analysis mapped the brevican gene (Bcan) to chromosome 3 between the microsatellite markers D3Mit22 and D3Mit11....

  18. Chromosomal localization of actin genes in the malaria mosquito Anopheles darlingi

    Science.gov (United States)

    BRIDI, L. C.; SHARAKHOVA, M. V.; SHARAKHOV, I. V.; CORDEIRO, J.; AZEVEDO, G. M.; TADEI, W. P.; RAFAEL, M. S.

    2012-01-01

    Physical and genetic maps have been used for chromosomal localization of genes in vectors of infectious diseases. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to precisely map genes of interest. We report physical mapping of two actin genes on polytene chromosomes of the major malaria vector in Amazon Anopheles darlingi. The clones with the actin genes sequences were obtained from a cDNA library constructed from RNA isolated from adult females and males of An. darlingi. Each of the two clones was mapped to a unique site on the chromosomal arm 2L in subdivisions 21A (clone pl05-A04) and 23B (clone pl17-G06). The obtained results together with previous mapping data provide a suitable basis for comparative genomics and for establishing chromosomal homologies among major malaria vectors. PMID:22804344

  19. Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

    Science.gov (United States)

    Nishi, Tatsuya; Onozato, Hiroyuki; Ohashi, Seiichi; Fukai, Katsuhiko; Yamada, Manabu; Morioka, Kazuki; Kanno, Toru

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus.

  20. Cloning, characterization and chromosomal location of a satellite DNA from the Pacific oyster, Crassostrea gigas

    Digital Repository Service at National Institute of Oceanography (India)

    Clabby, C.; Goswami, U.; Flavin, F.; Wilkins, N.P.; Houghton, J.A.; Powell, R.

    mixture was used to transform competent E. coli JM109 by electroporation. Recombinant clones were detected as white colonies on LB medium plates contain- ing 50 gg Km/ml, 40 gg XGal/ml and 0.2 mM IPTG and were screened by colony hybridization...

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

    OpenAIRE

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

    1992-01-01

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

  2. 阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析%Molecular Cloning and Characterization of a Rac1 Homologue cDNA from Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    傅玉才; 章家新; 郑晓虹; 刘红

    2004-01-01

    Objective To clone and characterize a Racl homologue from Trichomonas vaginalis for studying cell cycle of the organism. Methods A cDNA library derived from T. vaginalis mRNA was constructed into λ TriplEx2 phage vector. An expression sequence tag program was launched. Sequences of cDNA clones were analyzed using NCBI BLAST algorithms, and ClustalW and Treeview programs. Results A cDNA clone with a length of 714 base pairs was isolated. The sequence analysis showed that the cDNA clone has an open reading frame with 600 bp. The deduced amino acid sequence from the open reading frame contains 200 residuals and is most homologous to Rac1 subfamily of Rho GTPases with > 60% identity. The conserved sequence elements of Rho GTPases, such as GTP-binding sites, GTPase-activating protein (GAP) interaction motifs, GTP-dissociation inhibitors (GDI) interaction motifs, guanine nucleotide exchange factor (GEF) interaction elements, etc, were detected in the amino acid sequence. The phylogenetic analysis showed that the cDNA clone is grouped in the Rac subfamily and is more closely related to Rac1 proteins of protozoa. Conclusion The cDNA clone isolated belongs to Rac subfamily of Rho GTPases and is probably a Rac1 protein of T. vaginalis.%目的获得阴道毛滴虫Rac1蛋白的cDNA克隆,研究其在细胞周期中的调解作用.方法提取阴道毛滴虫总RNA,构建cDNA表达文库,随机分离cDNA克隆并测序.用在线生物分析软件NCBI BLAST、ClustalW以及Treeview等程序进行序列分析.结果获得一株有714 bp的cDNA克隆.序列分析表明,该克隆开放阅读框具600 bp,推测肽链具200个氨基酸.该肽链与Rho家族中Rac1鸟苷三磷酸(GTP)酶同源性最高(>60%),并具多种Rho GTP酶的保守基序,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等.进化树分析显示该克隆属于Rac亚家族GTP酶,与原虫Rac1蛋白最接近.结论该克隆

  3. Cloning, expression, and chromosomal assignment of the human mitochondrial intermediate peptidase gene (MIPEP).

    Science.gov (United States)

    Chew, A; Buck, E A; Peretz, S; Sirugo, G; Rinaldo, P; Isaya, G

    1997-03-15

    The mitochondrial intermediate peptidase of Saccharomyces cerevisiae (YMIP) is a component of the yeast mitochondrial protein import machinery critically involved in the biogenesis of the oxidative phosphorylation (OXPHOS) system. This leader peptidase removes specific octapeptides from the amino terminus of nuclear-encoded OXPHOS subunits and components of the mitochondrial genetic apparatus. To address the biologic role of the human peptidase [MIPEP gene, HMIP polypeptide], we have initiated its molecular and functional characterization. A full-length cDNA was isolated by screening a human liver library using a rat MIP (RMIP) cDNA as a probe. The encoded protein contained a typical mitochondrial leader peptide and showed 92 and 54% homology to RMIP and YMIP, respectively. A survey of human mitochondrial protein precursors revealed that, similar to YMIP, HMIP is primarily involved in the maturation of OXPHOS-related proteins. Northern analysis showed that the MIPEP gene is differentially expressed in human tissues, with the highest levels of expression in the heart, skeletal muscle, and pancreas, three organ systems that are frequently affected in OXPHOS disorders. Using fluorescence in situ hybridization, the MIPEP locus was assigned to 13q12. This information offers the possibility of testing the potential involvement of HMIP in the pathophysiology of nuclear-driven OXPHOS disorders.

  4. 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.

  5. Cloning, chromosomal localization, SNP detection and association analysis of the porcine IRS-1 gene.

    Science.gov (United States)

    Niu, P-X; Huang, Z; Li, C-C; Fan, B; Li, K; Liu, B; Yu, M; Zhao, S-H

    2009-11-01

    Insulin receptor substrate-1(IRS-1) gene is one member of the Insulin receptor substrate (IRS) gene family, which plays an important role in mediating the growth of skeletal muscle and the molecular metabolism of type 2 diabetes. Here, we cloned a 3,573 bp fragment of the partial CDS sequence of porcine IRS-1 gene by in silicon cloning strategy and RT-PCR method. The porcine IRS-1 gene was assigned to SSC15q25 by using IMpRH. Sequencing of PCR products from Duroc and Tibetan pig breeds identified one SNP in exon 1 of porcine IRS-1 gene (C3257A polymorphisms). Association analysis of genotypes with the growth traits, anatomy traits, meat quality traits and physiological biochemical indexes traits showed that different genotypes at locus 3,257 of IRS-1 have significant differences in carcass straight length in pigs (P = 0.0102 \\ 0.05).

  6. DNA sequences and composition from 12 BAC clones-derived MUSB SSR markers mapped to cotton (Gossypium Hirsutum L. x G. Barbadense L.)chromosomes 11 and 21

    Science.gov (United States)

    To discover resistance (R) and/or pathogen-induced (PR) genes involved in disease response, 12 bacterial artificial chromosome (BAC) clones from cv. Acala Maxxa (G. hirsutum) were sequenced at the Clemson University, Genomics Institute, Clemson, SC. These BACs derived MUSB single sequence repeat (SS...

  7. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes

    Science.gov (United States)

    The long terminal repeat (LTR) sequence of reticuloendotheliosis virus (REV) was inserted into the very virulent Marek’s disease virus (MDV) Md5 bacterial artificial chromosome clone. The insertion site was nearly identical to the REV LTR that was naturally inserted into the JM/102W strain of MDV fo...

  8. Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication.

    Science.gov (United States)

    Park, Soo-Jeong; Lee, Byung-Woo; Moon, Hyun-Woo; Sung, Haan Woo; Yoon, Byung-Il; Meng, Xiang-Jin; Kwon, Hyuk Moo

    2015-05-01

    A full-length infectious cDNA clone of the genotype 1 Korean avian hepatitis E virus (avian HEV) (pT11-aHEV-K) was constructed and its infectivity and pathogenicity were investigated in leghorn male hepatoma (LMH) chicken cells and broiler breeders. We demonstrated that capped RNA transcripts from the pT11-aHEV-K clone were translation competent when transfected into LMH cells and infectious when injected intrahepatically into the livers of chickens. Gross and microscopic pathological lesions underpinned the avian HEV infection and helped characterize its pathogenicity in broiler breeder chickens. The avian HEV genome contains a hypervariable region (HVR) in ORF1. To demonstrate the utility of the avian HEV infectious clone, several mutants with various deletions in and beyond the known HVR were derived from the pT11-aHEV-K clone. The HVR-deletion mutants were replication competent in LMH cells, although the deletion mutants extending beyond the known HVR were non-viable. By using the pT11-aHEV-K infectious clone as the backbone, an avian HEV luciferase reporter replicon and HVR-deletion mutant replicons were also generated. The luciferase assay results of the reporter replicon and its mutants support the data obtained from the infectious clone and its derived mutants. To further determine the effect of HVR deletion on virus replication, the capped RNA transcripts from the wild-type pT11-aHEV-K clone and its mutants were injected intrahepatically into chickens. The HVR-deletion mutants that were translation competent in LMH cells displayed in chickens an attenuation phenotype of avian HEV infectivity, suggesting that the avian HEV HVR is important in modulating the virus infectivity and pathogenicity.

  9. Cloning, structural characterization, and chromosomal localization of the gene encoding the human prostaglandin E(2) receptor EP2 subtype.

    Science.gov (United States)

    Smock, S L; Pan, L C; Castleberry, T A; Lu, B; Mather, R J; Owen, T A

    1999-09-17

    Northern blot analysis of human placental RNA using a probe to the 5' end of the human prostaglandin E(2) (PGE(2)) EP2 receptor subtype coding region revealed the existence of a high abundance, low molecular weight transcript. To investigate the origin of this transcript, and its possible relationship to the human EP2 mRNA, we have cloned and characterized the gene encoding the human PGE(2) EP2 receptor subtype, identified transcriptional initiation and termination sites in two tissues (spleen and thymus), and determined its chromosomal localization. The human EP2 gene consists of two exons separated by a large intron, utilizes a common initiation site in both spleen and thymus at 1113 bp upstream of the translation initiation site, and has 3' transcript termini at 1140 bp and 1149 bp downstream of the translation stop site in spleen and thymus respectively. Southern and fluorescence in situ hybridization analysis demonstrated the human EP2 gene to be a single copy gene located in band 22 of the long arm of chromosome 14 (14q22). Though our initial interest in this gene was to investigate potential differential splicing of the human EP2 gene in placenta, this work demonstrates that the atypical transcript observed in placenta probably arises from a distinct, yet related, gene. Knowledge of the sequence, structure, and transcription events associated with the human EP2 gene will enable a broader understanding of its regulation and potential role in normal physiology and disease.

  10. Rapid Emergence and Evolution of Staphylococcus aureus Clones Harboring fusC-Containing Staphylococcal Cassette Chromosome Elements.

    Science.gov (United States)

    Baines, Sarah L; Howden, Benjamin P; Heffernan, Helen; Stinear, Timothy P; Carter, Glen P; Seemann, Torsten; Kwong, Jason C; Ritchie, Stephen R; Williamson, Deborah A

    2016-04-01

    The prevalence of fusidic acid (FA) resistance amongStaphylococcus aureusstrains in New Zealand (NZ) is among the highest reported globally, with a recent study describing a resistance rate of approximately 28%. Three FA-resistantS. aureusclones (ST5 MRSA, ST1 MSSA, and ST1 MRSA) have emerged over the past decade and now predominate in NZ, and in all three clones FA resistance is mediated by thefusCgene. In particular, ST5 MRSA has rapidly become the dominant MRSA clone in NZ, although the origin of FA-resistant ST5 MRSA has not been explored, and the genetic context offusCin FA-resistant NZ isolates is unknown. To better understand the rapid emergence of FA-resistantS. aureus, we used population-based comparative genomics to characterize a collection of FA-resistant and FA-susceptible isolates from NZ. FA-resistant NZ ST5 MRSA displayed minimal genetic diversity and represented a phylogenetically distinct clade within a global population model of clonal complex 5 (CC5)S. aureus In all lineages,fusCwas invariably located within staphylococcal cassette chromosome (SCC) elements, suggesting that SCC-mediated horizontal transfer is the primary mechanism offusCdissemination. The genotypic association offusCwithmecAhas important implications for the emergence of MRSA clones in populations with high usage of fusidic acid. In addition, we found thatfusCwas colocated with a recently described virulence factor (tirS) in dominant NZS. aureusclones, suggesting a fitness advantage. This study points to the likely molecular mechanisms responsible for the successful emergence and spread of FA-resistantS. aureus.

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

    OpenAIRE

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

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately ...

  12. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA.

    Directory of Open Access Journals (Sweden)

    Matthew G Cottingham

    Full Text Available The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20. The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA, now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+ T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+ T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins, in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006. In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+ T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant

  13. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Konstantin A. Tsetsarkin

    2016-08-01

    Full Text Available An arthropod-borne virus, Zika virus (ZIKV, has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics.

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

    Science.gov (United States)

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

    2014-02-01

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

  15. Construction of an infectious cDNA clone of foot-and-mouth disease virus type O1BFS 1860 and its use in the preparation of candidate vaccine

    Indian Academy of Sciences (India)

    M Hema; D Chandran; S B Nagendrakumar; M Madhanmohan; V A Srinivasan

    2009-03-01

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA, RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines.

  16. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    Science.gov (United States)

    Tsetsarkin, Konstantin A.; Kenney, Heather; Chen, Rubing; Liu, Guangping; Manukyan, Hasmik; Whitehead, Stephen S.; Laassri, Majid

    2016-01-01

    ABSTRACT An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics. PMID:27555311

  17. Cloning and identification of full-length DCC cDNA and construction of its eukaryotic expression vector%人类DCC基因克隆及真核表达载体构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    翟保平; 李文涛; 张斌; 于洋; 李育红

    2011-01-01

    目的 克隆人类DCC基因并构建其真核表达载体pIRES2-AcGFPI/DCC.方法 从正常皮肤组织中提取总RNA,采用逆转录-聚合酶链反应(RT-PCR)方法扩增DCC基因全长cDNA(4351bp),克隆人pMD18-T载体并转化大肠杆菌JM109,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸测序分析,再将DCC基因定向克隆人pIRES2-AcGFP1载体中构建表达载体pIRES2-AcGFP1/DCC.结果 RT-PCR扩增后的产物在约4351 bp处出现明显的特异性条带,DCC基因的cDNA片段被成功插入真核表达载体pIRES2-AcGFP1质粒的多克隆位点,经鉴定与GenBank收录的DCC cDNA序列一致.结论 DCC基因的cDNA片段被成功克隆.%Objective To clone the full-length cDNA of human tumor suppressor DCC gene and construct its eukaryotic expression vector. Methods Total RNA was isolated from human foreskin tissue.Full-length DCC cDNA fragment (4351 bp) was amplified by reverse-transcription polymerase chain reaction (RT-PCR) and inserted into pMD18-T vector. The recombinant pMD18-T/DCC cDNA was transformed into E. coli JM109 host bacteria. The positive clones were confirmed by RT-PCR and doule-enzyme digestion assay. Orientation-based sub-cloning into pIRES2-AcGFP1 was performed as above followed by sequencing. Results Product of RT-PCR showed a clear specific band at 4341bp. pIRES2-AcGFP1/DCC was successfully constructed and transformed into E. coli JM109 host bacteria. Conclusion DCC gene cDNA has been inserted into eukaryotic expression vector pIRES2-AcGFP1 and successfully expressed.

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

    Institute of Scientific and Technical Information of China (English)

    庞娟; 祝逸平; 杨国玲

    2012-01-01

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

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

    Science.gov (United States)

    Kondo, M; Sutou, S

    1997-01-01

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

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

    NARCIS (Netherlands)

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

    2002-01-01

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

  1. Cloning and Sequence Analysis of the Full-length cDNA of a Novel yp05 Gene Associated With Citrinin Production in Monascus aurantiacus

    Institute of Scientific and Technical Information of China (English)

    YON-GHUA XIONG; YANG XU; WEI-HUA LAI; YAN-PIN LI; HUA WEI

    2007-01-01

    Objective To obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

  2. 家蚕铜锌超氧化物歧化酶cDNA的克隆与测序%Cloning and sequencing of the silkworm (Bombyx mori) copper zinc-superoxide dismutase cDNA

    Institute of Scientific and Technical Information of China (English)

    唐云明; 鲁成; 向仲怀; 许禾声

    2003-01-01

    The total RNA of the silkworm (Bombyx mori ) was obtained with chlorinated caesium density gradient cen-trifugation, mRNA was converted to cDNA by reverse transcription. This cDNA was used as a template for two PCR am-plifications by three primers. These were DP1: 5′-ATGGT (GT) GT (GT) AA (AG) GC (TC) GT-3′; DP2: 5′-ATGGT (GT) GT (GT) AA (AG) GC (TC) GT (GT) (CT) T-3′ and PA: 5′-GAGGACTCGAGCTCAAGC-3′. Northern blot identification of poly A+ mRNA extracted from silkworms by hybridization with the above amplified product detected a single mRNA transcript of approximate 500 bp. This showed that the amplified product was from silk-worm mRNA. Amplified products were separated by agarose gel electrophoresis and purified. The purified products were ligated to pUC19-T or pUCm-T vector and transformed into E. coli DHSct competent cells. Recombinant colonies were screened by X-gal. A clone of silkworm CuZn-SOD cDNA was thus obtained. DNA sequencing was done using the Sanger dideoxy method. The result of recombinant plasmid DNA sequencing using a DNA sequencer was 591 bps cloned. Se-quencing with DNAStar and DNAClub revealed that l - 3 bp of the 5′-extremity was the initiation codon ATG. 459 bp of the 5′-extremity translated 153 amino acids, 460 - 462 bp was the termination codon TAA which included the degenera-tion primer (DP2) derived from the N-terminal amino acid of the 5′end, PA of the 3′end and 73 bp poly A+ tail at the front of PA in the 3′end and so on.

  3. New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone.

    Science.gov (United States)

    Richards, Alexsia L; Sollars, Patricia J; Smith, Gregory A

    2016-08-31

    Infectious clones are fundamental tools for the study of many viruses, allowing for efficient mutagenesis and reproducible production of genetically-defined strains. For the large dsDNA genomes of the herpesviridae, bacterial artificial chromosomes have become the cloning vector of choice due to their capacity to house full-length herpesvirus genomes as single contiguous inserts. Furthermore, while maintained as plasmids in Escherichia coli, the clones can be mutated using robust prokaryotic recombination systems. An important consideration in the design of these clones is the means by which the vector backbone is removed from the virus genome upon delivery into mammalian cells. A common approach to vector excision is to encode loxP sites flanking the vector sequences and rely on Cre recombinase expression from a transformed cell line. Here we examine the efficiency of vector removal using this method, and describe a "self-excising" infectious clone of HSV-1 strain F that offers enhancements in virus production and utility. Insertion of a fluorescent protein expression cassette into the vector backbone of the HSV-1 strain F clone, pYEbac102, demonstrated that 2 serial passages on cells expressing Cre recombinase was required to achieve > 95 % vector removal from the virus population, with 3 serial passages resulting in undetectable vector retention. This requirement was eliminated by replacing the reporter coding sequence with the CREin gene, which consists of a Cre coding sequence disrupted by a synthetic intron. This self-excising variant of the infectious clone produced virus that propagated with wild-type kinetics in culture and lacked vector attenuation in a mouse neurovirulence model. Conversion of a herpesvirus infectious clone into a self-excising variant enables rapid production of viruses lacking bacterial vector sequences, and removes the requirement to initially propagate viruses in cells that express Cre recombinase. The self-excising bacterial

  4. 人UCA1基因新剪接变异体全长cDNA序列的克隆%Cloning of the full-length cDNA sequence of a novel human UCA1 spliced variant

    Institute of Scientific and Technical Information of China (English)

    王宇; 陈葳; 李旭

    2012-01-01

    Objective To clone the full-length cDNA sequence of novel UCA1 spliced isoforms for understanding the exact mechanism of this type of alternative splicing. Methods The full-length cDNA was amplified from BLZ-211 cells by using the in silicon sequence elongation technique, 5'-RACE and 3'-RACE techniques. Products of RT-PCR were sequenced and further assembled. Results The new UCA1 spliced isoform sequence was 2 202 bp. Conclusion A combination of the in silicon sequence elongation, 5'-RACE and 3'-RACE techniques is an effective way to obtain the full-length cDNA, which will guide further research on the mechanism of this type of alternative splicing.%目的 克隆新的UCA1剪接变异体全长cDNA序列,为研究其可变剪接机制奠定基础.方法 用电子克隆技术和cDNA序列末端快速扩增技术(rapid amplification of cDNA ends,RACE)扩增细胞系BLZ-211 cDNA并进行产物测序和序列拼接.结果 新克隆的UCA1剪接变异体全长cDNA序列为2 202 bp.结论 综合采用电子克隆技术与RACE技术是获得全长cDNA序列的有效方法,为该基因的后续可变剪接机制的研究奠定了基础.

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

    Institute of Scientific and Technical Information of China (English)

    王金玲; 王永; 刘鲁蜀; 陶永平

    2015-01-01

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

  6. cDNA and genomic cloning of human palmitoyl-protein thioesterase (PPT), the enzyme defective in infantile neuronal ceroid lipofuscinosis

    Energy Technology Data Exchange (ETDEWEB)

    Schriner, J.E.; Yi, W.; Hofmann, S.L. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)

    1996-06-15

    Palmitoyl-protein thioesterase (PPT) is a small glycoprotein that removes palmitate groups from cysteine residues in lipid-modified proteins. We recently reported mutations in PPT in patients with infantile neuronal ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder. INCL is characterized by the accumulation of proteolipid storage material in brain and other tissues, suggesting that the disease is a consequence of abnormal catabolism of acylated proteins. In the current paper, we report the sequence of the human PPT cDNA and the structure of the human PPT gene. The cDNA predicts a protein of 306 amino acids that contains a 25-amino-acid signal peptide, three N-linked glycosylation sites, and consensus motifs characteristic of thioesterases. Northern analysis of a human tissue blot revealed ubiquitous expression of a single 2.5-kb mRNA, with highest expression in lung, brain, and heart. The human PPT gene spans 25 kb and is composed of seven coding exons and a large eighth exon, containing the entire 3{prime}-untranslated region of 1388 bp. An Alu repeat and promoter elements corresponding to putative binding sites for several general transcription factors were identified in the 1060 nucleotides upstream of the transcription start site. The human PPT cDNA sequence and gene structure will provide the means for the identification of further causative mutations in INCL and facilitate genetic screening in selected high-risk populations. 31 refs., 5 figs., 1 tab.

  7. 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.

  8. 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.

  9. Molecular Cloning and Sequence Analysis on cDNA of Cystatin Gene from Tea Leaves%茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    王朝霞; 李叶云; 江昌俊; 余有本

    2005-01-01

    对多种已知植物巯基蛋白酶抑制剂(cystatin)基因的氨基酸序列进行比对分析,根据其高度保守的氨基酸序列设计一对简并引物,并从茶树品种龙井43鲜叶中提取总RNA,用RT-PCR法扩增出-204 bp的cDNA特异片段,然后通过3'/5'RACE的方法,分别扩增出3'端和5'端的序列,从而获得茶树巯基蛋白酶抑制剂基因的cDNA全长序列,所得序列全长627 bp,编码101个氨基酸,分子量约11.062 KDa.该基因在推测的氨基酸序列中含有巯基蛋白酶抑制剂家族中高度保守的、与其活性有关的QXVXG结构,且经Blast分析表明,该基因序列与其他植物巯基蛋白酶抑制剂基因的氨基酸序列同源性为54%~77%.%Two degenerate primers were designed according to the conserved region among the known plant cystatins. A cDNA fragment of 204 bp was amplified by RT-PCR (reverse transcription polymerase chain reaction)of total RNA extracted from fresh leaves of Tea plant (Camellia sinensis cv Longjing43). A full-length cDNA of the cystatin gene was obtained by 3'/5'RACE (rapid amplification of cDNA ends). The cDNA sequence of this 627 bp clone contained an open reading frame encoding a polypeptide of 101 amino acid residues with a predicable molecular mass of 11.026 KDa. The deduced amino acid sequence contained the motif QXVXG conserved among most members of the cystatin superfamily. By using the program of Blast on GenBank database, the sequence presented a high match with the cystatin genes from other plants, such as European chestnut, Cassava, Cowpea,Tomato, Soybean et al. All researched out sequences were all cystatins, so we can conclude that the cloned sequence is a member of cystatin gene from Tea plant.

  10. An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias.

    Science.gov (United States)

    Trinh, Quoclinh; Xu, Wentao; Shi, Hui; Luo, Yunbo; Huang, Kunlun

    2012-06-01

    A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO(4) and NH(2) groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3' ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments.

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

    Science.gov (United States)

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

    2014-10-01

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