Science.gov

Sample records for active cdna clones

  1. cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

    PubMed Central

    Ginsburg, D; Zeheb, R; Yang, A Y; Rafferty, U M; Andreasen, P A; Nielsen, L; Dano, K; Lebo, R V; Gelehrter, T D

    1986-01-01

    Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7. Images PMID:3097076

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

    SciTech Connect

    Meng, Baozhong; Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou; Dayan-Glick, Cathy; Mawassi, Munir

    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.

  3. Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning.

    PubMed

    Satoh, D; Horii, A; Ochiai, M; Ashida, M

    1999-03-12

    Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid. PMID:10066809

  4. cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds.

    PubMed

    Song, Xiaomin; Wang, Jing; Wu, Fang; Li, Xu; Teng, Maikun; Gong, Weimin

    2005-01-01

    SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal alpha-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far. PMID:15821865

  5. Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

    PubMed

    Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

    2016-10-01

    A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone. PMID:27139727

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

    PubMed

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

    2016-01-01

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

  7. Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.

    PubMed Central

    Davis, A R; Bos, T J; Nayak, D P

    1983-01-01

    We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein. Images PMID:6306656

  8. cDNA expression cloning in mammalian cells.

    PubMed

    Hoffman, B J

    2001-05-01

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

  9. Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor.

    PubMed Central

    Ny, T; Sawdey, M; Lawrence, D; Millan, J L; Loskutoff, D J

    1986-01-01

    A lambda gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the beta-migrating plasminogen activator inhibitor (beta-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 X 10(5) phages. Three clones (lambda 1.2, lambda 3, and lambda 9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for lambda 9.2, but not for lambda gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell beta-PAI and had beta-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human beta-PAI. Based on this alignment, the mature human beta-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with alpha 1-antitrypsin and antithrombin III, indicating that the beta

  10. Infectious Maize rayado fino virus from cloned cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.

    PubMed Central

    Cuenda, A; Alonso, G; Morrice, N; Jones, M; Meier, R; Cohen, P; Nebreda, A R

    1996-01-01

    Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6. Images PMID:8861944

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

    PubMed

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

    2016-04-01

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

  13. Rat peptide methionine sulphoxide reductase: cloning of the cDNA, and down-regulation of gene expression and enzyme activity during aging.

    PubMed Central

    Petropoulos, I; Mary, J; Perichon, M; Friguet, B

    2001-01-01

    Peptide methionine sulphoxide reductase (PMSR, EC 1.8.4.6), the msrA or pmsR gene product, is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins. Decreased expression and/or activity of the PMSR with age could explain, at least in part, the accumulation of oxidized protein observed upon aging. To test this hypothesis, the rat pmsR cDNA was cloned and sequenced. The recombinant protein was expressed, its catalytic activity checked with a synthetic substrate and polyclonal antibodies were raised against recombinant PMSR. The expression of the pmsR gene and protein as well as its catalytic activity were then analysed as a function of age in the rat brain and in two organs that express the most PMSR, liver and kidney. It appears that pmsR gene expression decreases with age in liver and kidney as early as 18 months, whereas protein level and protein activity are reduced in the three organs at the very end of the life of the rat (26 months). These results suggest that the down-regulation of PMSR can contribute to the accumulation of oxidized protein that has been associated with the aging process. PMID:11311146

  14. Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

    SciTech Connect

    Oshima, A.; Kyle, J.W.; Miller, R.D.; Hoffmann, J.W.; Powell, P.P.; Grubb, J.H.; Sly, W.S.; Tropak, M.; Guise, K.S.; Gravel, R.A.

    1987-02-01

    The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.

  15. Cloning and sequencing of chloroperoxidase cDNA.

    PubMed Central

    Fang, G H; Kenigsberg, P; Axley, M J; Nuell, M; Hager, L P

    1986-01-01

    An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA. A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322. The complete primary sequence of chloroperoxidase has been derived. We have also determined about 73% of the peptide sequence by amino acid sequencing. The DNA sequence data matches all of the available known peptide sequences. The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons. A putative signal peptide of 21 amino acids is proposed from DNA sequence data. The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences. Three cysteine residues are found in the protein sequence. A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam. No other heme protein homologues can be detected. We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group. A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E. coli or yeast. PMID:3774552

  16. Brain cDNA clone for human cholinesterase.

    PubMed Central

    McTiernan, C; Adkins, S; Chatonnet, A; Vaughan, T A; Bartels, C F; Kott, M; Rosenberry, T L; La Du, B N; Lockridge, O

    1987-01-01

    A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase (EC 3.1.1.8). Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase (EC 3.1.1.8) rather than acetylcholinesterase (EC 3.1.1.7). It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes, coded for cholinesterase. Images PMID:3477799

  17. cDNA cloning and gene expression of ascorbate oxidase in tobacco.

    PubMed

    Kato, N; Esaka, M

    1996-02-01

    A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Nothern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant. PMID:8624413

  18. Generation of an infectious Negev virus cDNA clone

    PubMed Central

    Gorchakov, Rodion V.; Tesh, Robert B.; Weaver, Scott C.

    2014-01-01

    The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus (NEGV) strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analogue. These results strongly suggest that NEGV, and likely other members within the genus, is a non-segmented, single-stranded, positive-sense RNA virus. PMID:24878640

  19. NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

    PubMed Central

    Andersson, M; Gunne, H; Agerberth, B; Boman, A; Bergman, T; Sillard, R; Jörnvall, H; Mutt, V; Olsson, B; Wigzell, H

    1995-01-01

    A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells. Images PMID:7737114

  20. Isolation and characterization of human defensin cDNA clones

    SciTech Connect

    Daher, K.A.; Lehrer, R.I.; Ganz, T.; Kronenberg, M. )

    1988-10-01

    Four clones that encode defensins, a group of microbicidal and cytotoxic peptides made by neutrophils, were isolated from an HL-60 human promyelocytic leukemia cDNA library. Analysis of these clones indicated that the defensins are made as precursor proteins, which must be cleaved to yield the mature peptides. Defensin mRNA was detected in normal bone marrow cells, but not in normal peripheral blood leukocytes. Defensin transcripts were also found in the peripheral leukocytes of some leukemia patients and in some lung and intestine tissues. Defensin mRNA content was augmented by treatment of HL-60 cells with dimethyl sulfoxide. These results define important aspects of the mechanism of synthesis and the tissue-specific expression of a major group of neutrophil granule proteins.

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

    SciTech Connect

    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. Human DNA ligase I cDNA: Cloning and functional expression in Saccharomyces cerevisiae

    SciTech Connect

    Barnes, D.E.; Kodama, Kenichi; Tomkinson, A.E.; Lindahl, T.; Lasko, D.D. ); Johnston, L.H. )

    1990-09-01

    Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomuces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

  3. cDNA cloning and structural characterization of a lectin from the mussel Crenomytilus grayanus with a unique amino acid sequence and antibacterial activity.

    PubMed

    Kovalchuk, Svetlana N; Chikalovets, Irina V; Chernikov, Oleg V; Molchanova, Valentina I; Li, Wei; Rasskazov, Valery A; Lukyanov, Pavel A

    2013-10-01

    An amino acid sequence of GalNAc/Gal-specific lectin from the mussel Crenomytilus grayanus (CGL) was determined by cDNA sequencing. CGL consists of 150 amino acid residues, contains three tandem repeats with high sequence similarities to each other (up to 73%) and does not belong to any known lectins family. According to circular dichroism results CGL is a β/α-protein with the predominance of β-structure. CGL was predicted to adopt a ß-trefoil fold. The lectin exhibits antibacterial activity and might be involved in the recognition and clearance of bacterial pathogens in the shellfish. PMID:23886951

  4. Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

    PubMed

    Jikuya, Hiroyuki; Takano, Jun; Kikuno, Reiko; Hirosawa, Makoto; Nagase, Takahiro; Nomura, Nobuo; Ohara, Osamu

    2003-02-28

    To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility. PMID:12693554

  5. cDNA cloning and chromosomal mapping of a novel human GAP (GAP1M), GTPase-activating protein of Ras

    SciTech Connect

    Li, Shaowei; Nakamura, Shun; Hattori, Seisuke

    1996-08-01

    We have previously isolated a novel Ras GTPase-activating protein (Ras GAP), Gapl{sup m}, from rat brain. Gap1{sup m} is considered to be a negative regulator of the Ras signaling pathways, like other Ras GAPs, neurofibromin, which is a gene product of the neurofibromatosis type I gene, and p120GAP. In this study we have isolated a human cDNA of this Gap and mapped the gene. The gene encodes a protein of 853 amino acids that shows 89% sequence identity to rat Gapl{sup m}. The human gene was mapped to chromosome 3 by PCR analysis on a panel of human-mouse hybrid cells. FISH analysis refined the location of the gene further to 3q22-q23. 11 refs., 2 figs.

  6. Isolation and partial characterization of cDNA clone of human ceruloplasmin receptor.

    PubMed

    Sasina, L K; Tsymbalenko, N V; Platonova, N A; Puchkova, L V; Voronina, O V; Gyulikhandanova, N E; Gaitskhoki, V S

    2000-05-01

    An individual clone, presumably carrying a 3 bp fragment of ceruloplasmin receptor cDNA was isolated from the expression library of human placenta cDNA using polyclonal specific antibodies to ceruloplasmin receptors. EcoR1-hydrolysate of isolated DNA was cloned in a pTZ19 bacterial vector and sequenced in the forward and reverse direction. The comparison of the revealed sequence with known sequences of human genome revealed its high similarity to ceruloplasmin cDNA. PMID:10977961

  7. Method enabling fast partial sequencing of cDNA clones.

    PubMed

    Nordström, T; Gharizadeh, B; Pourmand, N; Nyren, P; Ronaghi, M

    2001-05-15

    Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed. PMID:11355860

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

    PubMed

    Ryabov, Eugene V

    2008-01-01

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

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

    PubMed

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

    2007-06-01

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

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

    SciTech Connect

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M. )

    1989-11-01

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

  11. Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide.

    PubMed Central

    Hayes, J D; Judah, D J; Neal, G E; Nguyen, T

    1992-01-01

    Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to

  12. Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver.

    PubMed Central

    Bladergroen, B A; Houweling, M; Geelen, M J; van Golde, L M

    1999-01-01

    CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development. PMID:10493918

  13. cDNA cloning and promoter analysis of rat caspase-9.

    PubMed Central

    Nishiyama, J; Yi, X; Venkatachalam, M A; Dong, Z

    2001-01-01

    Caspase-9 is the apex caspase of the mitochondrial pathway of apoptosis, which plays a critical role in apoptotic initiation and progression. However, gene regulation of caspase-9 is largely unknown. This is in part due to the lack of information on the gene promoter. Here we have cloned the full-length cDNA of rat caspase-9 and have isolated promoter regions of this gene. The rat caspase-9 cDNA of 2058 bp predicts a protein of 454 amino acids, which contains a caspase-recruitment domain ('CARD') at the N-terminus and enzymic domains at the C-terminus. The enzyme's active site, with a characteristic motif of QACGG, was also identified. Overall, rat and human caspase-9 have 71% identity. With the cDNA sequence, we subsequently isolated the proximal 5'-flanking regions of rat caspase-9 by the procedure of genomic walking. The 2270 bp genomic segment is 'TATA-less', but contains several GC boxes. Elements binding known transcription factors such as Sp-1, Pit-1, CCAAT-enhancer-binding protein (C/EBP), glucocorticoid receptor and hypoxia-inducible factor 1 (HIF-1) were also identified. When cloned into reporter gene vectors, the genomic segment showed significant promoter activity, indicating that the 5'-flanking regions isolated by genomic walking contain the gene promoter of rat caspase-9. Of significance is that the cloned promoter segments were activated by severe hypoxia, conditions inducing caspase-9 transcription. Thus, the genomic sequences reported here contain not only the basal promoter of rat caspase-9 but also regulatory elements responsive to pathophysiological stimuli including hypoxia. PMID:11695991

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

    PubMed

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

    1999-03-01

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

  15. In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

    PubMed

    Wolf, D; Laver-Rudich, Z; Rotter, V

    1985-08-01

    The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons. PMID:3018534

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

    PubMed Central

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

    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 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. PMID:26633465

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

    PubMed

    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. PMID:26633465

  18. Isolation and characterization of cDNA clones for human apolipoprotein A-I.

    PubMed Central

    Breslow, J L; Ross, D; McPherson, J; Williams, H; Kurnit, D; Nussbaum, A L; Karathanasis, S K; Zannis, V I

    1982-01-01

    We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states. PMID:6294659

  19. mu opiate receptor: cDNA cloning and expression.

    PubMed Central

    Wang, J B; Imai, Y; Eppler, C M; Gregor, P; Spivak, C E; Uhl, G R

    1993-01-01

    mu opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands. Expressing cells bind morphine, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin (DAMGO), and [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu opiate receptor that avidly recognizes analgesic and euphoric opiate drugs and opioid peptides. Images Fig. 1 Fig. 3 PMID:8234282

  20. cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3.

    PubMed

    Knight, L P; Primiano, T; Groopman, J D; Kensler, T W; Sutter, T R

    1999-07-01

    The aflatoxin B1 (AFB1) aldehyde metabolite of AFB1 may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction. Aflatoxin B1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reductases of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB1 dihydrodiol by forming AFB1 dialcohol. Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult human liver cDNA library. The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively. Recombinant rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia coli as hexa-histidine tagged fusion proteins. These proteins catalyzed the reduction of several model carbonyl-containing substrates. The NADPH-dependent formation of AFB1 dialcohol by recombinant human AKR7A3 was confirmed by liquid chromatography coupled to electrospray ionization mass spectrometry. Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein were shown to detect nanogram amounts of rat and human AKR7A protein. The amount of AKR7A-related protein in hepatic cytosols of 1, 2-dithiole-3-thione-treated rats was 18-fold greater than in cytosols from untreated animals. These antibodies detected AKR7A-related protein in normal human liver samples ranging from 0.3 to 0.8 microg/mg cytosolic protein. Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver and in several extrahepatic tissues, with relatively high levels in the stomach, pancreas, kidney and liver. Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB1 dihydrodiol at pH 7.4, the catalytic efficiency of this reaction (k2/K, per M/s) equals or exceeds those reported for other enzymes, for example cytochrome P450s and glutathione S-transferases, known to metabolize AFB1 in vivo. These findings indicate that, depending on the extent of AFB1 dihydrodiol formation, AKR

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

    SciTech Connect

    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.

  2. Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

    PubMed Central

    Calhoun, D H; Bishop, D F; Bernstein, H S; Quinn, M; Hantzopoulos, P; Desnick, R J

    1985-01-01

    Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation. Images PMID:2997789

  3. A compilation of partial sequences of randomly selected cDNA clones from the rat incisor.

    PubMed

    Matsuki, Y; Nakashima, M; Amizuka, N; Warshawsky, H; Goltzman, D; Yamada, K M; Yamada, Y

    1995-01-01

    The formation of tooth organs is regulated by a series of developmental programs. We have initiated a genome project with the ultimate goal of identifying novel genes important for tooth development. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3- to 4-week-old rats, sequenced cDNA clones, and classified their sequences by homology search through the GenBank data base and the PIR protein data base. Here, we report partial DNA sequences obtained by automated DNA sequencing on 400 cDNA clones randomly selected from the library. Of the sequences determined, 51% represented sequences of new genes that were not related to any previously reported gene. Twenty-six percent of the clones strongly matched genes and proteins in the data bases, including amelogenin, alpha 1(I) and alpha 2(I) collagen chains, osteonectin, and decorin. Nine percent of clones revealed partial sequence homology to known genes such as transcription factors and cell surface receptors. A significant number of the previously identified genes were expressed redundantly and were found to encode extracellular matrix proteins. Identification and cataloging of cDNA clones in these tissues are the first step toward identification of markers expressed in a tissue- or stage-specific manner, as well as the genetic linkage study of tooth anomalies. Further characterization of the clones described in this paper should lead to the discovery of novel genes important for tooth development. PMID:7876422

  4. Cloning and expression of human neuron-specific enolase cDNA in Escherichia coli.

    PubMed

    Pavlov, K A; Gurina, O I; Antonova, O M; Semenova, A V; Chekhonin, V P

    2011-12-01

    cDNA fragment encoding neuron-specific enolase was amplified from the cDNA library of human brain. Then the fragment was cloned for expression in E. coli using the vector pET28-a. High level of neuron-specific enolase expression was confirmed by SDS-PAAG electrophoresis and immunochemical identity by immunoblot analysis. The constructed producer strain is the cheapest source of neuron-specific enolase suitable for the use in diagnostic applications. PMID:22808461

  5. Expression cloning of a rat brain somatostatin receptor cDNA.

    PubMed Central

    Kluxen, F W; Bruns, C; Lübbert, H

    1992-01-01

    We have used an expression-cloning strategy to isolate a cDNA encoding a somatostatin (somatotropin release-inhibiting factor, SRIF) receptor from rat cortex and hippocampus. A positive clone was identified by autoradiography after binding of radiolabeled SRIF to COS-1 cells previously transfected with pools of cDNA clones. The deduced amino acid sequence of the receptor displays sequence and structural homology to the family of G-protein-coupled receptors. The affinity of various SRIF analogs to the expressed receptor resembles their effects on growth hormone release from pituitary cells. In addition, the distribution of the mRNA in various tissues corresponds to that described for native SRIF receptors. Therefore, we conclude that we have isolated a rat brain SRIF receptor cDNA. Images PMID:1374909

  6. A Novel Alliinase from Onion Roots. Biochemical Characterization and cDNA Cloning1

    PubMed Central

    Lancaster, Jane E.; Shaw, Martin L.; Joyce, Meeghan D. Pither; McCallum, John A.; McManus, Michael T.

    2000-01-01

    We have purified a novel alliinase (EC 4.4.1.4) from roots of onion (Allium cepa L.). Two isoforms with alliinase activity (I and II) were separated by concanavalin A-Sepharose and had molecular masses of 52.7 (I) and 50.5 (II) kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 51 (I) and 57.5 (II) kD by gel filtration fast-protein liquid chromatography. Isoform I had an isoelectric point of 9.3, while isoform II had isoelectric points of 7.6, 7.9, 8.1, and 8.3. The isoforms differed in their glycosylation. Both contained xylose/fucose containing complex-type N-linked glycans, and isoform II also contained terminal mannose structures. Both isoforms had activity with S-alk(en)yl-l-cysteine sulfoxides. Unlike other allium alliinases, A. cepa root isoforms had cystine lyase activity. We cloned a gene from A. cepa root cDNA and show that it codes for A. cepa root alliinase protein. Homology to other reported allium alliinase genes is 50%. The gene coded for a protein of mass 51.2 kD, with two regions of deduced amino acid sequence identical to a 25- and a 40-amino acid region, as determined experimentally. The A. cepa root alliinase cDNA was expressed mainly in A. cepa roots. The structure and function of the alliinase gene family is discussed. PMID:10759524

  7. Cloning and sequence analysis of cDNA for human cathepsin D.

    PubMed Central

    Faust, P L; Kornfeld, S; Chirgwin, J M

    1985-01-01

    An 1110-base-pair cDNA clone for human cathepsin D was obtained by screening a lambda gt10 human hepatoma G2 cDNA library with a human renin exon 3 genomic fragment. Poly(A)+ RNA blot analysis with this cathepsin D clone demonstrated a message length of about 2.2 kilobases. The partial clone was used to screen a size-selected human kidney cDNA library, from which two cathepsin D recombinant plasmids with inserts of about 2200 and 2150 base pairs were obtained. The nucleotide sequences of these clones and of the lambda gt10 clone were determined. The amino acid sequence predicted from the cDNA sequence shows that human cathepsin D consists of 412 amino acids with 20 and 44 amino acids in a pre- and a prosegment, respectively. The mature protein region shows 87% amino acid identity with porcine cathepsin D but differs in having nine additional amino acids. Two of these are at the COOH terminus; the other seven are positioned between the previously determined junction for the light and heavy chains of porcine cathepsin D. A high degree of sequence homology was observed between human cathepsin D and other aspartyl proteases, suggesting a conservation of three-dimensional structure in this family of proteins. Images PMID:3927292

  8. A cDNA clone encoding a peptide highly specific for hepatitis C infection.

    PubMed

    Arima, T; Mori, C; Takamizawa, A; Shimomura, H; Tsuji, T

    1990-04-01

    A random primed lambda gt11-cDNA library was constructed from donors plasma presumably infected by blood-borne non-A, non-B hepatitis (hepatitis C:HC) agent and immunoscreened with serum pooled from patients with acute or chronic HC. Twelve lambda gt11-cDNA clones encoding antigens associated with HC infection in Japan as well as in the USA were isolated. Of these one clone consisting of 114 nucleotides and showing a discrete band on an immunoblot analysis, was extensively studied. The clone is not derived from the host DNA encoding one polypeptide specific and highly sensitive for serum from patients with HC and has no homology to the nucleotide sequences of known human viruses including hepatitis A,B and D viruses, Ebstein-Barr virus, coxsackievirus, immunodeficiency virus type 1 or Japanese encephalitis virus. These results suggest that this clone is derived from the genome of HC agent. PMID:1693349

  9. Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

    PubMed

    Gholizadeh, A; Kohnehrouz, B Baghban; Santha, I M; Lodha, M L; Kapoor, H C

    2005-09-01

    A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714. PMID:16266271

  10. Characterization of cDNA clones selected by the GeneMark analysis from size-fractionated cDNA libraries from human brain.

    PubMed

    Hirosawa, M; Nagase, T; Ishikawa, K; Kikuno, R; Nomura, N; Ohara, O

    1999-10-29

    We have conducted a sequencing project of human cDNAs which encode large proteins in brain. For selection of cDNA clones to be sequenced in this project, cDNA clones have been experimentally examined by in vitro transcription/translation prior to sequencing. In this study, we tested an alternative approach for picking up cDNA clones having a high probability of carrying protein coding region. This approach exploited 5'-end single-pass sequence data and the GeneMark program for assessing protein-coding potential, and allowed us to select 74 clones out of 14,804 redundant cDNA clones. The complete sequence data of these 74 clones revealed that 45% of them encoded proteins consisting of more than 500 amino acid residues while all the clones thus selected carried possible protein coding sequences as expected. The results indicated that the GeneMark analysis of 5'-end sequences of cDNAs offered us a simple and effective means to select cDNA clones with protein-coding potential although the sizes of the encoded proteins could not be predicted. PMID:10574461

  11. Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA.

    PubMed Central

    Paterson, R G; Hiebert, S W; Lamb, R A

    1985-01-01

    cDNAs encoding the mRNAs for the fusion protein (F) and the hemagglutinin/neuraminidase protein (HN) of the paramyxovirus simian virus 5 have been inserted into a eukaryotic expression vector under the control of the simian virus 40 late promoter. The F and HN proteins synthesized in recombinant infected cells are indistinguishable in terms of electrophoretic mobility and glycosylation from the proteins synthesized in simian virus 5-infected cells. In addition, the expressed F and HN proteins have been shown to be anchored in the plasma membrane in a biologically active form by indirect live cell immunofluorescence, the F-mediated formation of syncytia, and the ability of HN to cause the hemadsorption of erythrocytes to the infected cell surface. Images PMID:3865176

  12. Isolation and characterization of two cDNA clones of anaerobically induced lactate dehydrogenase from barley roots

    SciTech Connect

    Hondred, D.; Hanson, A.D. )

    1990-05-01

    In barley roots during hypoxia, five lactate dehydrogenase (LDH) isozymes accumulate with a concomitant increase in enzyme activity ({approximately}20-fold). These isozymes are thought to be tetramers resulting from the random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH have been isolated from a {lambda}gt11 library using antiserum raised against barley LDH purified {approximately}3,000-fold and using nucleic acid probes synthesized by the polymerase chain reaction. Two cDNA clones were obtained (1,305 and 1,166 bp). The deduced amino acid sequences of the two barley LDHs are 96% identical to each other, and 50% and 40% identical to vertebrate and bacterial LDHs, respectively. Northern blots identified a single mRNA band ({approximately}1.5 kb) whose level rose 8-fold during hypoxia.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences

    PubMed Central

    Owens, Robert A.; Cress, Dean E.

    1980-01-01

    Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC)·oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [32P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for Ava I, Hae III, Hpa II, and Sma I. The additional Ava I, Hpa II, and Sma I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by Hae III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells. Images PMID:16592877

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

    PubMed

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

    2011-06-01

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

  16. cDNA cloning and expression of potato polyphenol oxidase.

    PubMed

    Hunt, M D; Eannetta, N T; Yu, H; Newman, S M; Steffens, J C

    1993-01-01

    Polyphenol oxidases (PPOs) of plants are copper metalloproteins which catalyze the oxidation of mono- and o-diphenols to o-diquinones. Although PPOs are believed to be primarily responsible for the deleterious browning of many fruit and vegetable crops and are thought to be involved in plant-pest interactions, direct evidence for these roles is lacking. We report the cloning of two PPO cDNAs from Solanum tuberosum leaves. These cDNAs exhibit 97% and 98% sequence similarity at the DNA and deduced amino acid levels, respectively. Putative copper-binding regions of both cDNAs are very similar to those of mammalian, bacterial and Neurospora tyrosinases. Both leaf PPO cDNAs appear to encode polypeptides which are processed to a mature molecular weight of 57,000. In potato leaves, petioles, roots, and flowers, PPO is encoded by ca. 2 kb transcripts. Leaf PPO mRNA is developmentally regulated and only detectable in young foliage. In contrast, the protein profile of immunologically detectable PPO remains constant from the apical node through the eleventh leaf node. PMID:7678763

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

    PubMed

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

    2014-02-01

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

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

    SciTech Connect

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. )

    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.

  19. Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus.

    PubMed

    Li, Xundong; Zhuo, Kan; Luo, Mei; Sun, Longhua; Liao, Jinling

    2011-06-01

    The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2-70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus. PMID:21371475

  20. Detection of spurious interruptions of protein-coding regions in cloned cDNA sequences by GeneMark analysis.

    PubMed

    Hirosawa, M; Ishikawa, K; Nagase, T; Ohara, O

    2000-09-01

    cDNA is an artificial copy of mRNA and, therefore, no cDNA can be completely free from suspicion of cloning errors. Because overlooking these cloning errors results in serious misinterpretation of cDNA sequences, development of an alerting system targeting spurious sequences in cloned cDNAs is an urgent requirement for massive cDNA sequence analysis. We describe here the application of a modified GeneMark program, originally designed for prokaryotic gene finding, for detection of artifacts in cDNA clones. This program serves to provide a warning when any spurious split of protein-coding regions is detected through statistical analysis of cDNA sequences based on Markov models. In this study, 817 cDNA sequences deposited in public databases by us were subjected to analysis using this alerting system to assess its sensitivity and specificity. The results indicated that any spurious split of protein-coding regions in cloned cDNAs could be sensitively detected and systematically revised by means of this system after the experimental validation of the alerts. Furthermore, this study offered us, for the first time, statistical data regarding the rates and types of errors causing protein-coding splits in cloned cDNAs obtained by conventional cloning methods. PMID:10984451

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

    PubMed

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

    1995-04-01

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

  2. Cloning and expression of Brassica napus beta-carbonic anhydrase cDNA.

    PubMed

    Deng, Qiu-Hong; Li, Mao-Teng; Yu, Long-Jiang

    2009-01-01

    A new full-length beta-carbonic anhydrase cDNA was obtained from Brassica napus by homologous cloning. The cDNA has an open-reading frame of 996 nucleotides, encoding 331 amino acids with a calculated molecular weight of 35,692 Da and an estimated pI value of 5.459. The deduced amino acid sequence of beta-carbonic anhydrase from Brassica napus shared significant identity with beta-carbonic anhydrases from Brassica carinata, Arabidopsis thaliana, and Thlaspi caerulescens (97.9%, 94%, and 93.5% identity, respectively). This cDNA was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-32a(+). The expression band corresponded to the calculated mass plus the N-terminal fusion protein derived from the vector. PMID:20158161

  3. Horse cDNA clones encoding two MHC class I genes

    SciTech Connect

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

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

    PubMed

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

    2012-12-01

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

  5. Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin.

    PubMed

    Masuda, Y; Nirasawa, S; Nakaya, K; Kurihara, Y

    1995-08-19

    A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and was present specifically in the pulp. PMID:7665074

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

    SciTech Connect

    McCue, K.F.; Hanson, A.D. )

    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.

  7. Isolation and analysis of a cDNA clone encoding an S. guttatum alternataive oxidase protein

    SciTech Connect

    Rhoads, D.M.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Antibodies that recognize the 35, 36, and 37 kilodalton (kDa) alternative oxidase proteins were used to isolate a cDNA proteins were used to isolate a cDNA clone of a nuclearly encoded protein of Sauromatum guttatum. The amino acid sequence deduced from clone pAOSG81 revealed a protein with a predicted molecular mass of 44 kDa, while a 42 kDa protein is immunoprecipitated from in vitro translation products made using S. guttatum poly A+ RNA. The protein contains a 60-65 amino acid transit peptide which is predicted to form amphiphilic helices. We have also identified regions of the mature 42 kDa protein which are likely to be membrane associated. Clone pAOSG81 is being used to screen a genomic library. The genomic clone encoding the 42 kDa protein will be used to investigate the salicylic-acid-controlled transcriptional regulation of the S. guttatum alternative oxidase proteins.

  8. Selection and sequence analysis of a cDNA clone encoding a known chorion protein of the A family.

    PubMed Central

    Tsitilou, S G; Regier, J C; Kafatos, F C

    1980-01-01

    Using as criteria the size, abundance and developmental specificity of hybridizing mRNA sequences, we have selected from our chorion cDNA library a clone corresponding to a specific chorion protein, A4--cl. Comparison between the clone sequence and the largely known sequence of A4--cl validates the use of the cDNA library for sequence analysis of the chorion multigene families. The two major chorion protein families, A and B, share certain structural similarities. Images PMID:7433133

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

    PubMed

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

    1988-07-01

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

  10. Cloning of the cDNA and gene for a human D sub 2 dopamine receptor

    SciTech Connect

    Grady, D.K.; Makam, H.; Stofko, R.E.; Bunzow, J.R.; Civelli, O. ); Marchionni, M.A.; Alfano, M.; Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Server, A.C. )

    1989-12-01

    A clone encoding a human D{sub 2} dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D{sub 2} receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed.

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

    SciTech Connect

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-02-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambdagt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16/sup +/ natural killer cells and CD3/sup +/, CD16/sup -/ T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells.

  12. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

    PubMed

    Whelan, S P; Ball, L A; Barr, J N; Wertz, G T

    1995-08-29

    Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions. PMID:7667300

  13. Cloning and characterization of human liver cDNA encoding a protein S precursor

    SciTech Connect

    Hoskins, J.; Norman, D.K.; Beckmann, R.J.; Long, G.L.

    1987-01-01

    Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approx. = 0.01%. Blot hybridization of electrophoretically fractionated poly(A)/sup +/ RNA revealed a major mRNA approx. = 4 kilobases long and two minor forms of approx. = 3.1 and approx. = 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid leader peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal ..gamma..-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approx. = 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function.

  14. cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.

    PubMed

    Wu, Fang; Yan, Ming; Li, Yikun; Chang, Shaojie; Song, Xiaomin; Zhou, Zhaocai; Gong, Weimin

    2003-12-19

    SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16. PMID:14680830

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

    PubMed

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

    2016-06-01

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

  16. Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

    PubMed Central

    Gonda, T J; Gough, N M; Dunn, A R; de Blaquiere, J

    1985-01-01

    We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb. Images Fig. 5. PMID:2998780

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

    PubMed

    Zhao, T S; Xia, Y H

    2016-01-01

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

  18. Sequence and expression of an Eisenia-fetida-derived cDNA clone that encodes the 40-kDa fetidin antibacterial protein.

    PubMed

    Lassegues, M; Milochau, A; Doignon, F; Du Pasquier, L; Valembois, P

    1997-06-15

    Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal antifetidin serum, we have cloned a cDNA that encoded the 40-kDa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates. PMID:9219536

  19. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.

    PubMed

    Yoneyama, T; Brewer, J M; Hatakeyama, K

    1997-04-11

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. PMID:9092499

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

    PubMed

    San, Cha Thye; Shah, Farida Habib

    2005-12-01

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

  1. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  2. Partial sequence analysis of 130 randomly selected maize cDNA clones.

    PubMed Central

    Keith, C S; Hoang, D O; Barrett, B M; Feigelman, B; Nelson, M C; Thai, H; Baysdorfer, C

    1993-01-01

    As part of a project to identify novel maize (Zea mays L. cv B73) genes functionally, we have partially sequenced 130 randomly selected clones from a maize leaf cDNA library. Data base comparisons revealed seven previously sequenced maize cDNAs and 18 cDNAs with sequence similarity to related maize genes or to genes from other organisms. One hundred five cDNAs show little or no similarity to previously sequenced genes. Our results also establish the suitability of this library for large-scale sequencing in terms of its large insert size, proper insert orientation, and low duplication rate. PMID:8278499

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

    SciTech Connect

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III ); Billheimer, J.T. )

    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.

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

    PubMed Central

    Finocchiaro, G; Taroni, F; Rocchi, M; Martin, A L; Colombo, I; Tarelli, G T; DiDonato, S

    1991-01-01

    We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), 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 NH2-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 NH2-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-hamster somatic cell hybrids. Images PMID:1988962

  5. Characterization of cDNA clones for the human c-yes gene.

    PubMed Central

    Sukegawa, J; Semba, K; Yamanashi, Y; Nishizawa, M; Miyajima, N; Yamamoto, T; Toyoshima, K

    1987-01-01

    Three c-yes cDNA clones were obtained from poly(A)+ RNA of human embryo fibroblasts. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-yes mRNA, which could encode a polypeptide of 543 amino acids with a relative molecular weight (Mr) of 60,801. The predicted amino acid sequence of the protein has no apparent membrane-spanning region or suspected ligand binding domain and closely resembles pp60c-src. Comparison of the sequences of c-yes and v-yes revealed that the v-yes gene contains most of the c-yes coding sequence except the region encoding its extreme carboxyl terminus. The region missing from the v-yes protein is the part that is highly conserved in cellular gene products of the protein-tyrosine kinase family. PMID:2436037

  6. Putative diazepam binding inhibitor peptide: cDNA clones from rat.

    PubMed Central

    Mocchetti, I; Einstein, R; Brosius, J

    1986-01-01

    cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes. Images PMID:3463960

  7. Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

    PubMed Central

    Wu, S; Kriz, A L; Widholm, J M

    1994-01-01

    The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2012-06-01

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

  10. Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

    PubMed

    Mattsson, J G; Ljunggren, E L; Bergström, K

    2001-05-01

    The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies. PMID:11393829

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

    NASA Astrophysics Data System (ADS)

    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.

  12. A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases.

    PubMed

    Dominguez-Puigjaner, E; LLop, I; Vendrell, M; Prat, S

    1997-07-01

    A cDNA clone (Ban17), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening. Northern analysis showed that Ban17 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit. Accumulation of the Ban17 transcript can be induced in green banana fruit by exogenous application of ethylene. The demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening. The deduced amino acid sequence derived from the Ban17 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia. Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Ban17 might play a role in the loss of mesocarp firmness during fruit ripening. PMID:9232883

  13. Characterization and cDNA cloning of androgenic gland hormone of the terrestrial isopod Armadillidium vulgare.

    PubMed

    Okuno, A; Hasegawa, Y; Ohira, T; Katakura, Y; Nagasawa, H

    1999-10-22

    The sex differentiation in crustaceans is known to be controlled by a peptide hormone called androgenic gland hormone (AGH). AGH was extracted and purified from the androgenic glands (AGs) of the male isopod Armadillidium vulgare by high-performance liquid chromatography. AGH consisted of two peptide chains and their N-terminal amino acid sequences were determined. A cDNA encoding AGH was cloned by PCR and sequenced. The cDNA had an open reading frame of 432 bp, which encoded a preproAGH consisting of a signal peptide (21 residues), B chain (44 residues), C peptide (46 residues), and A chain (29 residues). Through processing, the A and B chains might form a heterodimer interlinked by disulfide bonds. The A chain possessed a putative N-linked glycosylation site. A Northern blot analysis using the cDNA as a probe detected a hybridization signal with 0.8 kb in the RNA preparation only from the AGs. PMID:10529379

  14. Isolation and characterization of cDNA clones for human erythrocyte. beta. -spectrin

    SciTech Connect

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-11-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical ..cap alpha.. (M/sub r/ 240,000) and ..beta.. (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific ..beta..-spectrin cDNA clone that encodes parts of the ..beta..-9 through ..beta..-12 repeat segments. This cDNA was used as a hybridization probe to assign the ..beta..-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte ..beta..-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the ..beta..-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

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

    PubMed

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

    1993-12-01

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

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

    SciTech Connect

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. )

    1988-07-01

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

  17. Human type VII collagen: cDNA cloning and chromosomal mapping of the gene

    SciTech Connect

    Parente, M.G.; Chung, L.C.; Ryynaenen, J.; Monli Chu; Uitto, J. ); Woodley, D.T.; Wynn, K.C.; Bauer, E.A. ); Mattei, M.G. )

    1991-08-15

    A human keratinocyte cDNA expression library in bacteriophage {lambda}gt11 was screened with the purified IgG fraction of serum from a patient with epidermolysis bullosa acquisita, which had a high titer of anti-type VII collagen antibodies. Screening of {approx}3 {times} 10{sup 5} plaques identified 8 positive clones, the largest one (K-131) being {approx}1.9 kilobases in size. Dideoxynucleotide sequencing of K-131 indicated that it consisted of 1875 base pairs and contained an open reading frame coding for a putative N-terminal noncollagenous domain of 439 amino acids and a collagenous domain was characterized by repeating Gly-Xaa-Yaa sequences that were interrupted in several positions by insertions or deletions of 1-3 amino acids. The deduced amino acid sequence also revealed a peptide segment that had a high degree of identity with a published type VII collagen protein sequence. The results mapped the COL7A1 to the locus 3p21. The cDNA clones characterized in this study will be valuable for understanding the protein structure and gene expression of type VII collagen present in anchoring fibrils and its aberrations in the dystrophic forms of heritable epidermolysis bullosa.

  18. Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.

    PubMed Central

    Long, E O; Wake, C T; Strubin, M; Gross, N; Accolla, R S; Carrel, S; Mach, B

    1982-01-01

    cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines. Images PMID:6818545

  19. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence

    SciTech Connect

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. ); Chapline, C.; Crabb, J.W. )

    1991-10-22

    Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

  20. Cloning and characterization of a cDNA encoding hexokinase from tomato.

    PubMed

    Menu, T; Rothan, C; Dai, N; Petreikov, M; Etienne, C; Destrac-Irvine, A; Schaffer, A; Granot, D; Ricard, B

    2001-01-01

    Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant yeast (Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers. PMID:11164592

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-06-01

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

  3. Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15.

    PubMed Central

    Phaneuf, D; Labelle, Y; Bérubé, D; Arden, K; Cavenee, W; Gagné, R; Tanguay, R M

    1991-01-01

    Type 1 hereditary tyrosinemia (HT) is an autosomal recessive disease characterized by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH; E.C.3.7.1.2). We have isolated human FAH cDNA clones by screening a liver cDNA expression library using specific antibodies and plaque hybridization with a rat FAH cDNA probe. A 1,477-bp cDNA was sequenced and shown to code for FAH by an in vitro transcription-translation assay and sequence homology with tryptic fragments of purified FAH. Transient expression of this FAH cDNA in transfected CV-1 mammalian cells resulted in the synthesis of an immunoreactive protein comigrating with purified human liver FAH on SDS-PAGE and having enzymatic activity as shown by the hydrolysis of the natural substrate fumarylacetoacetate. This indicates that the single polypeptide chain encoded by the FAH gene contains all the genetic information required for functional activity, suggesting that the dimer found in vivo is a homodimer. The human FAH cDNA was used as a probe to determine the gene's chromosomal localization using somatic cell hybrids and in situ hybridization. The human FAH gene maps to the long arm of chromosome 15 in the region q23-q25. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 8 PMID:1998338

  4. cDNA cloning and mRNA expression of canine pancreatic and duodenum homeobox 1 (Pdx-1).

    PubMed

    Takemitsu, Hiroshi; Yamamoto, Ichiro; Lee, Peter; Ohta, Taizo; Mori, Nobuko; Arai, Toshiro

    2012-10-01

    Pancreatic and duodenal homeobox 1 (Pdx-1) is a critical insulin transcription factor expressed by pancreatic β-cells, and is crucial in the early stage of pancreas development. Unfortunately, nothing concerning Pdx-1 in canine has been elucidated yet. In this study, full length canine Pdx-1 cDNA was cloned and it was 1498 bp in length, consisting of a 99 bp 5'-untranslated region (UTR), a 849 bp coding region, and a 550 bp 3'-UTR region. A deduced 282 amino acid sequence of canine PDX-1 displayed high overall sequence identity with human, bovine, and mouse PDX-1. qRT-PCR analysis revealed that a high level of Pdx1 mRNA expression is exists in the duodenum and pancreas of canines. In addition, functional canine insulin promoter-luciferase reporter constructs with various canine insulin promoter region fragments revealed that our Pdx-1 isolated cDNA sequence encodes for a functionally active PDX-1 protein. Significant promoter activity was observed within the -583 bp 5'-upstream region of canine insulin gene with Chinese hamster ovary cells. In addition, Pdx-1 appears to have a synergistic effect with beta cell transactivator 2 (BETA2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), which also have important roles in the activation of the insulin gene promoter. Our results confirm that similar to humans, Pdx1 plays an important role in expression of insulin gene in canines. PMID:22172402

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

    PubMed

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2004-02-27

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

  6. Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

    PubMed

    Gholizadeh, Ashraf; Santha, Ittiaparambu Mana; Kohnehrouz, Bahram Baghban; Lodha, Madan Lal; Kapoor, Harish Chander

    2005-12-01

    Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems. PMID:15842197

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

    PubMed

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

    1998-07-01

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

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

    PubMed Central

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

    1999-01-01

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

  9. Cloning and sequencing of a cDNA for Akazara scallop troponin T.

    PubMed

    Inoue, A; Ojima, T; Nishita, K

    1996-10-01

    A cDNA clone encoding troponin T of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle has been isolated and sequenced. The complete sequence deduced consists of 314 amino acid residues with a molecular weight of 37,206. Akazara scallop troponin T contains 55 amino acid residues more and 82 residues fewer than rabbit skeletal muscle troponin T and Drosophila melanogaster troponin T, respectively, showing almost the lowest sequence homology with rabbit troponin T (26%) but the highest homology with Drosophila troponin T (33%). Further, high sequence homology was seen in the functional regions: residues 33-120 and 174-227, corresponding respectively to residues 71-158 and 197-250 of rabbit troponin T (tropomyosin-binding regions); and residues 200-204, corresponding to 223 227 of rabbit troponin T (troponin I-binding region). In residues 1-70 (tropomyosin-binding region), however, only six residues are identical with rabbit troponin T. PMID:8947849

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

    PubMed

    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. PMID:25818933

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

    PubMed

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

    2014-01-01

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

  12. Cloning of a Locusta cDNA encoding a precursor peptide for two structurally related proteinase inhibitors.

    PubMed

    Kromer, E; Nakakura, N; Lagueux, M

    1994-03-01

    Two peptides of respectively 35 and 36 residues were recently isolated from Locusta migratoria and their full structural characteristics were established by Edman degradation and mass spectrometry. These peptides were subsequently shown to have a proteinase inhibiting activity. We report here the cloning and characterization of a cDNA encoding a 92-residue precursor with three distinct domains: (I) a typical signal peptide of 19 residues; (II) the peptide sequence of the 35-residue inhibitor separated by a Lys-Arg dipeptide cleavage site from (III) the peptide sequence of the 36-residue inhibitor. We show by Northern blot analysis that the gene encoding this precursor is mainly transcribed in the cells of the fat body. PMID:8019577

  13. cDNA Cloning and Molecular Modeling of Procerain B, a Novel Cysteine Endopeptidase Isolated from Calotropis procera

    PubMed Central

    Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

    2013-01-01

    Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed. PMID:23527269

  14. Cloning a cDNA encoding an alternatively spliced protein of BRCA2-associated factor 35.

    PubMed

    Wang, Chiang; McCarty, Ida M; Balazs, Louisa; Li, Yi; Steiner, Mitchell S

    2002-07-01

    Inheritance of mutations in the breast cancer susceptibility gene, BRCA2, predisposes humans to breast and ovarian cancers. Inherited mutations in the BRCA2 gene are also known to cause susceptibility to prostate cancer. BRCA2 protein exists in a large multi-protein complex from which a novel structural DNA binding protein BRCA2-associated factor 35 (BRAF35) has been isolated. We have cloned a novel cDNA encoding an alternatively spliced protein of BRAF35, designated as BRAF25. BRAF25 transcript is present in various human cells. We have precisely mapped the BRAF25 cDNA sequence to the genomic chromosome 19 sequence. Analysis of the predicted sequence of BRAF25 identified a protein of 215 amino acids. BRAF25 contains a truncated high mobility group domain, a kinesin-like coiled-coil domain and multiple Src homology 2 (SH2) motifs. Western blot analysis using antibodies specific for BRAF25 revealed the presence of BRAF25 in human prostate cancer cells. PMID:12083779

  15. cDNA cloning and sequencing of rat alpha sub 1 -macroglobulin

    SciTech Connect

    Waermegaard, B.; Martin, N.; Johansson, S. )

    1992-03-03

    cDNA clones coding for the plasma protease inhibitor {alpha}{sub 1}-macroglobulin were isolated from a rat liver library. The obtained cDNA sequence contained 4701 nucleotides and had an open reading frame coding for a 1500 amino acid long protein, including a 24 amino acid signal peptide. The identity of the deduced protein sequence as {alpha}{sub 1}-macroglobulin was established by comparison with published peptide sequences of the protein. The mature protein shares 53% and 57% overall amino acid identity with the two other identified members of the rat {alpha}-macroglobulin family, {alpha}{sub 1}-inhibitor 3 and {alpha}{sub 2}-macroglobulin. A sequence typical for an internal thiol ester was identified. Of the 24 cysteines, 23 are conserved with {alpha}{sub 2}-macroglobulin. However, instead of the two most C-terminal cysteines in {alpha}{sub 2}-macroglobulin, which forms a disulfide bridge in the receptor binding domain, {alpha}{sub 1}-macroglobulin contains phenylalanine. One MRNA species hybridizing with the {alpha}{sub 1}-macroglobulin probe was observed in rat and mouse liver RNA ({approximately} 6.2 kb), whereas no corresponding transcript was detected in RNA from human liver.

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

    PubMed

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

    2014-02-01

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

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

    PubMed

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

    2015-04-15

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

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

    PubMed

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

    2016-06-01

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

  19. Construction and characterization of an infectious cDNA clone of Echovirus 25.

    PubMed

    Hou, Wangheng; Yang, Lisheng; Li, Shuxuan; Yu, Hai; Xu, Longfa; He, Delei; Chen, Mengyuan; He, Shuizhen; Ye, Xiangzhong; Que, Yuqiong; Shih, James Wai Kuo; Cheng, Tong; Xia, Ningshao

    2015-07-01

    Echovirus 25 (E-25) is a member of the enterovirus family and a common pathogen that induces hand, foot, and mouth disease (HFMD), meningitis, skin rash, and respiratory illnesses. In this study, we constructed and characterized an infectious full-length E-25 cDNA clone derived from the XM0297 strain, which was the first subgenotype D6 strain isolated in Xiamen, China. The 5'-Untranslated Regions (5'-UTR), P3 (3A-3B, 3D) and P3 (3C) regions of this E-25 (XM0297) strain were highly similar to EV-B77, E-16 and E-13, respectively. Our data demonstrate that the rescued E-25 viruses exhibited similar growth kinetics to the prototype virus strain XM0297. We observed the rescued viral particles using transmission electron microscope (TEM) and found them to possess an icosahedral structure, with a diameter of approximately 30 nm. The cross neutralization test demonstrated that the E-25 (XM0297) strain immune serum could not neutralize EV-A71, CV-A16 or CV-B3; likewise, the EV-A71 and CV-A16 immune serum could not neutralize E-25 (XM0297). The availability of this infectious clone will greatly enhance future virological investigations and possible vaccine development against E-25. PMID:26004198

  20. Cloning yeast actin cDNA leads to an investigative approach for the molecular biology laboratory.

    PubMed

    Black, Michael W; Tuan, Alice; Jonasson, Erin

    2008-05-01

    The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the classroom because of the preparation, expense, and logistics involved in independent student projects. The authors have designed a 10-week lab series that mimics the research environment by tying separate fundamental lab techniques to a common goal: to build a plasmid with yeast actin cDNA cloned in a particular orientation. In the process of completing this goal, a problem arises in that students are unable to obtain the target plasmid and instead only recover the gene cloned in the opposite orientation. To address this problem, students identify four plausible hypotheses and work in teams to address them by designing and executing experiments. This project reinforces the utility and flexibility of techniques covered earlier in the class and serves to develop their skills in experimental design and analysis. As the project is focused on one problem, the diversity of experimental approaches is limited and may be prepared in advance with little additional expense in reagents or technical support. PMID:21591194

  1. COMPLETE CDNA CLONING AND POLYMORPHISMS AT PORCINE BPI: ASSOCIATIONS WITH BACTERIAL LOAD AND IMMUNE RESPONSE TRAITS IN PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The bactericidal permeability-increasing (BPI) gene encodes a neutrophil protein with inhibitory/killing functions against gram-negative bacteria. We investigated BPI as a candidate gene for resistance to Salmonella cholerasuis (SC) in pigs. We cloned and sequenced a full-length BPI cDNA and iden...

  2. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  3. Screening of a peanut (Arachis hypogaea L.) cDNA library to isolate a Bowman-Birk trypsin inhibitor clone.

    PubMed

    Boateng, Judith A; Viquez, Olga M; Konan, Koffi N; Dodo, Hortense W

    2005-03-23

    Peanut crop losses due to insect and pest infestation cost peanut farmers nearly 20% of their annual yields. The conventional use of chemicals to combat this problem is costly and toxic to humans and livestock and leads to the development of resistance by target insects. Transgenic plants expressing a trypsin inhibitor gene in tobacco and cowpea have proven to be efficient for resistance against insects. Therefore, a transgenic peanut overexpressing a trypsin inhibitor gene could be an alternative solution to the use of toxic chemicals. Five Bowman-Birk trypsin inhibitor (BBTI) proteins were previously isolated from peanut. However, to date, neither cDNA nor genomic DNA sequences are available. The objective of this research was to screen a peanut cDNA library to isolate and sequence at least one full-length peanut BBTI cDNA clone. Two heterologous oligonucleotides were constructed on the basis of a garden pea (Pisum sativa) trypsin inhibitor nucleotide sequence and used as probes to screen a peanut lambda gt-11 cDNA library. Two positive and identical cDNA clones were isolated, subcloned into a pBluescript vector, and sequenced. Sequence analysis revealed a full-length BBTI cDNA of about 243 bp, with a start codon ATG at position +1 and a stop codon TGA at position +243. In the 3' end, two poly adenylation signals (AATAAA) were identified at positions +261 and +269. The isolated cDNA clone encodes a protein of 80 amino acid residues including a leader sequence of 11 amino acids. The deduced amino acid sequence is 100% identical to published sequences of peanut BBTI AI, AII, BI, and BIII and 81% identical to BII. PMID:15769131

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

    SciTech Connect

    Kelley, M.R. ); Venugopal, S.; Harless, J.; Deutsch, W.A. . Dept. of Biochemistry)

    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 apurinicapyrimidine (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-polyacrymalide gel electrophoresis of Drosophilia 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-klb mRNA also hybridized to the AP3 cDNA, but species was restricted to the early stages of development.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

    Shah; Cha

    2000-05-29

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

  7. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    SciTech Connect

    Hirono, A.; Beutler, E. )

    1988-06-01

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3{prime} end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C{sup 33} {yields} G, G{sup 202} {yields} A, and A{sup 376} {yields} G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

  8. cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera.

    PubMed

    Titarenko, E; Chrispeels, M J

    2000-10-01

    We report the characterization and cDNA cloning of two alpha-amylase isozymes from larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeConte). Larvae raised on artificial media have very low levels of amylase activity, and much higher levels are found in larvae raised on maize seedlings. At pH 5.7, the optimum pH for enzyme activity, the alpha-amylases are substantially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the reverse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode alpha-amylase-like polypeptides. Expression of one of the two cDNAs in insect cells with a baculovirus vector shows that this cDNA encodes an active amylase with a mobility that corresponds to that of one of the two isozymes present in larval extracts. The expressed enzyme is substantially inhibited by the same two inhibitors. We also show that expression in Arabidopsis of the cDNA that encodes the amylase inhibitor AI-1 of the common bean results in the accumulation of active inhibitor in the roots, and the results are discussed with reference to the possibility of using amylase inhibitors as a strategy to genetically engineer maize plants that are resistant to Western corn rootworm larvae. PMID:10899464

  9. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

    PubMed Central

    Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

    1995-01-01

    Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

  10. Construction of a normalized directionally cloned cDNA library from adult heart and analysis of 3040 clones by partial sequencing.

    PubMed

    Tanaka, T; Ogiwara, A; Uchiyama, I; Takagi, T; Yazaki, Y; Nakamura, Y

    1996-07-01

    Large-scale sequencing of clones from cDNA libraries derived from specific tissues is a rapid and efficient way of discovering novel genes expressed in those tissues. However, because the heart is continually contracting and relaxing, it strongly expresses muscle-contractile genes and/or mitochondrial genes, a bias that reduces the efficiency of this method. To improve the efficiency of identifying novel genes expressed in the heart, we constructed a normalized directionally cloned cDNA library from adult heart and partially sequenced 3040 clones. Comparisons of these sequence data with known DNA sequences in the database revealed that 57.1% of the clones matched human genes already known, 23.4% were identical or almost identical to human expressed sequence tags (ESTs), 14.2% bore no significant homology to any sequences in the database, and 1.2% represented repetitive sequences. The remaining 4.1% showed some homology with known genes, and Northern blot analysis of several clones in this category revealed that most of them were expressed mainly in the heart and skeletal muscle. After redundancy was excluded, the 3040 clones accounted for 1395 distinctive ESTs, 446 of which exhibited no match to any known sequence. Our results suggest that our normalized library is less redundant than standard libraries and is a useful resource for cataloging genes expressed in the heart. PMID:8661126

  11. Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland.

    PubMed

    Roberto, Patrícia G; Kashima, Simone; Soares, Andreimar M; Chioato, Lucimara; Faça, Victor M; Fuly, André L; Astolfi-Filho, Spartaco; Pereira, José O; França, Suzelei C

    2004-09-01

    The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities. PMID:15294287

  12. Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

    PubMed Central

    Johnson, Karyn N.; Ball, L. Andrew

    2001-01-01

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

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

    PubMed

    Johnson, K N; Ball, L A

    2001-12-01

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

  14. Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases.

    PubMed Central

    Arimitsu, E; Aoki, S; Ishikura, S; Nakanishi, K; Matsuura, K; Hara, A

    1999-01-01

    Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins. PMID:10477285

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

    PubMed

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

    2014-03-01

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

  16. Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs

    PubMed Central

    Córdoba, Laura; Feagins, Alicia R.; Opriessnig, Tanja; Cossaboom, Caitlin M.; Dryman, Barbara A.; Huang, Yao-Wei

    2012-01-01

    Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future. PMID:22837416

  17. cDNA cloning of the developmentally regulated lamin LIII of Xenopus laevis.

    PubMed Central

    Stick, R

    1988-01-01

    Lamins are nucleoskeletal proteins which form intermediate type filaments in close association with the inner nuclear envelope membrane. Based on molecular and biochemical properties the lamins were grouped as type-A and type-B lamins, respectively. I have cloned the cDNA encoding lamin LIII of Xenopus which is the lamin protein present in oocyte nuclei and in cleavage nuclei. The data presented here indicate that a pool of maternal lamin LIII RNA is synthesized very early in oogenesis and that it continues to be present until gastrulation when the vast majority of the LIII RNA is degraded. Despite the similarities shared by all lamin proteins, the lamin LIII sequence neither possesses the features diagnostic for either type-A or type-B lamins nor does it show greater sequence similarity to one of the lamin types than to the other and thus it may represent a third type of lamin protein which may reflect its special function in oogenesis and early development. Images PMID:3181134

  18. Cloning and characterization of p8 homolog cDNA in the Atlantic halibut (Hippoglossus hippoglossus).

    PubMed

    Wang, Jia-Qing; Li, Lin-Chun; Lin, Sheng-Guo; Wang, Zhi-Ping

    2010-06-01

    The p8 gene encodes a transcription factor known to modulate cell growth, division, and apoptosis and influences gene expression. In this study, an Atlantic halibut (Hippoglossus hippoglossus) homolog of the p8 gene was cloned, sequenced, and characterized. The full-length p8 cDNA consists of 601 bp and encodes 76 amino acids with a molecular mass of 9 kD. The bHLH region is well conserved between Atlantic halibut and other animals. Analysis by RT-PCR showed that the p8 transcript is constitutively expressed in 9 of the 12 tissues tested: pancreas, intestine, stomach, gill, head kidney, heart, liver, ovary, and spleen. A predicted microRNA target site was found in the 3'UTR of Atlantic halibut p8 mRNA. We speculate that the target site may pair to microRNA molecules because the target site resides in a big loop, a space large enough for the binding of microRNA molecules. PMID:20454949

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

    PubMed

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

    2010-01-01

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

  20. Isolation of cDNA clones from within the spinal muscular atrophy (SMA) disease gene region

    SciTech Connect

    McLean, M.; Roy, N.; Tamai, K.

    1994-09-01

    Spinal muscular atrophy (SMA) is a recessive neuromuscular disease characterized by death of spinal cord {alpha} motor neurons, resulting in skeletal muscle atrophy. The critical SMA disease gene region on 5q13.1 contains families of microsatellite repeat sequences which exist at multiple subloci that are dispersed over a 100 to 200 kbp region. We have detected significant linkage disequilibrium between SMA type 1, the most severe form of the disorder, and two subloci of one such microsatellite, the CATT-1 family of microsatellites. Furthermore, a recombination event in a chromosome of an individual with SMA type 1 mapping between the members of two other extended microsatellite families, including CMS-1, has been observed. Combining this with previously reported recombinants refines the critical SMA region to approximately 300 kbp. P1 artificial chromosome (PAC), YAC and cosmid clones which possess both CMS-1 alleles which bracket this recombination event, as well as CATT-1 alleles showing linkage disequilibrium with SMA, have been used to probe cDNA libraries from human and other mammalian sources in search of genes within this interval; three of these cDNAs are currently being tested as candidates for the SMA gene.

  1. Synaptophysin: molecular organization and mRNA expression as determined from cloned cDNA.

    PubMed Central

    Leube, R E; Kaiser, P; Seiter, A; Zimbelmann, R; Franke, W W; Rehm, H; Knaus, P; Prior, P; Betz, H; Reinke, H

    1987-01-01

    Synaptophysin is a major glycoprotein of Mr approximately 38,000 (in deglycosylated form: Mr approximately 34,000) characteristic of a certain class of small (30-80 nm diameter) neurosecretory vesicles, including presynaptic vesicles, but also vesicles of various neuroendocrine cells of both neuronal and epithelial phenotype. Using synaptophysin-specific antibodies we have isolated cDNA clones from rat nervous tissue libraries, which identify an approximately 2.5-kb mRNA in rat and human cells, including neuroendocrine tumours, that contains a reading frame for a polypeptide of 307 amino acids with a total mol. wt of 33 312. The deduced amino acid sequence, which was partly confirmed by comparison with sequences of two tryptic peptides obtained from purified synaptophysin, revealed four hydrophobic regions of 24 amino acids each, which are characterized, according to conformation prediction analyses, by marked alpha-helicity. The sequence shows a single potential N-glycosylation site, which is assigned to the vesicle interior, and a carboxy-terminal tail of 89 amino acids which contains glycine-rich tetrapeptide repeats, the epitope of monoclonal antibody SY38, and a number of collagenase-sensitive sites accessible on the surface of the intact vesicles. These features suggest that the polypeptide spans the vesicle membrane four times, with both N and C termini located on the outer, i.e. cytoplasmic, surface of the vesicles. Images Fig. 2. Fig. 6. PMID:3123215

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

    PubMed

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

    2010-09-01

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

  3. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family

    SciTech Connect

    Hanks, S.K.

    1987-01-01

    Mixed /sup 32/P-labeled oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identify with rabbit skeletal muscle phosphorylase kinase ..gamma..-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

  4. Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase.

    PubMed

    Chen, D; Zangl, A L; Zhao, Q; Markley, J L; Zheng, J; Bird, I M; Magness, R R

    2001-04-25

    Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy. To test this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells. Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced. Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb. Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 3'-untranslated region. Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved. We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a [His]6-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1. Incubation of exogenous [His]6-tagged oCav-1 with resting UAEC extracts led to the formation of a [His]6-tagged oCav-1-eNOS complex. In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, [His]6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited. eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co

  5. Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase.

    PubMed

    Abedinia, M; Layfield, R; Jones, S M; Nixon, P F; Mattick, J S

    1992-03-31

    Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wernicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species. PMID:1567394

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

    PubMed

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

    2016-01-01

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

  7. Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

    PubMed Central

    Zhang, W; Wagner, B J; Ehrenman, K; Schaefer, A W; DeMaria, C T; Crater, D; DeHaven, K; Long, L; Brewer, G

    1993-01-01

    The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466, 1991) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUF1. Using antisera specific for these polypeptides, we demonstrate that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation. Images PMID:8246982

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

    PubMed Central

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

    2016-01-01

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

  9. Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds.

    PubMed

    Games, Patrícia D; Dos Santos, Izabela S; Mello, Erica O; Diz, Mariângela S S; Carvalho, André O; de Souza-Filho, Gonçalo A; Da Cunha, Maura; Vasconcelos, Ilka M; Ferreira, Beatriz Dos S; Gomes, Valdirene M

    2008-12-01

    The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani. PMID:18786582

  10. Purification, characterization and cDNA cloning of soluble carbonic anhydrase from Chlorella sorokiniana grown under ordinary air.

    PubMed

    Satoh, A; Iwasaki, T; Odani, S; Shiraiwa, Y

    1998-11-01

    Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO2 was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)-1. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A. These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About 60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized. The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH2-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an alpha-type CA with salt-sensitive, periplasm-locating and acidic properties and very different from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively. PMID:9821693

  11. Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic peptide domains.

    PubMed

    Lei, Weiwei; Zhang, Yong; Yu, Guoyu; Jiang, Ping; He, Yingying; Lee, Wenhui; Zhang, Yun

    2011-04-01

    The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs. PMID:21334357

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

    SciTech Connect

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro Univ. of Tokyo )

    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.

  13. Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula1

    PubMed Central

    Xie, De-Yu; Jackson, Lisa A.; Cooper, John D.; Ferreira, Daneel; Paiva, Nancy L.

    2004-01-01

    Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species. PMID:14976232

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

    SciTech Connect

    Chen, J.; Varner, J.E.

    1985-07-01

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

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

    PubMed

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

    2004-02-01

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

  16. Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae).

    PubMed

    Kwon, Hyojung; Bang, Kyeongrin; Lee, Minsup; Cho, Saeyoull

    2014-09-01

    A full-length lysozyme cDNA from Gryllotalpa orientalis was cloned and sequenced. The deduced amino acid sequence of the lysozyme protein was 143 amino acids in length, with a calculated molecular mass of 15.84 kDa and an isoelectric point of 4.74. Sequence motifs, together with alignment and phylogenetic results, confirmed that G. orientalis lysozyme belongs to the C (chicken)-type lysozyme family of proteins. The protein sequence of lysozyme from G. orientalis showed high identity to that of Drosophila melanogaster (51.7 %); however, in contrast to D. melanogaster lysozyme, G. orientalis lysozyme was immune inducible and expressed in a wide range of tissues. Expression of G. orientalis lysozyme mRNA was highest at 8 h post-infection and subsequently decreased with time after bacterial infection. We also expressed G. orientalis lysozyme protein in vitro using the pET expression system. Compared with the negative control, over-expressed G. orientalis lysozyme showed antimicrobial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis by radial diffusion assay, with minimal inhibitory concentration values of 30.3 and 7.55 µM, respectively. These results indicate that G. orientalis lysozyme may have stronger antimicrobial activity than other lysozymes against a broad range of microorganisms. PMID:24929538

  17. Ovine aquaporin-2: cDNA cloning, ontogeny and control of renal gene expression.

    PubMed

    Butkus, A; Earnest, L; Jeyaseelan, K; Moritz, K; Johnston, H; Tenis, N; Wintour, E M

    1999-06-01

    The aim of this study was to test the hypothesis that the relative insensitivity of the ovine fetal kidney to arginine vasopressin (AVP) is due to low levels of expression of the gene for aquaporin-2 (AQP2) which encodes the AVP-regulated water channel. We report the cloning of the cDNA for the ovine AQP2 which has a major transcript at 4.2 kilobases (kb) and a minor transcript at 1.5 kb, resembling the human gene transcripts. At 40-60 days' (term = 145-150 days'), mRNA levels are very low, detectable only by reverse transcription-polymerase chain reaction (RT-PCR). By Northern blot analysis AQP2 mRNA is detectable at 75 days'. The ratio of AQP2/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA increases approximately 2.4-fold between 100 and 140 days' when it is about 41% of adult values. Both glucocorticoids and the renin-angiotensin system are involved in maturation of renal function. When fetuses at 75 or 85 days of gestation were exposed to high levels of dexamethasone for 2-3 days, mRNAs for both GAPDH and AQP2 doubled, but the ratio was unchanged. Angiotensin I, infused for 3 days at 115-120 days' gestation, increased the AQP2/GAPDH mRNA ratios by twofold (major transcript) and sixfold (minor transcript), which were highly significant (P<0.001). The increasing sensitivity of the ovine fetal kidney to AVP, from 100-140 days of gestation, is largely due to increasing AQP2 gene expression over this period. PMID:10412857

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

    PubMed

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

    2006-08-01

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

  19. Isolation of a rat liver Golgi mannosidase II clone by mixed oligonucleotide-primed amplification of cDNA.

    PubMed Central

    Moremen, K W

    1989-01-01

    A clone encoding Golgi mannosidase II (MII; GlcNAc-transferase I-dependent alpha 1,3(alpha 1,6) mannosidase), an enzyme involved in asparagine-linked oligosaccharide processing, was isolated from a rat liver lambda gt11 cDNA library by a method that employs a modification of the polymerase chain reaction. Specific oligonucleotide primers were designed from two regions of protein sequence and were combined in an amplification reaction with a single-stranded cDNA preparation derived from rat liver poly(A)+ RNA. Based upon mapping of the protein sequences 42 kDa apart on the MII polypeptide, the procedure was expected to generate an approximately 1150-base-pair amplification product representing a segment of the MII gene between the two primer regions. The size of the amplified product (1170 base pairs) was in close agreement with this predicted fragment size. The authenticity of the amplified fragment was confirmed by the agreement of the DNA sequence with additional protein sequence data. A 1474-base-pair clone was isolated from a cDNA library by plaque hybridization using the amplification fragment as a radiolabeled probe. The nucleotide sequence of this clone predicts a single continuous open reading frame and, based upon a polypeptide molecular mass of 117 kDa for the enzyme subunit, is consistent with the clone representing approximately 50% of the coding sequence of MII. Both the clone and the amplification product hybridized to a rat liver mRNA of approximately 8 kilobases, a message size approximately 4.7 kilobases larger than the size of the predicted open reading frame. This extensive non-coding information on the MII message is a feature common to two other Golgi processing enzymes, both of which contain most of the non-coding information on the 3' end of their messages. The function of these disproportionately large untranslated regions is not clear. Images PMID:2748583

  20. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-).

    PubMed Central

    Hirono, A; Beutler, E

    1988-01-01

    Glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) A(-) is a common variant in Blacks that causes sensitivity to drug-and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3' end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C33----G, G202----A, and A376----G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The base substitution at position 376, identical to the substitution that has been reported in G6PD A(+), was present in all G6PD A(-) samples and none of the control G6PD B(+) samples examined. The substitution at position 202 was found in four of the five G6PD A(-) samples and no normal control sample. At position 33 guanine was found in all G6PD A(-) samples and seven G6PD B(+) control samples and is, presumably, the usual nucleotide found at this position. The finding of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein. Images PMID:2836867

  1. Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple mRNA species.

    PubMed Central

    Johnson, K R; Nauseef, W M; Care, A; Wheelock, M J; Shane, S; Hudson, S; Koeffler, H P; Selsted, M; Miller, C; Rovera, G

    1987-01-01

    Myeloperoxidase is a component of the microbicidal network of polymorphonuclear leukocytes. The enzyme is a tetramer consisting of two heavy and two light subunits. A large proportion of humans demonstrate genetic deficiencies in the production of myeloperoxidase. As a first step in analyzing these deficiencies in more detail, we have isolated cDNA clones for myeloperoxidase from an expression library of the HL-60 human promyelocytic leukemia cell line. Two overlapping plasmids (pMP02 and pMP062) were identified as myeloperoxidase cDNA clones based on the detection with myeloperoxidase antiserum of 70 kDa protein expressed in pMP02-containing bacteria and a 75 kDa polypeptide produced by hybridization selection and translation using pMP062 and HL-60 RNA. Formal identification of the clones was made by matching the predicted amino acid sequences with the amino terminal sequences of the heavy and light subunits. Both subunits are encoded by one mRNA in the following order: pre-pro-sequences--light subunit--heavy subunit. The molecular weight of the predicted primary translation product is 83.7 kDa. Northern blots reveal two size classes of hybridizing RNAs (approximately 3.0-3.3 and 3.5-4.0 kilobases) whose expression is restricted to cells of the granulocytic lineage and parallels the changes in enzymatic activity observed during differentiation. Images PMID:3031585

  2. Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase.

    PubMed Central

    Sakumi, K; Shiraishi, A; Hayakawa, H; Sekiguchi, M

    1991-01-01

    cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase. The rat cDNA encodes a protein with 209 amino acid residues. The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites. When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced. The rat DNA methyltransferase thus expressed could complement the biological defects of the E. coli cell caused by lack of its own DNA methyltransferases; e.g. increased sensitivity to alkylating agents in terms of both cell death and mutation induction. Images PMID:1945835

  3. Cloning and expression of a human kidney cDNA for an /alpha//sub 2/-adrenergic receptor subtype

    SciTech Connect

    Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1988-09-01

    An /alpha//sub 2/-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet /alpha//sub 2/-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet /alpha//sub 2/-adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the /alpha//sub 2/-adrenergic ligand (/sup 3/H)rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the /alpha//sub 2/B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet /alpha//sub 2/-adrenergic receptor (/alpha//sub 2/A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective /alpha/-adrenergic ligands.

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

    SciTech Connect

    McAllister, G.; Amara, S.G.; Lerner, M.R. )

    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.

  5. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

    PubMed Central

    Zibert, A; Maass, G; Strebel, K; Falk, M M; Beck, E

    1990-01-01

    A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed. Images PMID:2159523

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

    PubMed

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

    1988-09-01

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

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

    PubMed

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

    2015-12-15

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

  8. Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

    PubMed Central

    Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

    2010-01-01

    Background 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. Methodology 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 ∼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. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

  9. Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

    PubMed Central

    Schiebel, W; Pélissier, T; Riedel, L; Thalmeir, S; Schiebel, R; Kempe, D; Lottspeich, F; Sänger, H L; Wassenegger, M

    1998-01-01

    A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. PMID:9836747

  10. cDNA cloning and cadmium-induced expression of metallothionein mRNA in the zebra mussel Dreissena polymorpha.

    PubMed

    Engelken, J; Hildebrandt, A

    1999-01-01

    Using pooled degenerate oligonucleotides inferred from the N-terminal amino acid sequence of Dreissena polymorpha metallothionein and a Cys-X-Cys motif characteristic for known metallothioneins, a 150-bp metallothionein-specific reverse transcription PCR product was generated. The PCR product was used to screen a Dreissena polymorpha cDNA library, and a complete metallothionein cDNA sequence from Dreissena was identified. Four clones with the identical sequence were detected, supporting the idea of a single metallothionein gene in Dreissena. The sequence contains a 141-bp 5' untranslated region and a 572-bp 3' untranslated region with two polyadenylation signals. The coding region spans 219 bp. The deduced amino acid sequence shows 21 cysteine residues present in the metallothionein-typical motifs. Induction studies were performed with 50 microg Cd2+/L for up to 16 days. The exposed mussels show a sevenfold higher metallothionein mRNA level compared with uninduced control mussels. PMID:10505795

  11. Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primary structure.

    PubMed

    Födinger, M; Ortner, S; Plaimauer, B; Wiedermann, G; Scheiner, O; Duchêne, M

    1993-06-01

    Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica. PMID:8341328

  12. Molecular cloning of cDNA for BRab from the brain of Bombyx mori and biochemical properties of BRab expressed in Escherichia coli.

    PubMed

    Uno, T; Ueno, M; Nakajima, A; Shirai, Y; Aizono, Y

    1998-10-01

    From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTP gamma S and [3H]-GDP with association constants of 1.5 x 10(6) M-1 and 0.58 x 10(6) M-1, respectively. The binding of [35S]-GTP gamma S was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTP gamma S and [3H]-GDP were exchanged with GTP gamma S most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP. PMID:9836423

  13. Nucleolin from Xenopus laevis: cDNA cloning and expression during development.

    PubMed

    Caizergues-Ferrer, M; Mariottini, P; Curie, C; Lapeyre, B; Gas, N; Amalric, F; Amaldi, F

    1989-03-01

    Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis. PMID:2656405

  14. Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification.

    PubMed

    Uno, T; Nakasuji, A; Hara, W; Aizono, Y

    2000-04-01

    A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch. PMID:10737920

  15. Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C.

    PubMed Central

    Dewaste, V; Pouillon, V; Moreau, C; Shears, S; Takazawa, K; Erneux, C

    2000-01-01

    Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin. PMID:11085927

  16. cDNA cloning, identification, tissue localisation, and transcription profile of a transglutaminase from white shrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus.

    PubMed

    Yeh, Maw-Sheng; Liu, Chun-Hung; Hung, Chia-Wen; Cheng, Winton

    2009-12-01

    Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134); tiger shrimp, Penaeus monodon (AAL78166); and Pacifastacus leniusculus (AF336805). The 2638-bp cDNA contained an open reading frame (ORF) of 2172 bp, a 55-bp 5'-untranslated region (UTR), and a 411-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (757 aa) was 84.9 kDa with an estimated pI of 5.2. The L. vannamei TG (abbreviated LvTG) contains a typical transglutaminase-like homologue, a putative integrin-binding motif (RGD), and four calcium-binding sites; a catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two subgroups, STGS1 and STGS2, and LvTG is more closely related to STGS1 than to STGS2. LvTG mRNA and TG activities were detected in all tested tissues of L. vannamei, with LvTG mainly being synthesised in haemocytes. However, the pattern of LvTG mRNA expression was not directly correlated with TG activity. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant decrease of TG activity at 3 h and a significant increase of LvTG mRNA expression at 6 h followed by a notable decrease from 12 to 24 h, which indicated that cloned LvTG was involved in the immune response of shrimp. The results also imply that more than one type of TG may be involved in the defense response in L. vannamei. PMID:19782141

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

    PubMed

    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. PMID:26323263

  18. cDNA cloning and disruption of the major vault protein alpha gene (mvpA) in Dictyostelium discoideum.

    PubMed

    Vasu, S K; Kedersha, N L; Rome, L H

    1993-07-25

    Vaults are large cytoplasmic ribonucleoprotein particles found in nearly all eukaryotic cells. Dictyostelium vaults contain two major proteins, MVP alpha (94.2 kDa) and MVP beta (approximately 92 kDa). Using an anti-rat vault antibody, we screened a Dictyostelium cDNA expression library and isolated a 2.8-kilobase pair clone that contained a single full-length reading frame. The identity of the clone was established by the presence of a predicted 20-amino acid sequence identical to that found in a peptide sequenced from purified MVP alpha. We have disrupted the single copy gene using homologous recombination and have demonstrated a loss of MVP alpha. Although the cells still produce MVP beta, they do not contain characteristic vault particles, suggesting that MVP alpha is required for normal vault structure. These cells should be a valuable tool for elucidating the function of vaults. PMID:8340365

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

    PubMed

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

    2001-05-01

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

  20. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O sup 6 -alkylguanine

    SciTech Connect

    Tano, K.; Shiota, S.; Collier, J.; Foote, R.S.; Mitra, S. )

    1990-01-01

    O{sup 6}-Methylguanine-DNA methyltransferase a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O{sup 6}-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-like cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada{sup {minus}}) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGNT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A){sup +} RNA transcript.

  1. Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA.

    PubMed Central

    Chebath, J; Merlin, G; Metz, R; Benech, P; Revel, M

    1983-01-01

    Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs. Images PMID:6186990

  2. Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning.

    PubMed

    Ariizumi, K; Shen, G L; Shikano, S; Xu, S; Ritter, R; Kumamoto, T; Edelbaum, D; Morita, A; Bergstresser, P R; Takashima, A

    2000-06-30

    Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1. PMID:10779524

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

    SciTech Connect

    Travis, G.H.; Sutcliffe, J.G.

    1988-03-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors 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 cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

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

    NASA Technical Reports Server (NTRS)

    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.

  5. cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis.

    PubMed

    Zhang, Qingli; Li, Fuhua; Zhang, Xiaojun; Dong, Bo; Zhang, Jiquan; Xie, Yusu; Xiang, Jianhai

    2008-05-01

    Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pI of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). PMID:18353680

  6. Construction of an infectious cDNA clone of Enterovirus 71: insights into the factors ensuring experimental success.

    PubMed

    Lazouskaya, Natallia V; Palombo, Enzo A; Poh, Chit-Laa; Barton, Peter A

    2014-03-01

    Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus. PMID:24361875

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. cDNA, genomic sequence cloning and overexpression of giant panda (Ailuropoda melanoleuca) mitochondrial ATP synthase ATP5G1.

    PubMed

    Hou, W-R; Hou, Y-L; Ding, X; Wang, T

    2012-01-01

    The ATP5G1 gene is one of the three genes that encode mitochondrial ATP synthase subunit c of the proton channel. We cloned the cDNA and determined the genomic sequence of the ATP5G1 gene from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively. The cloned cDNA fragment contains an open reading frame of 411 bp encoding 136 amino acids; the length of the genomic sequence is of 1838 bp, containing three exons and two introns. Alignment analysis revealed that the nucleotide sequence and the deduced protein sequence are highly conserved compared to Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, and Sus scrofa. The homologies for nucleotide sequences of the giant panda ATP5G1 to those of these species are 93.92, 92.21, 92.46, 93.67, and 92.46%, respectively, and the homologies for amino acid sequences are 90.44, 95.59, 93.38, 94.12, and 91.91%, respectively. Topology prediction showed that there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, five N-myristoylation sites, and one ATP synthase c subunit signature in the ATP5G1 protein of the giant panda. The cDNA of ATP5G1 was transfected into Escherichia coli, and the ATP5G1 fused with the N-terminally GST-tagged protein gave rise to accumulation of an expected 40-kDa polypeptide, which had the characteristics of the predicted protein. PMID:23007995

  9. A new potassium channel toxin from the sea anemone Heteractis magnifica: isolation, cDNA cloning, and functional expression.

    PubMed

    Gendeh, G S; Young, L C; de Medeiros, C L; Jeyaseelan, K; Harvey, A L; Chung, M C

    1997-09-23

    A new potassium channel toxin, HmK, has been isolated from the sea anemone Heteractis magnifica. It inhibits the binding of [125I]-alpha-dendrotoxin (a ligand for voltage-gated K channels) to rat brain synaptosomal membranes with a Ki of about 1 nM, blocks K+ currents through Kv 1.2 channels expressed in a mammalian cell line, and facilitates acetylcholine release at the avian neuromuscular junction. HmK comprises of 35 amino acids (Mr 4055) with the sequence R1TCKDLIPVS10ECTDIRCRTS20MKYRLNLCRK30TCGSC35. A full assignment of the disulfide linkages was made by using partial reduction with tri(2-carboxyethyl)phosphine (TCEP) at acid pH and rapid alkylation with iodoacetamide. The disulfide bridges were identified as Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. A cDNA clone encoding HmK was isolated using RT-PCR from the total RNA obtained from sea anemone tentacles, while the 5'- and 3'-flanking regions of the cDNA were amplified by RACE. The full-length cDNA was 563 bp long and contained a sequence encoding a signal peptide of 39 amino acids. The coding region for matured HmK toxin was cloned and expressed as a glutathione S-transferase (GST) fusion product in the cytoplasm of Escherichia coli. After affinity purification and cleavage, the recombinant toxin was shown to be identical to native HmK in its N-terminal sequence, chromatographic behavior, and binding to dendrotoxin binding sites on rat brain membranes. PMID:9298966

  10. Cloning and analysis of human gastric mucin cDNA reveals two types of conserved cysteine-rich domains.

    PubMed Central

    Klomp, L W; Van Rens, L; Strous, G J

    1995-01-01

    Human gastric mucin was isolated by successive CsCl-gradient ultracentrifugation in the presence of guanidinium hydrochloride to prevent degradation of the polypeptide moieties of the molecules. The amino acid sequence of a tryptic fragment of this molecule was identical to that of a tryptic fragment of tracheobronchial mucin. An oligonucleotide based on this sequence hybridized specifically to human stomach mRNA and was subsequently used to screen a human stomach lambda ZAPII cDNA library. The largest of 10 positive clones encoded 850 amino acid residues, including the tryptic fragment, with high amounts of threonine, serine and proline residues. Interestingly, cysteine accounted for almost 8% of the amino acid residues. The 3' part of the sequence was very similar but not identical to the 3' region of human tracheobronchial cDNA. No tandem repeated sequences were present and the deduced polypeptide sequence contained two potential N-linked glycosylation sites. Four cysteine-rich clusters were detected, one of which was apparently homologous to the D-domains present in other mucins and in von Willebrand factor. The arrangement of the cysteines in three other cysteine-rich clusters was conserved in the human gastric mucin cDNA in a similar fashion as in two domains in the MUC2 gene product. The cysteine-rich domains were separated by short stretches of non-repetitive amino acid residues with a very high content of threonine and serine residues. These data suggest that the encoded polypeptide of this clone may be involved in disulphide-bond-mediated oligomerization of the mucin, and provide new insights into the molecular organization of mammalian apomucins. Images Figure 1 PMID:8948439

  11. Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus (CLRDV) results in systemic infection in cotton and the model plant Nicotiana benthamiana.

    PubMed

    Delfosse, Verónica C; Casse, María F; Agrofoglio, Yamila C; Kresic, Iván Bonacic; Hopp, Horacio E; Ziegler-Graff, Véronique; Distéfano, Ana J

    2013-07-01

    Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse

  12. Isolation and characterization of human cDNA clones encoding the. alpha. and the. alpha. prime subunits of casein kinase II

    SciTech Connect

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs E.G. )

    1990-09-11

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two {alpha} or {alpha}{prime} subunits (or one of each) and two {beta} subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell {lambda}gt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5{prime} untranslated region) and followed by 871 bp (3{prime} untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5{prime} untranslated region) and followed by 550 bp (3{prime} untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of {alpha} and {alpha}{prime} subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the {alpha} and {alpha}{prime} subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II ({alpha} and {alpha}{prime}) and that the sequence of these subunits is largely conserved between the bovine and the human.

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

    PubMed

    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

  14. Induction of human beta-interferon synthesis with poly(rI . rC) in mouse cells transfected with cloned cDNA plasmids.

    PubMed Central

    Pitha, P M; Ciufo, D M; Kellum, M; Raj, N B; Reyes, G R; Hayward, G S

    1982-01-01

    Human genomic DNA and plasmids carrying portions of the cDNA gene for human beta-interferon have been introduced into mouse Ltk- cells by cotransfection with a herpes simplex virus thymidine kinase (TK) gene. One plasmid contains 840 base pairs of human DNA complementary to pre-beta-interferon mRNA inserted into pBR322, whereas the other plasmids have hybrid genes containing only the 560-base pair coding region inserted under the transcriptional control of the TK promoter. Constitutive interferon production could not be detected in any of the mouse TK+ cell lines tested. Nevertheless, synthesis of interferon could be induced by poly(rI . rC) treatment in at least 16 of these cell lines, including clones transfected with genomic DNA, the beta-interferon cDNA, and the TK-beta-interferon cDNA hybrid gene. The interferon produced was specific for human cells and could be neutralized by antiserum against human beta-interferon. In contrast to human fibroblast cells, in which the synthesis of induced beta-interferon is transient, the poly(rI . rC)-induced TK+ lines continued to produce beta-interferon for prolonged periods of time and did not respond to superinduction conditions. Therefore, in transfected mouse cells, the coding DNA sequence from the human beta-interferon gene, without any of the adjacent 3' or 5' flanking human DNA sequences, was sufficient both to direct synthesis of biologically active product and to respond to the specific induction system that operates in human cells. However, the mechanism that switches off the synthesis of induced interferon in human cells appears not to operate in mouse cells transfected with beta-interferon cDNA. PMID:6956863

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

    PubMed

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

    1990-08-01

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

  16. Generation of expressed sequence tags of random root cDNA clones of Brassica napus by single-run partial sequencing.

    PubMed Central

    Park, Y S; Kwak, J M; Kwon, O Y; Kim, Y S; Lee, D S; Cho, M J; Lee, H H; Nam, H G

    1993-01-01

    Two hundred thirty-seven expressed sequence tags (ESTs) of Brassica napus were generated by single-run partial sequencing of 197 random root cDNA clones. A computer search of these root ESTs revealed that 21 ESTs show significant similarity to the protein-coding sequences in the existing data bases, including five stress- or defense-related genes and four clones related to the genes from other kingdoms. Northern blot analysis of the 10 data base-matched cDNA clones revealed that many of the clones are expressed most abundantly in root but less abundantly in other organs. However, two clones were highly root specific. The results show that generation of the root ESTs by partial sequencing of random cDNA clones along with the expression analysis is an efficient approach to isolate genes that are functional in plant root in a large scale. We also discuss the results of the examination of cDNA libraries and sequencing methods suitable for this approach. PMID:8029332

  17. cDNA cloning and expression of Blo t 11, the Blomia tropicalis allergen homologous to paramyosin.

    PubMed

    Ramos, J D; Cheong, N; Lee, B W; Chua, K Y; Nge, C; Wah, L B; Yan, C K

    2001-12-01

    Blomia tropicalis is an important mite species in many parts of the world and the most predominant mite species in tropical countries. The prevalence of sensitization to this species has probably been underestimated because commercial extracts are largely unavailable. Identification and characterization of B. tropicalis allergens is an important step toward understanding the role of this species in allergic sensitization and could provide appropriate reagents for diagnostic and therapeutic procedures. This paper describes the isolation, sequence analysis, expression and allergenicity of a cDNA gene coding for a B. tropicalis allergen with homology to paramyosin, a high-molecular-weight allergen previously identified in Dermatophagoides farinae. The full-length Blo t 11 cDNA gene was isolated by cDNA library screening, 5'-rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of CLUSTAL W, CGC and BLAST program packages. The cDNA gene was expressed as a GST fusion protein in Escherichia coli and purified by affinity chromatography using the glutathione Sepharose column. Allergenicity of the rBlo t 11 was tested by human IgE dot blot immunoassay. Blo t 11 is a 3,111-bp cDNA gene with a 2,625-bp open reading frame coding for an 875-amino acid protein, exhibiting significant homology with different invertebrate paramyosins. The human IgE dot blot immunoassay showed that the rBlo t 11 reacted positively to 52% (33/63) of sera from asthmatic patients. Blo t 11 is the homolog of Der f 11 exhibiting potentially important allergenic activity. PMID:11815735

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

    SciTech Connect

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

    1987-06-01

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

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

    SciTech Connect

    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.

  20. Cloning, characterization, and expression analysis of LGP2 cDNA from goose, Anser cygnoides.

    PubMed

    Wei, L; Song, Y; Cui, J; Qu, N; Wang, N; Ouyang, G; Liao, M; Jiao, P

    2016-10-01

    Laboratory of genetics and physiology 2 ( LGP2: ) is a homologue of the retinoic acid inducible gene-I and melanoma differentiation associated gene 5 that lacks the caspase activation and recruitment domain required for signaling. It plays a pivotal role in host immune response. In this study, we cloned and characterized the full-length open reading frame ( ORF: ) sequence of LGP2 in the Qingyuan goose (Anser cygnoides) and evaluated the mRNA expression of this gene post infection with an H5N1 highly pathogenic avian influenza virus ( HPAIV: ). The full-length goose LGP2 ORF (2,028 bp) encoded a polypeptide of 675 amino acids. The deduced amino acid sequence contained 5 main overlapping structural domains-2 DEAD/DEAH box helicase domains, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily C-terminal domain and one C-terminal regulatory domain. Quantitative real-time PCR analysis indicated that goose LGP2 was constitutively expressed in all 19 investigated tissues, but the expression level was different among them. It was high expressed in the trachea, jejunum, bursa, kidney and heart, but low in the glandular stomach, lung, liver, spleen, crop and muscular stomach. A significant increase in the transcription of LGP2 was detected in the brain, spleen and lungs of geese post infection with H5N1 HPAIV versus uninfected tissues. These findings indicated that goose LGP2 was an important receptor that is involved in the host antiviral innate immune defense to H5N1 HPAIV in geese. PMID:27143779

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

    PubMed Central

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

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache’s reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level. PMID:27046474

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

    PubMed

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

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level. PMID:27046474

  3. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.

    PubMed Central

    Uéda, K; Fukushima, H; Masliah, E; Xia, Y; Iwai, A; Yoshimoto, M; Otero, D A; Kondo, J; Ihara, Y; Saitoh, T

    1993-01-01

    A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD. Images Fig. 1 Fig. 3 Fig. 5 PMID:8248242

  4. cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli.

    PubMed

    Araya-Garay, José Miguel; Feijoo-Siota, Lucía; Veiga-Crespo, Patricia; Villa, Tomás González

    2011-11-01

    Lycopene beta-cyclase (β-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic β-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, β-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-β from Ficus carica (Lyc-β Fc), which codes for the enzyme lycopene β-cyclase (β-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. β-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to β-carotene as a result of the enzyme's action. The β-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the β-carotene. The lycopene to β-carotene conversion rate was 90%. The experiments carried out in this work showed that β-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to β-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing β-LCY in E. coli, we have obtained a new gene for β-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica. PMID:21792589

  5. [Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

    PubMed

    Yang, Wen-ting; Liu, Xiu-ming; Wan, Qiu; Yao, Na; Wang, Nan; Zhang, Xue-meng; Jiao, Zhong-da; Li, Hai-yan; Li, Xiao-kun

    2015-02-01

    Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids. PMID:26137682

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

    PubMed

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

    1989-01-01

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

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

    PubMed

    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. PMID:19019007

  8. Cloning of the canine GALC cDNA and identification of the mutation causing globoid cell leukodystrophy in West Highland White and Cairn terriers

    SciTech Connect

    Victoria, T.; Rafi, M.A.; Wenger, D.A.

    1996-05-01

    Globoid cell leukodystrophy, or Krabbe disease, is a severe, autosomal recessive disorder resulting from a deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. In addition to the human patients, there are several naturally occurring animal models for this disease, including the twitcher mouse, West Highland White terriers (WHWT), and Cairn terriers. All species have deficient GALC activity and have the characteristic pathological findings in the nervous system. We now describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in both terrier breeds. The 2007-bp open reading frame is 88% identical to that in human, and the deduced amino acid sequence is about 90% identical. However, the 3{prime}-untranslated region is about 1 kb shorter than that in the human. Two nucleotide changes were found in affected dogs, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells demonstrated that the A rapid test for the identification of the genotype at that position has been developed, and over 100 WHWT and Cairn terriers have been screened. This will allow breeders to mate their dogs selectivity and will permit the establishment of a colony of dogs for use in therapy trials. 30 refs., 4 figs., 2 tabs.

  9. Identification and cDNA cloning of a protein abundantly expressed during apple fruit development.

    PubMed

    Yamada, K; Mori, H; Yamaki, S

    1999-02-01

    A 60 kDa protein (MF-60) abundantly appearing in matured apple fruit was detected by SDS-PAGE of the soluble protein. It was partially purified through Butyl-Toyopearl and DEAE-cellulose. Its partial amino acid sequences were determined to isolate a full-length cDNA. MF-60 cDNA (mf-60) consisting of 1,825 bp containing an open reading frame of 1,524 bp and encoding a 54.2 kDa polypeptide. The deduced polypeptide of mf-60 has 81.1% identity to turgor-responsive protein 26 g from wilted garden pea shoot. Northern blot and Western blot analyses showed that the levels of the protein and the transcript of MF-60 changed in parallel through the developmental season; they were very low in young fruit at 36 DAF and 60 DAF, started to increase at 85 DAF, and then remained at a higher level from 114 DAF to 176 DAF. These results suggested that MF-60 functions are connected with fruit development but not with the fruit ripening induced by ethylene. PMID:10202815

  10. Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation.

    PubMed Central

    White, P C; New, M I; Dupont, B

    1984-01-01

    We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Images PMID:6609358

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

    PubMed

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

    1995-01-01

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

  12. Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts

    SciTech Connect

    Lin, C.S.; Leavitt, J.

    1988-01-01

    The authors isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an ..cap alpha../sub fast/-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle ..cap alpha..-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle ..cap alpha..-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle ..cap alpha..-tropomyosin and the smooth muscle ..cap alpha..-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.

  13. Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones.

    PubMed Central

    Lee, R C; Burton, R A; Hrmova, M; Fincher, G B

    2001-01-01

    A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH(4))(2)SO(4) and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65 kDa and releases L-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (k(cat)) of 6.9 s(-1) and a catalytic efficiency factor (k(cat)/K(m)) of 0.76 (ml x s(-1) x mg(-1)). Whereas the hydrolysis of alpha-L-arabinofuranosyl residues linked to C(O)3 of backbone (1-->4)-beta-xylosyl residues proceeds at the fastest rate, alpha-L-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain. PMID:11336650

  14. Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

    PubMed Central

    LaPolla, R J; Mayne, K M; Davidson, N

    1984-01-01

    A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences. Images PMID:6096870

  15. Partial Purification and Characterization of Bacillus thuringiensis Cry1A Toxin Receptor A from Heliothis virescens and Cloning of the Corresponding cDNA

    PubMed Central

    Oltean, Daniela I.; Pullikuth, Ashok K.; Lee, Hyun-Ku; Gill, Sarjeet S.

    1999-01-01

    Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins. PMID:10543783

  16. Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2.

    PubMed Central

    Paris, J; Le Guellec, R; Couturier, A; Le Guellec, K; Omilli, F; Camonis, J; MacNeill, S; Philippe, M

    1991-01-01

    Fertilization of Xenopus laevis eggs triggers a period of rapid cell division comprising 12 nearly synchronous mitoses. Protein synthesis is required for these divisions, and new proteins appear after fertilization. Others proteins however, which are synthesized in the unfertilized egg, are no longer made in the early embryo. To identify such proteins, a differential screen of an egg cDNA library gave nine clones corresponding to mRNAs that are deadenylylated soon after fertilization. The sequence of one of these clones (Eg1) revealed a high homology to p34cdc2, the kinase subunit of maturation-promoting factor. Only 12 amino acids in the deduced amino acid sequence were unique to Eg1 when its sequence was compared to all other known examples of cdc2. Despite this strong similarity, however, Eg1 was unable to complement a yeast cdc2- mutant in Schizosaccharomyces pombe or a cdc28 mutant of Saccharomyces cerevisiae. Four Eg1 transcripts, two major and two minor, were found in Xenopus oocytes and early embryos. These RNAs appeared very early (stage I) in oogenesis and their level remained constant until the midblastula transition, at which time they declined. Eg1 RNA is found in the poly(A)+ fraction of oocytes only between the time of meiotic maturation and fertilization--that is to say, in the unfertilized egg. At fertilization the RNA loses its poly(A) tail and at the same time leaves the polyribosomes. Images PMID:1704128

  17. Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin.

    PubMed Central

    Kincaid, R L; Nightingale, M S; Martin, B M

    1988-01-01

    A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A beta-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain approximately 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain. Images PMID:2848250

  18. Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3.

    PubMed

    Fitter, S; Tetaz, T J; Berndt, M C; Ashman, L K

    1995-08-15

    Platelet-endothelial cell tetra-span antigen (PETA-3) was originally identified as a novel human platelet surface glycoprotein, gp27, which was detected by a monoclonal antibody (MoAb), 14A2.H1. Although this glycoprotein is present in low abundance on the platelet surface, MoAb 14A2.H1 stimulates platelet aggregation and mediator release. We now report isolation of a cDNA clone encoding PETA-3 from a library derived from the megakaryoblastic leukemia cell line MO7e. The clone encodes an open reading frame of 253 amino acids that displays 25% to 30% amino acid sequence identity with several members of the newly defined Tetraspan, or Transmembrane 4 superfamily. These proteins consist of four conserved putative transmembrane domains with a large divergent extracellular loop between the third and fourth membrane-spanning regions. PETA-3 has a single consensus sequence for N-linked glycosylation located in this extracellular loop. A single PETA-3 RNA transcript (1.6 kb) was detected in RNA isolated from MO7e cells, bone marrow stromal cells, the C11 endothelial cell line, and several myeloid leukemia cell lines. No transcript was detected in the lymphoblastoid cell lines MOLT-4 and BALM-1. This pattern correlates well with previous protein expression data. Northern blot analysis of RNA from a range of human tissues indicated that the transcript was present in most tissues, the notable exception being brain. PMID:7632941

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

    PubMed

    Udo, Hiroshi

    2015-01-01

    One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells. PMID:26422141

  20. Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli.

    PubMed

    Rathinasabapathi, Bala; Wu, Shan; Sundaram, Sabarinath; Rivoal, Jean; Srivastava, Mrittunjai; Ma, Lena Q

    2006-12-01

    Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI's role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells

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

    EPA Science Inventory

    cDNA Cloning of Fathead minnow (Pimephales promelas) Estrogen and Androgen Receptors for Use in Steroid Receptor Extrapolation Studies for Endocrine Disrupting Chemicals.

    Wilson, V.S.1,, Korte, J.2, Hartig P. 1, Ankley, G.T.2, Gray, L.E., Jr 1, , and Welch, J.E.1. 1U.S...

  2. Cloning and mapping of a novel human cDNA homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene

    SciTech Connect

    Isomura, Minoru; Okui, Keiko; Nakamura, Yusuke

    1996-02-15

    This article reports on the isolation and localization to human chromosome 7q34 of a human cDNA clone that encodes a protein which is homologous to DROER, the enhancer of the Drosophila melanogaster rudimentary gene. The structure and expression of this gene is also discussed. 12 refs., 3 figs.

  3. Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes.

    PubMed Central

    Long, E O; Wake, C T; Gorski, J; Mach, B

    1983-01-01

    At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence. PMID:11894954

  4. Molecular cloning, sequence, and expression of a human GDP-L-fucose:. beta. -D-galactoside 2-. alpha. -L-fucosyltransferase cDNA that can form the H blood group antigen

    SciTech Connect

    Larsen, R.D.; Ernst, L.K.; Nair, R.P.; Lowe, J.B. )

    1990-09-01

    The authors have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:{beta}D-galactoside 2-{alpha}-L-fucosyltransferase. Although this fragment determined expression of an {alpha}(1,2)FT whose kinetic properties mirror those of the human H blood group {alpha}(1,2)FT, their precise nature remained undefined. They describe here the molecular cloning, sequence, and expression of a human of cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, the cDNA directs expression of cell surface H structures and a cognate {alpha}(1,2)FT activity with properties analogous to the human H blood group {alpha}(1,2)FT. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an {alpha}(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identified DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned {alpha}(1,2)FT cDNA represents the product of the human H blood group locus.

  5. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction.

    PubMed

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin; Orton, Richard J; Blome, Sandra; Höper, Dirk; Beer, Martin; Rasmussen, Thomas Bruun

    2015-01-01

    RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants. PMID

  6. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

    PubMed Central

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin; Orton, Richard J; Blome, Sandra; Höper, Dirk; Beer, Martin; Rasmussen, Thomas Bruun

    2015-01-01

    RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants. PMID

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

    PubMed

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

    1991-02-01

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

  8. Molecular cloning of a cDNA for human {triangle}{sup 1}-pyrroline-5-carboxylate (P5C) dehydrogenase, the gene defective in type 2 hyperprolinemia

    SciTech Connect

    Hu, C.A.; Lin, W.; Valle, D.

    1994-09-01

    P5C dehydrogenase (EC 1.5.1.12) is a mitochondrial matrix NAD(P) dependent enzyme catalyzing the conversion of P5C, derived from either proline or ornithine, to glutamate. This reaction is an important component in the pathway interconnecting the urea cycle with the tricarboxylic acid cycle. Deficiency of P5C dehydrogenase causes type 2 hyperprolinemia (HPII), an autosomal recessive disorder characterized by seizures, hyperprolinemia and accumulation of P5C. To investigate the molecular basis of HPII and the pathophysiology of gyrate atrophy, a disorder of ornithine metabolism, we have cloned a cDNA for P5C dehydrogenase. Utilizing published sequences of peptides from purified human P5C dehydrogenase and the nucleotide sequence of yeast P5C dehydrogenase, we designed degenerate PCR primers to amplify cDNAs from a HepG2 cDNA library. We identified an amplified fragment of the correct size that encoded one of the many peptides and used it to clone near full length clones of the corresponding cDNA. The longest is 1.8 kb with a 1,485 bp ORF encoding a protein corresponding to the C terminal 495 residues of yeast P5C dehydrogenase. The predicted amino acid sequence of this clone has 100% identity to published sequence of human P5C dehydrogenase peptides and 42% identity with the corresponding sequence of the yeast enzyme. This cDNA detects a 2.3 kb transcript in Northern blots of fibroblast RNA. We conclude we have cloned a near full length cDNA for human P5C dehydrogenase. Studies investigating the molecular basis of HPII are in progress.

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

    PubMed

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

    2015-02-01

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

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

    SciTech Connect

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

    PubMed

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

    2009-07-01

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

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

    NASA Astrophysics Data System (ADS)

    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).

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

    PubMed

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    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 His(69), Asp(117), and Ser(216). 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. PMID:27399771

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

    PubMed

    Funk, C D; Funk, L B; Kennedy, M E; Pong, A S; Fitzgerald, G A

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A2, 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 its gene regulation. PMID:1907252

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

    SciTech Connect

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. )

    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.

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

    PubMed Central

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    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. PMID:27399771

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

    PubMed

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

    2012-12-01

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

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

    PubMed

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

    1995-03-01

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

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

    SciTech Connect

    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. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.

    PubMed

    Bokar, J A; Shambaugh, M E; Polayes, D; Matera, A G; Rottman, F M

    1997-11-01

    The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to

  1. Screening and cDNA Cloning of Kv1 Potassium Channel Toxins in Sea Anemones

    PubMed Central

    Yamaguchi, Yoshikazu; Hasegawa, Yuichi; Honma, Tomohiro; Nagashima, Yuji; Shiomi, Kazuo

    2010-01-01

    When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of 125I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3′RACE and 5′RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ1.3-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34–63% identities with the other type 1 potassium channel toxins. PMID:21339955

  2. Cloning of cDNA encoding human rapsyn and mapping of the RAPSN gene locus to chromosome 11p11.2-p11.1

    SciTech Connect

    Buckel, A.; Beeson, D.; Vincent, A.

    1996-08-01

    We have isolated and sequenced cDNA clones for the human 43-kDa acetylcholine receptor-associated protein rapsyn. The cDNA encodes a 412-amino-acid protein that has a predicted molecular mass of 46,330 Da and shows 96% sequence identity with mouse rapsyn. Analysis of PCR amplifications, first from somatic cell hybrids and subsequently from radiation hybrids, localizes the human RAPSN gene locus to chromosome 11p11.2-p11.1 in close proximity to ACP2. 12 refs., 2 figs.

  3. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

    SciTech Connect

    Aris, J.P.; Blobel, G. )

    1991-02-01

    The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is {approximately}1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain {approximately}75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

  4. Vitamin D receptor alleles: Cloning and characterization of the VDR gene and RT-PCR of VDR cDNA

    SciTech Connect

    Javed, A.A.; Huang, Y.; Bombard, A.T.

    1994-09-01

    Vitamin D{sub 3} receptors (VDR) function as regulators through the action of the ligand 1{alpha}, 25-dihydroxy vitamin D{sub 3}. The receptor specifically finds its ligand and exerts it effect on the regulation of the expression of target genes. It has been shown that mutations in the VDR gene affect the function of the receptors and cause a corresponding disorder state. Recently, it has been reported that common allelic variations found normally in the Caucasian (Australian) population pose varying degrees of risk for osteoporosis. We present here the cloning of the VDR gene and RT-PCR of VDR cDNA. Studies are in progress to establish allele frequency in the Black, Hispanic and Caucasian populations to systematically study the influence of allele types and to develop a risk profile for osteoporosis. The present method for detection of various alleles is based on RFLP analysis. We are developing PCR-based methods for the rapid detection and typing of alleles.

  5. Molecular cloning of the precursor cDNA for schistostatins, locust allatostatin-like peptides with myoinhibiting properties.

    PubMed

    Vanden Broeck, J; Veelaert, D; Bendena, W G; Tobe, S S; De Loof, A

    1996-09-18

    The cDNA encoding the precursor polypeptide for schistostatins, allatostatin-like peptides which have been shown to inhibit peristaltic movements of the lateral oviducts of Schistocerca gregaria, has been cloned and sequenced. Translation of this sequence reveals the presence of a pre-proschistostatin consisting of 283 amino acids. It contains ten different peptide sequences which are flanked by dibasic cleavage sites and C-terminal amidation signals. Eight of these peptides were identical to the schistostatins (or Scg-ASTs) that were previously purified from Schistocerca gregaria brain extracts. Two novel peptide sequences were discovered. One of these is the first AST-like peptide which has a C-terminal valine residue. Two peptides contain within their sequence an internal dibasic site which suggests a possible role for alternative processing and/or degradation. The schistostatin precursor differs from cockroach pre-proallatostatins in size, in sequence and in organization. It contains a lower number of peptides (10 versus 13 or 14) which are interrupted only once by an acidic spacer region (versus four in Diploptera punctata and Periplaneta americana). Northern analysis showed the presence of a 2.4 kb mRNA band in the locust central nervous system and midgut. This indicates that schistostatins, like other ASTs, are a good example of insect brain/gut peptides. PMID:8902849

  6. Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence.

    PubMed

    Kato, A; Takeda-Yoshikawa, Y; Hayashi, M; Kondo, M; Hara-Nishimura, I; Nishimura, M

    1998-02-01

    Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypeptide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other microbody proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells. PMID:9559562

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

    PubMed

    Kiriake, Aya; Shiomi, Kazuo

    2011-11-01

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

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

    SciTech Connect

    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.

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

    PubMed

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

    2016-07-01

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

  10. Chicken chromobox proteins: cDNA cloning of CHCB1, -2, -3 and their relation to W-heterochromatin.

    PubMed

    Yamaguchi, K; Hidema, S; Mizuno, S

    1998-07-10

    Three clones for chicken chromobox proteins were obtained from liver and ovary cDNA libraries. pCHCB1 and pCHCB2 encode polypeptides showing 96 and 95% identity with mouse M31 and M32, respectively, which are homologues of Drosophila heterochromatin protein 1 (HP1), and pCHCB3 encodes a polypeptide whose sequences of chromobox and C-terminal region show high-level similarities with those of mouse M33, Drosophila polycomb (Pc) protein, and Xenopus Pc homologue. When these cDNAs were expressed in female chicken embryonic fibroblasts (CEFs) as GFP-fused or HA-tagged proteins, all three proteins were found to be localized in nuclei. Among them, CHCB1 associates with brightly stained spots with 4', 6-diamidino-2-phenylindole (DAPI), suggesting its accumulation on heterochromatins. One of those spots was identified as W-heterochromatin. When CHCB1 lacking the N-terminal basic/acidic region or a part of the chromobox region was overexpressed in CEFs, W-heterochromatin became partially or extensively decondensed in the majority of nuclei. Overexpression of CHCB3 lacking a part of the chromobox did not cause decondensation of W-heterochromatin. Specific antisera raised against a part of CHCB1 or CHCB2, produced in Escherichia coli, detected protein species having apparent molecular masses of 25 kDa or 22 plus 23 kDa, respectively, in the subnuclear fraction containing the majority of chromatin from female chicken MSB-1 cells. PMID:9665828

  11. Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

    PubMed

    Reymond, I; Sergeant, A; Tappaz, M

    1996-06-01

    The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported. PMID:8679699

  12. Molecular cloning of the cDNA coding for mouse aldehyde oxidase: tissue distribution and regulation in vivo by testosterone.

    PubMed Central

    Kurosaki, M; Demontis, S; Barzago, M M; Garattini, E; Terao, M

    1999-01-01

    The cDNA coding for mouse aldehyde oxidase (AO), a molybdoflavoprotein, has been isolated and characterized. The cDNA is 4347 nt long and consists of an open reading frame predicting a polypeptide of 1333 amino acid residues, with 5' and 3' untranslated regions of 13 and 335 nt respectively. The apparent molecular mass of the translation product in vitro derived from the corresponding cRNA is consistent with that of the monomeric subunit of the AO holoenzyme. The cDNA codes for a catalytically active form of AO, as demonstrated by transient transfection experiments conducted in the HC11 mouse mammary epithelial cell line. The deduced primary structure of the AO protein contains consensus sequences for two distinct 2Fe-2S redox centres and a molybdopterin-binding site. The amino acid sequence of the mouse AO has a high degree of similarity with the human and bovine counterparts, and a significant degree of relatedness to AO proteins of plant origin. Northern blot and in situ hybridization analyses demonstrate that hepatocytes, cardiocytes, lung endothelial or epithelial cells and oesophagus epithelial cells express high levels of AO mRNA. In the various tissues and organs considered, the level of AO mRNA expression is not strictly correlated with the amount of the corresponding protein, suggesting that the synthesis of the AO enzyme is under translational or post-translational control. In addition, we observed sex-related regulation of AO protein synthesis. In the liver of male animals, despite similar amounts of AO mRNA, the levels of the AO enzyme and corresponding polypeptide are significantly higher than those in female animals. Treatment of female mice with testosterone increases the amounts of AO mRNA and of the relative translation product to levels similar to those in male animals. PMID:10377246

  13. Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA.

    PubMed Central

    Khromykh, A A; Westaway, E G

    1994-01-01

    Completion of the Kunjin virus (KUN) RNA sequence showed that it is the longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. The 3' noncoding sequence of 624 nucleotides included a unique insertion sequence of 46 bases adjacent to the stop codon, but otherwise it had properties similar to those of RNAs of closely related flaviviruses. A full-length KUN cDNA clone which could be stably propagated in Escherichia coli DH5 alpha was constructed; SP6 polymerase RNA transcripts from amplified cDNA were infectious when transfected into BHK-21 cells. A mutational change abolishing the BamHI restriction site at position 4049, leading to a conservative amino acid change of Arg-175 to Lys in the NS2A protein, was introduced into the cDNA during construction and was retained in the recovered virus. Extra terminal nucleotides introduced during cloning of the cDNA were shown to be present in the in vitro RNA transcripts but absent in the RNA of recovered virus. Although recovered virus differed from the parental KUN by a smaller plaque phenotype and delayed growth rate in BHK-21 cells and mice, it was very similar as assessed by several other criteria, such as peak titer during growth in cells, infectivity titer in cells and in mice, rate of adsorption and penetration in cells, replication at 39 degrees C, and neurovirulence after intraperitoneal injection in mice. The KUN stably cloned cDNA will provide a useful basis for future studies in defining and characterizing functional roles of all the gene products. Images PMID:8207832

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

    SciTech Connect

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. )

    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.

  15. Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus.

    PubMed

    MacFarlane, S A; Wallis, C V; Taylor, S C; Goulden, M G; Wood, K R; Davies, J W

    1991-05-01

    Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses. PMID:2024460

  16. CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

    PubMed Central

    Yokota, K; Kagawa, S; Shimizu, Y; Akioka, H; Tsurumi, C; Noda, C; Fujimuro, M; Yokosawa, H; Fujiwara, T; Takahashi, E; Ohba, M; Yamasaki, M; DeMartino, G N; Slaughter, C A; Toh-e, A; Tanaka, K

    1996-01-01

    The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome. Images PMID:8816993

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

    PubMed Central

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

    2009-01-01

    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 EST collection denotes an

  18. A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?

    PubMed

    Neumann, Gabriele; Whitt, Michael A; Kawaoka, Yoshihiro

    2002-11-01

    Since the first generation of a negative-sense RNA virus entirely from cloned cDNA in 1994, similar reverse genetics systems have been established for members of most genera of the Rhabdo- and Paramyxoviridae families, as well as for Ebola virus (Filoviridae). The generation of segmented negative-sense RNA viruses was technically more challenging and has lagged behind the recovery of nonsegmented viruses, primarily because of the difficulty of providing more than one genomic RNA segment. A member of the Bunyaviridae family (whose genome is composed of three RNA segments) was first generated from cloned cDNA in 1996, followed in 1999 by the production of influenza virus, which contains eight RNA segments. Thus, reverse genetics, or the de novo synthesis of negative-sense RNA viruses from cloned cDNA, has become a reliable laboratory method that can be used to study this large group of medically and economically important viruses. It provides a powerful tool for dissecting the virus life cycle, virus assembly, the role of viral proteins in pathogenicity and the interplay of viral proteins with components of the host cell immune response. Finally, reverse genetics has opened the way to develop live attenuated virus vaccines and vaccine vectors. PMID:12388800

  19. Brief report: genome sequence and construction of an infectious cDNA clone of Ribgrass mosaic virus from Chinese cabbage in Korea.

    PubMed

    Ryu, So-Young; Hong, Jin-Sung; Rhee, Sun-Ju; Lee, Gung Pyo

    2012-04-01

    Ribgrass mosaic virus (RMV) has severely decreased the production and lowered quality of Chinese cabbage co-infected with Turnip mosaic virus (63.4%) in Korea. The complete genome sequence of RMV isolated from Brassica rapa ssp. pekinensis was determined. The full genome consisted of 6,304 nucleotides and showed sequence identities of 91.5-94.2% with the corresponding genome of other RMV strains. Full-length cDNA of RMV-Br was amplified by RT-PCR with a 5'-end primer harboring a T7 promoter sequence and a 3'-end RMV specific primer. Subsequently, the full-length cDNA was cloned into plasmid vectors. Capped transcripts synthesized from the cDNA clone were highly infectious and caused characteristic symptoms in B. rapa ssp. pekinensis and several indicator plants, similar to wild type RMV. Since there has not been found RMV resistant Chinese cabbage yet and the virus has been prevalent already throughout the natural fields of Korea, the identification of full sequence and development of infectious clone would help developing breeding program for RMV resistant crops. PMID:22143325

  20. Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31) in NIH-3T3 Cell Line

    PubMed Central

    Salehi-Lalemarzi, Hamed; Shanehbandi, Dariush; Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Baradaran, Behzad; Movassaghpour, Ali Akbar; Kazemi, Tohid

    2015-01-01

    Purpose: PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. Methods: CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. Conclusion: Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins. PMID:26236664

  1. Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants.

    PubMed

    Mischkale, Katrin; Reimann, Ilona; Zemke, J; König, P; Beer, Martin

    2010-04-21

    Based on their genomic sequences, two genotypes of Bovine viral diarrhea virus (BVDV) can be differentiated, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). The complete genomic sequence of the highly virulent BVDV-2 strain 890 was cloned as cDNA to establish the infectious cDNA clone p890FL. In vitro-synthesised full-length RNA of p890FL was transfected into bovine cells and infectious virus could be recovered (v890FL). In vitro, recombinant v890FL showed similar growth characteristics as wild type virus v890WT. However, infection experiments in calves revealed an attenuation of recombinant v890FL in comparison to the parental isolate. Both leukocytopenia and fever were less pronounced in v890FL-infected calves. Nevertheless, viremia and virus shedding were comparable between recombinant and parental BVDV 890. Furthermore, mutants with partial deletions of the genomic region encoding for the autoprotease N(pro) (p890DeltaN(pro)) or the capsid protein (p890DeltaC) were constructed and characterised. In order to generate pseudovirions, replicon p890DeltaC was efficiently trans-complemented on a helper cell line. In summary, the newly developed construct p890FL represents the first infectious full-length cDNA clone for the BVDV-2 strain 890 and offers a useful tool for further studies on the pathogenesis of BVDV-2 and the development of novel recombinant BVDV-2 specific vaccine candidates. PMID:19875251

  2. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus

    SciTech Connect

    Kwon, B.S.; Haq, A.K.; Pomerantz, S.H.; Halaban, R.

    1987-11-01

    Screening of a lambdagt11 human melanocyte cDNA library with antibodies against hamster tyrosinase resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were form related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.

  3. Thyroid hormone-regulated brain mitochondrial genes revealed by differential cDNA cloning.

    PubMed Central

    Vega-Núñez, E; Menéndez-Hurtado, A; Garesse, R; Santos, A; Perez-Castillo, A

    1995-01-01

    Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit III). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain. Images PMID:7635984

  4. Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase: identification of the biotin carboxylase and biotin-carrier domains.

    PubMed Central

    Song, J; Wurtele, E S; Nikolau, B J

    1994-01-01

    Soybean genomic clones were isolated based on hybridization to probes that code for the conserved biotinylation domain of biotin-containing enzymes. The corresponding cDNA was isolated and expressed in Escherichia coli through fusion to the bacterial trpE gene. The resulting chimeric protein was biotinylated in E. coli. Antibodies raised against the chimeric protein reacted specifically with an 85-kDa biotin-containing polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carboxylase (EC 6.4.1.4) activity in cell-free extracts of soybean leaves. Thus, the isolated soybean gene and corresponding cDNA code for the 85-kDa biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase. The nucleotide sequence of the cDNA and portions of the genomic clones was determined. Comparison of the deduced amino acid sequence of the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase with sequences of other biotin enzymes suggests that this subunit contains the functional domains for the first half-reaction catalyzed by all biotin-dependent carboxylases--namely, the carboxylation of biotin. These domains are arranged serially on the polypeptide, with the biotin carboxylase domain at the amino terminus and the biotin-carboxyl carrier domain at the carboxyl terminus. Images PMID:8016064

  5. Molecular cloning and expression of the porcine trigeminal ganglion cDNA encoding a 5-ht(1F) receptor.

    PubMed

    Bhalla, Pankaj; Sharma, Hari S; Wurch, Thierry; Pauwels, Petrus J; Saxena, Pramod R

    2002-02-01

    Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht(1F)) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht(1F) receptor sequences from other species, including human. The recombinant porcine 5-ht(1F) receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [3H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 (N-methyl-3 [pyrrolidin 2(R)-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan =5HT>eletriptan>sumatriptan>frovatriptan =avitriptan>dihydroergotamine>zolmitriptan>5-carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl 4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1'-methyl-5-[2'-methyl-4'(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4'-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-(S,R) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht(1F) receptor (Spearman correlation coefficient; r(s)=0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht(1F) receptor. Using RT-PCR technique, the expression of porcine 5-ht(1F) receptor mRNA was observed in

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

    PubMed

    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. PMID:22940178

  7. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  8. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

    PubMed Central

    Shi, J; Dixon, R A; Gonzales, R A; Kjellbom, P; Bhattacharyya, M K

    1995-01-01

    An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein. Images Fig. 1 Fig. 2 PMID:7753826

  9. Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli.

    PubMed

    Hartwig, S; Frister, T; Alemdar, S; Li, Z; Krings, U; Berger, R G; Scheper, T; Beutel, S

    2014-05-01

    Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co(2+)-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition. PMID:24576659

  10. Purification, identification, characterization, and cDNA cloning of a high molecular weight extracellular superoxide dismutase of hamster that transiently increases in plasma during arousal from hibernation.

    PubMed

    Akita, Kenji; Hanaya, Toshiharu; Arai, Shigeyuki; Ohta, Tsunetaka; Okamoto, Iwao; Fukuda, Shigeharu

    2007-02-01

    We previously studied antioxidant profiles in the plasma of hibernating Syrian hamsters and found a transient increase of a superoxide radical-scavenging activity during the arousal phase. In this report, we purified and identified the high molecular weight superoxide dismutase (SOD)-like factor from the plasma of arousing hamsters. The cyanide-sensitive 240 kDa SOD-like factor showed a significant homology to mammalian extracellular SOD (EC-SOD) reported, although the molecular mass of EC-SOD was 135 kDa. The cDNA cloning revealed that the 240 kDa SOD-like factor was identical to the hamster ortholog of EC-SOD. It consisted of 245 amino acid residues including a signal sequence of 20 amino acid residues. Five cysteine residues that would participate in inner- and inter-subunit bonds were well conserved among species. Interestingly, there were four potential N-glycosylation sites in hamster EC-SOD, whereas there is only one site in other species. The amino acid sequence analysis indicated that three of the four sites were modified. These results suggest that the anomalistically high molecular weight of hamster EC-SOD is ascribed, at least in part, to the addition of extra sugar chains. Furthermore, results obtained here also propose the involvement of EC-SOD in the antioxidative defense of hibernating hamsters. PMID:17157046

  11. cDNA cloning and 1.75 A crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds.

    PubMed

    Cavada, Benildo S; Moreno, Frederico B B; da Rocha, Bruno A M; de Azevedo, Walter F; Castellón, Rolando E R; Goersch, Georg V; Nagano, Celso S; de Souza, Emmanuel P; Nascimento, Kyria S; Radis-Baptista, Gandhi; Delatorre, Plínio; Leroy, Yves; Toyama, Marcos H; Pinto, Vicente P T; Sampaio, Alexandre H; Barettino, Domingo; Debray, Henri; Calvete, Juan J; Sanz, Libia

    2006-09-01

    Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. PMID:16934035

  12. Clofibrate-induced cytochrome P450-lauric acid omega hydroxylase(P450LA omega):purification, cDNA cloning, sequence and regulation

    SciTech Connect

    Hardwick, J.P.; Song, B.J.; Gonzalez, F.J.

    1986-05-01

    A cytochrome P450 that hydroxylates lauric acid at the 12 position (P450LA omega) was isolated from liver microsomes of clofibrate treated rats. P450LA omega was immunologically distinct from P450s a,b,c,d,e,f,g,h,j,PB1, and PCN1. Polyclonal antibody against P450LA omega was utilized to screen a gt11 cDNA library. A clone (pP450LA omega), was isolated and its sequence determined. The P450LA omega mRNA is a minimum 2387 nts in length and codes for a P450 of Mr.58,222 daltons. This protein shares less than 35% amino acid similarity with P450s b,c,d,e,f,PB1, and PCN1; however, it does contain a hydrophobic amino terminal peptide and a conserved sequence surrounding the Cys residue at position 456, which is similar to other microsomal P450s. P450LA omega is present at high levels in untreated rat kidney and is induced by clofibrate in both kidney and liver. This induction is the result of an accumulation of mRNA through a rapid transcriptional activation of the P450LA gene. Southern blotting data suggest the presence of 2 or 3 genes in the P450LA omega family. This P450 gene family may be associated with arachidonic acid and prostraglandin metabolism in kidney and other tissues.

  13. cDNA cloning and complete primary structure of the alpha subunit of a leukocyte adhesion glycoprotein, p150,95.

    PubMed Central

    Corbi, A L; Miller, L J; O'Connor, K; Larson, R S; Springer, T A

    1987-01-01

    The leukocyte adhesion receptors, p150,95, Mac-1 and LFA-1 are integral membrane glycoproteins which contain distinct alpha subunits of 180,000-150,000 Mr associated with identical beta subunits of 95,000 Mr in alpha beta complexes. p150,95 alpha subunit tryptic peptides were used to specify oligonucleotide probes and a cDNA clone of 4.7 kb containing the entire coding sequence was isolated from a size-selected myeloid cell cDNA library. The 4.7-kb cDNA clone encodes a signal sequence, an extracellular domain of 1081 amino acids containing 10 potential glycosylation sites, a transmembrane domain of 26 amino acids, and a C-terminal cytoplasmic tail of 29 residues. The extracellular domain contains three tandem homologous repeats of approximately 60 amino acids with putative divalent cation-binding sites, and four weaker repeats which lack such binding sites. The cDNA clone hybridizes with a mRNA of 4.7 kb which is induced during in vitro differentiation of myeloid cell lines. The p150,95 alpha subunit is homologous to the alpha subunits of receptors which recognize the RGD sequence in extracellular matrix components, as has previously been shown for the beta subunits, supporting the concept that receptors involved in both cell-cell and cell-matrix interactions belong to a single gene superfamily termed the integrins. Distinctive features of the p150,95 alpha subunit include an insertion of 126 residues N-terminal to the putative metal binding region and a deletion of the region in which the matrix receptors are proteolytically cleaved during processing. Images Fig. 2. PMID:3327687

  14. Molecular Cloning and Characterization of cDNA Encoding a Putative Stress-Induced Heat-Shock Protein from Camelus dromedarius

    PubMed Central

    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. PMID:21845074

  15. Isolation of Dihydroflavonol 4-Reductase cDNA Clones from Angelonia x angustifolia and Heterologous Expression as GST Fusion Protein in Escherichia coli

    PubMed Central

    Gosch, Christian; Nagesh, Karthik Mudigere; Thill, Jana; Miosic, Silvija; Plaschil, Sylvia; Milosevic, Malvina; Olbricht, Klaus; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at −80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast. PMID:25238248

  16. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones.

    PubMed Central

    Newman, T; de Bruijn, F J; Green, P; Keegstra, K; Kende, H; McIntosh, L; Ohlrogge, J; Raikhel, N; Somerville, S; Thomashow, M

    1994-01-01

    High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequences similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing project is provided. PMID:7846151

  17. cDNA cloning and characterization of the antibacterial peptide cecropin 1 from the diamondback moth, Plutella xylostella L.

    PubMed

    Jin, Fengliang; Sun, Qiang; Xu, Xiaoxia; Li, Linmiao; Gao, Gang; Xu, Yingjie; Yu, Xiaoqiang; Ren, Shunxiang

    2012-10-01

    Cecropins are linear cationic antibacterial peptides that have potent activities against microorganisms. In the present study, a 480bp full-length cDNA encoding diamondback moth (Plutella xylostella) cecropin 1 (designated as Px-cec1) was obtained using RT-PCR. A Northern blot analysis showed that the Px-cec1 transcript was predominantly expressed in fat bodies, hemocytes, midgut and epidermis with the highest expression level in fat bodies. The expression of Px-cec1 mRNA in fat bodies was significantly increased 24h after microbial challenge, with the highest induced expression by Staphylococcus aureus. A circular dichroism (CD) analysis revealed that the recombinant Px-cec1 mainly contained α-helixes. Antimicrobial assays demonstrated that recombinant Px-cec1 exhibited a broad spectrum of anti-microbial properties against fungi, Gram-positive and Gram-negative bacteria, but it did not exhibit hemolytic activity against human erythrocytes. Furthermore, Px-cec1 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy and transmission electron microscopy. These results demonstrated that Px-cec1 exerts its antibacterial activity by acting on the cell membrane to disrupt bacterial cell structures. PMID:22921836

  18. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.

    PubMed Central

    Cutting, G R; Lu, L; O'Hara, B F; Kasch, L M; Montrose-Rafizadeh, C; Donovan, D M; Shimada, S; Antonarakis, S E; Guggino, W B; Uhl, G R

    1991-01-01

    Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family. Images PMID:1849271

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

    PubMed Central

    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

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

    PubMed

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

    2016-01-01

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

  1. Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus.

    PubMed Central

    Richardson, C D; Berkovich, A; Rozenblatt, S; Bellini, W J

    1985-01-01

    A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA. Images PMID:3838350

  2. Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.

    PubMed Central

    Parvari, R; Ziv, E; Lentner, F; Tel-Or, S; Burstein, Y; Schechter, I

    1987-01-01

    cDNA libraries of chicken spleen and Harder gland (a gland enriched with immunocytes) constructed in pBR322 were screened by differential hybridization and by mRNA hybrid-selected translation. Eleven L-chain cDNA clones were identified from which VL probes were prepared and each was annealed with kidney DNA restriction digests. All VL probes revealed the same set of bands, corresponding to about 15 germline VL genes of one subgroup. The nucleotide sequences of six VL clones showed greater than or equal to 85% homology, and the predicted amino acid sequences were identical or nearly identical to the major N-terminal sequence of L-chains in chicken serum. These findings, and the fact that the VL clones were randomly selected from normal lymphoid tissues, strongly indicate that the bulk of chicken L-chains is encoded by a few germline VL genes, probably much less than 15 since many of the VL genes are known to be pseudogenes. Therefore, it is likely that somatic mechanisms operating prior to specific triggering by antigen play a major role in the generation of antibody diversity in chicken. Analysis of the constant region locus (sequencing of CL gene and cDNAs) demonstrate a single CL isotype and suggest the presence of CL allotypes. Images Fig. 1. Fig. 2. Fig. 4. PMID:3107981

  3. Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene.

    PubMed Central

    Fung, Y K; Shackleford, G M; Brown, A M; Sanders, G S; Varmus, H E

    1985-01-01

    The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase. Images PMID:3018519

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

    SciTech Connect

    Vorechovsky, I.; Zhou, J.N.; Hammarstroem, L.

    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.

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

    PubMed

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

    2009-05-01

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

  6. Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.

    PubMed Central

    Frommer, W B; Hummel, S; Riesmeier, J W

    1993-01-01

    To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism. Images Fig. 1 PMID:8327465

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

    PubMed

    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. PMID:25264343

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

    PubMed Central

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

    2007-01-01

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

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

    SciTech Connect

    Miyamura, Tatsuo; Saito, Izumu ); Katayama, Tohru ); Kikuchi, Shu; Tateda, Akira ); Houghton, M.; Choo, Quilim; Kuo, G. )

    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.

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

    PubMed

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

    2009-11-01

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

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

    NASA Technical Reports Server (NTRS)

    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.

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

    SciTech Connect

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E.

    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.

  13. Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites.

    PubMed

    Kümmerer, Beate Mareike; Grywna, Klaus; Gläsker, Sabine; Wieseler, Janett; Drosten, Christian

    2012-09-01

    Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research. PMID:22673932

  14. Cloning of Human IgG Fc cDNA and Expression of Whole Human Anti-HBsAg Antibody in CHO Cells.

    PubMed

    Tong, Yi-Gang; Xu, Jing; Liu, Guo-Qi; Zhang, Yong-Guo; Cheng, Wan-Rong; Liu, Shu-Ling; Wang, Hai-Tao

    2000-01-01

    Messenger RNA was extracted from human peripheral lymphocytes and first strand cDNA was prepared by reverse-transciption. The cDNA of Fc fragment of human IgG1 was then obtained by PCR and was cloned into the pGEM T-vector. The DNA sequences encoding signal peptides of both light and heavy chains were synthesized and cloned respectively. For construction of the light chain expression plasmid, the light chain signal sequence was linked with the light chain variable and constant regions (VL-CL) which had been cloned previously by screening of phage display libraries with HBsAg. The resulting full-lenth light chain sequence was then inserted into pcDNA3.1, a mammalian expression vector. For construction of the heavy chain expression plasmid, the heavy chain signal sequence, the variable region, the first constant region (VH-CH1, cloned previously by screening of phage display libraries with HBsAg) and Fc fragment sequence were ligated to form a full-length heavy chain ORF, which was then cloned into another mammalian expression vector, pCI-DHFR1. CHO(dhfr(-)) cells were cotransfected with the above light and heavy chain expression plasmids, and cell clones expressing human anti-HBsAg antibodies were selected by G418 and methotrexate (MTX). The recombinant human antibodies were purified with protein L affinity chromatography from the cell culture medium. As human serum IgG, the recombinant IgG exhibited only one band with a molecular weight of more than 100 kD in non-reducing SDS-PAGE in reducing SDS-PAGE, however, it turned out to be two bands of approximately 50 kD and 25 kD respectively. Western-blot analysis demonstrated that the whole IgG in the non-reducing SDS-PAGE, and the heavy chain in the reducing SDS-PAGE both reacted with goat anti-human Fc antiserum. PMID:12075430

  15. Cloning

    MedlinePlus

    Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

  16. Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis.

    PubMed

    Tanida, Hiroaki; Tahara, Eiji; Mochizuki, Miwa; Yamane, Yukiko; Ryoji, Masaru

    2005-12-01

    Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function. PMID:16302973

  17. Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype

    PubMed Central

    Boot, Hein J.; ter Huurne, A. Agnes H. M.; Hoekman, Arjan J. W.; Peeters, Ben P. H.; Gielkens, Arno L. J.

    2000-01-01

    Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young

  18. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    SciTech Connect

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-11-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  19. Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones

    SciTech Connect

    Nguyen, C.; Rocha, D.; Granjeaud, S.

    1995-09-01

    High-throughput measurement of hybridization signatures obtained using complex probes prepared from poly(A){sup +} RNA and high-density cDNA colony filters is described. The performance of the system, elimination of artifacts, and verification of the validity of the data are discussed. cDNAs corresponding to sequences present at levels of approximately 0.01% in the complex probe can be detected. Good correlation is observed between expression profiles determined by this method and by Northern blotting. The method is applied to a preliminary investigation of differential expression in three cell types present in the murine thymus. 41 refs., 3 figs., 1 tab.

  20. Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

    PubMed Central

    Reymond, P; Kunz, B; Paul-Pletzer, K; Grimm, R; Eckerskorn, C; Farmer, E E

    1996-01-01

    Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication. PMID:8989883

  1. A partial cDNA clone of trypomastigote decay-accelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF.

    PubMed Central

    Tambourgi, D V; Kipnis, T L; da Silva, W D; Joiner, K A; Sher, A; Heath, S; Hall, B F; Ogden, G B

    1993-01-01

    Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982). Images PMID:7689538

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

    NASA Astrophysics Data System (ADS)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

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

  3. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.

    PubMed Central

    Thelander, L; Berg, P

    1986-01-01

    Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced. Images PMID:3025593

  4. cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects

    PubMed Central

    Suntravat, Montamas; Jia, Ying; Lucena, Sara E.; Sánchez, Elda E.; Pérez, John C.

    2013-01-01

    A 5′ truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5′-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, C. atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC50 of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC50s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders. PMID:23313448

  5. Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates.

    PubMed

    Haruta, Miyoshi; Pedersen, Jens A.; Constabel, C. Peter

    2001-08-01

    The biochemical anti-herbivore defense of trembling aspen (Populus tremuloides Michx.) was investigated in a molecular analysis of polyphenol oxidase (PPO; EC 1.10.3.2). A PPO cDNA was isolated from a trembling aspen wounded leaf cDNA library and its nucleotide sequence determined. Southern analysis indicated the presence of two PPO genes in the trembling aspen genome. Expression of PPO was found to be induced after herbivory by forest tent caterpillar, by wounding, and by methyl jasmonate treatment. Wound induction was systemic, and occurred in unwounded leaves on wounded plants. This pattern of expression is consistent with a role of this enzyme in insect defense. A search for potential PPO substrates in ethanolic aspen leaf extracts using electron spin resonance (ESR) found no pre-existing diphenolic compounds. However, following a brief delay and several additions of oxygen, an ESR signal specific for catechol was detected. The source of this catechol was most likely the aspen phenolic glycosides tremulacin or salicortin which decomposed during ESR experiments. This was subsequently confirmed in experiments using pure salicortin. PMID:11473716

  6. The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri.

    PubMed

    Ni, Duojiao; Song, Linsheng; Gao, Qiang; Wu, Longtao; Yu, Yundong; Zhao, Jianmin; Qiu, Limei; Zhang, Huan; Shi, Fangfang

    2007-11-01

    Cu, Zn superoxide dismutases (SODs) are metalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu, Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu, Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. luteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. PMID:17574438

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

    PubMed

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

    2002-02-01

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

  8. Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain.

    PubMed Central

    Hogervorst, F; Kuikman, I; von dem Borne, A E; Sonnenberg, A

    1990-01-01

    The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins. Images Fig. 2. Fig. 3. PMID:2311578

  9. The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding.

    PubMed Central

    Spritz, R A; Strunk, K; Surowy, C S; Hoch, S O; Barton, D E; Francke, U

    1987-01-01

    We have isolated and sequenced cDNA clones encoding the human U1-70K snRNP protein, and have mapped this locus (U1AP1) to human chromosome 19. The gene produces two size classes of RNA, a major 1.7-kb RNA and a minor 3.9-kb RNA. The 1.7-kb species appears to be the functional mRNA; the role of the 3.9-kb RNA, which extends further in the 5' direction, is unclear. The actual size of the hU1-70K protein is probably 52 kd, rather than 70 kd. The protein contains three regions similar to known nucleic acid-binding proteins, and it binds RNA in an in vitro assay. Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing of at least four alternative exon segments. This suggests that multiple forms of the hU1-70K protein may exist, possibly with different functions in vivo. Images PMID:2447561

  10. Human acidic ribosomal phosphoproteins P0, P1, and P2: Analysis of cDNA clones, in vitro synthesis, and assembly

    SciTech Connect

    Rich, B.E.; Steitz, J.A.

    1987-11-01

    cDNA clones encoding three antigenically related human ribosomal phosophoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identifies of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.

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

    PubMed

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

    1997-07-15

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

  12. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway

    SciTech Connect

    Bernhard, W.R.; Thoma, S.; Botella, J.; Somerville, C.R. )

    1991-01-01

    A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.

  13. cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit in the Mediterranean fruit fly Ceratitis capitata.

    PubMed

    Verras, Meletios; Gourzi, Polyxeni; Kalosaka, Katerina; Zacharopoulou, Antigone; Mintzas, Anastassios C

    2008-03-01

    In the present study, we report the cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit from the Mediterranean fruit fly Ceratitis capitata (medfly). Using an RT-PCR fragment that corresponds to the amino-terminal region of the Drosophila melanogaster 20S proteasome alpha5 subunit, we isolated a 987-bp cDNA that encodes the complete coding region of the medfly ortholog, which was named CcPSMA5. CcPSMA5 consists of 241 amino acids and has a predicted molecular weight of 26.4 kDa and pI 4.75. Comparison of the CcPSMA5 amino acid sequence with the sequences of all known 20S proteasome alpha5 subunits from different organisms indicated that the medfly 20S proteasome alpha5 subunit has the strongest homology to that of Drosophila. In situ hybridization showed that the CcPSMA5 gene is mapped in the region 44B of chromosome 4. Northern blot hybridization analysis showed that the CcPSMA5 mRNA has a size of approximately 1.2 kb. High levels of the CcPSMA5 mRNA were detected in freshly laid eggs, indicating that they were maternally deposited. The mRNA expression pattern during medfly development suggests that the CcPSMA5 gene is upregulated before mid-embryogenesis and at the onset of metamorphosis. PMID:18163525

  14. Cloning and Distribution of Ca2+-activated K+ channels in Lobster Panulirus interruptus

    PubMed Central

    Ouyang, Qing; Patel, Vinay; Vanderburgh, Jacqueline; Harris-Warrick, Ronald M.

    2010-01-01

    Large conductance Ca2+-activated potassium (BK) channels play important roles in controlling neuronal excitability. We cloned the PISlo gene encoding BK channels from the spiny lobster, Panulirus interruptus. This gene shows 81–98% sequence identity to Slo genes previously found in other organisms. We isolated a number of splice variants of the PISlo cDNA within Panulirus interruptus nervous tissue. Sequence analysis indicated that there are at least 7 alternative splice sites in PISlo, each with multiple alternative segments. Using immunohistochemistry, we found that the PISlo proteins are distributed in the synaptic neuropil, axon and soma of STG neurons. PMID:20682332

  15. Cloning and distribution of Ca2+-activated K+ channels in lobster Panulirus interruptus.

    PubMed

    Ouyang, Q; Patel, V; Vanderburgh, J; Harris-Warrick, R M

    2010-10-27

    Large conductance Ca(2+)-activated potassium (BK) channels play important roles in controlling neuronal excitability. We cloned the PISlo gene encoding BK channels from the spiny lobster, Panulirus interruptus. This gene shows 81-98% sequence identity to Slo genes previously found in other organisms. We isolated a number of splice variants of the PISlo cDNA within Panulirus interruptus nervous tissue. Sequence analysis indicated that there are at least seven alternative splice sites in PISlo, each with multiple alternative segments. Using immunohistochemistry, we found that the PISlo proteins are distributed in the synaptic neuropil, axon and soma of stomatogastric ganglion (STG) neurons. PMID:20682332

  16. Differential regulation by a peroxisome proliferator of the different multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific multifunctional protein 2.

    PubMed Central

    Caira, F; Clémencet, M C; Cherkaoui-Malki, M; Dieuaide-Noubhani, M; Pacot, C; Van Veldhoven, P P; Latruffe, N

    1998-01-01

    After our previous report on the cloning of two cDNA species in guinea pig, both encoding the same hepatic 79 kDa multifunctional protein 1 (MFP-1) [Caira, Cherkaoui-Malki, Hoefler and Latruffe (1996) FEBS Lett. 378, 57-60], here we report the cloning of a cDNA encoding a second multifunctional peroxisomal protein (MFP-2) in guinea-pig liver. This 2356 nt cDNA encodes a protein of 735 residues (79.7 kDa) whose sequence shows 83% identity with rat MFP-2 [Dieuaide-Noubhani, Novikov, Baumgart, Vanhooren, Fransen, Goethals, Vandekerckhove, Van Veldhoven and Mannaerts (1996) Eur. J. Biochem. 240, 660-666]. In parallel, we studied the effect of ciprofibrate, a hypolipaemic agent also known as peroxisome proliferator in rodent, on the expression of MFP-1 and MFP-2 (2.6 kb) in rats and guinea pigs. By Northern blotting analysis we demonstrated that three MFP-1-related mRNA species are expressed in the guinea-pig liver. The expression of two of them (3.5 and 2.6 kb) is slightly increased by ciprofibrate, whereas the 3.0 kb MFP-1 mRNA is, unlike the rat one, strongly down-regulated in guinea pigs treated with ciprofibrate. In a similar way, the hepatic expression of the guinea-pig 2.6 kb MFP-2 mRNA is also down-regulated in guinea pigs treated with ciprofibrate. These results demonstrate (1) that in contrast with the unique 3.0 kb MFP-1 rat mRNA, at least three hepatic MFP-1-related mRNA species are co-expressed in guinea pig; and (2) that, opposed to the accepted idea of non-responsiveness of the guinea pig to ciprofibrate, this drug affects MFP-1 and MFP-2 gene expression in this species. Also, the mRNA species for acyl-CoA oxidase and thiolase, two other enzymes of the peroxisomal beta-oxidation pathway that are induced severalfold in responsive species are down-regulated in guinea pig. This paper is the first, to our knowledge, reporting the down-regulation of the expression of genes encoding enzymes involved in the peroxisomal beta-oxidation of fatty acids (MFP-1) and

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

    PubMed

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

    1998-06-01

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

  18. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar

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

    PubMed

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

    1999-07-30

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

  20. The bark of Robinia pseudoacacia contains a complex mixture of lectins.Characterization of the proteins and the cDNA clones.

    PubMed Central

    Van Damme, E J; Barre, A; Smeets, K; Torrekens, S; Van Leuven, F; Rougé, P; Peumans, W J

    1995-01-01

    Two lectins were isolated from the inner bark of Robinia pseudoacacia (black locust). The first (and major) lectin (called RPbAI) is composed of five isolectins that originate from the association of 31.5- and 29-kD polypeptides into tetramers. In contrast, the second (minor) lectin (called RPbAII) is a hometetramer composed of 26-kD subunits. The cDNA clones encoding the polypeptides of RPbAI and RPbAII were isolated and their sequences determined. Apparently all three polypeptides are translated from mRNAs of approximately 1.2 kb. Alignment of the deduced amino acid sequences of the different clones indicates that the 31.5- and 29-kD RPbAI polypeptides show approximately 80% sequence identity and are homologous to the previously reported legume seed lectins, whereas the 26-kD RPbAII polypeptide shows only 33% sequence identity to the previously described legume lectins. Modeling the 31.5-kD subunit of RPbAI predicts that its three-dimensional structure is strongly related to the three-dimensional models that have been determined thus far for a few legume lectins. Southern blot analysis of genomic DNA isolated from Robinia has revealed that the Robinia bark lectins are the result of the expression of a small family of lectin genes. PMID:7716244

  1. Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.

    PubMed Central

    Arredondo-Peter, R; Moran, J F; Sarath, G; Luan, P; Klucas, R V

    1997-01-01

    Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin. PMID:9193085

  2. 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

    DOEpatents

    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.

  3. 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

    DOEpatents

    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.

  4. Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein).

    PubMed

    Mondal, Shankar P; Cook, R Frank; Chelvarajan, R Lakshman; Henney, Pamela J; Timoney, Peter J; Balasuriya, Udeni B R

    2016-04-01

    Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques. PMID:26711457

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

    PubMed

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

    2015-08-01

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

  6. Cloning and sequence analysis of cDNA coding for a lectin from Helianthus tuberosus callus and its jasmonate-induced expression.

    PubMed

    Nakagawa, R; Yasokawa, D; Okumura, Y; Nagashima, K

    2000-06-01

    Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus. cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum. The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314). When introduced into E. coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity. The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins. When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner. The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus. In view of these results, HTA I is considered to be a jasmonate-induced protein. PMID:10923797

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

    NASA Astrophysics Data System (ADS)

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

    1981-02-01

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

  8. High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme.

    PubMed Central

    Wang, Y H; Citron, B A; Ribeiro, P; Kaufman, S

    1991-01-01

    A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase. Images PMID:1681542

  9. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    NASA Astrophysics Data System (ADS)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  10. cDNA cloning of FRIL, a lectin from Dolichos lablab, that preserves hematopoietic progenitors in suspension culture.

    PubMed

    Colucci, G; Moore, J G; Feldman, M; Chrispeels, M J

    1999-01-19

    Ex vivo culture of hematopoietic stem cells is limited by the inability of cytokines to maintain primitive cells without inducing proliferation, differentiation, and subsequent loss of repopulating capacity. We identified recently in extracts of kidney bean and hyacinth bean a mannose-binding lectin, called FRIL, and provide here evidence that this protein appears to satisfy properties of a stem cell preservation factor. FRIL was first identified based on its ability to stimulate NIH 3T3 cells transfected with Flt3, a tyrosine kinase receptor central to regulation of stem cells. Molecular characterization from polypeptide sequencing and identification of the cDNA of hyacinth bean FRIL shows 78% amino acid identity with a mannose-binding lectin of hyacinth beans. Treatment of primitive hematopoietic progenitors in suspension culture with purified hyacinth FRIL alone is able to preserve cells for 1 month without medium changes. In vitro progenitor assays for human hematopoietic cells cultured 3 weeks in FRIL displayed small blast-like colonies that were capable of serial replating and persisted even in the presence of cytokines known to induce differentiation. These results suggest that FRIL is capable of preserving primitive progenitors in suspension culture for prolonged periods. FRIL's clinical utility involving procedures for stem cell transplantation, tumor cell purging before autologous transplantation, and ex vivo cultures used for expansion and stem cell gene therapy currently are being explored. PMID:9892687

  11. Cloned cDNA to cholecystokinin mRNA predicts an identical preprocholecystokinin in pig brain and gut.

    PubMed Central

    Gubler, U; Chua, A O; Hoffman, B J; Collier, K J; Eng, J

    1984-01-01

    Molecular cloning has established the structure of preprocholecystokinin in porcine cerebral cortex and duodenal mucosa. This precursor is 114 amino acids long, is identical in brain and gut, contains all the cholecystokinin (CCK) peptides previously isolated, and has the characteristics of a prohormone. It contains a putative amino-terminal signal peptide, basic processing sites, and a carboxyl-terminal amidation signal. The CCK mRNAs from brain and gut are approximately 850 nucleotides long and differ by only a few single base changes. This analysis establishes by a strict criterion that CCK is synthesized in both brain and gut and that the different distributions of molecular forms of CCK in the two tissues are most probably a consequence of tissue-specific posttranslational events. Images PMID:6205394

  12. Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

    PubMed

    Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

    1996-08-01

    The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region. PMID:8771778

  13. Molecular cloning and analysis of a pea cDNA that is expressed in darkness and very rapidly induced by gibberellic acid.

    PubMed

    Li, H Y; Guo, Z F; Zhu, Y X

    1998-09-01

    Using cDNA representational difference analysis (cDNA RDA), we isolated a cDNA named GDA-1 from a cDNA library constructed with mRNA from short-day (SD) grown G2 pea apical tissue. The amino acid sequence deduced from GDA-1 shares partial identity with the B2 protein which is expressed during embryogenesis of carrot cells. Northern analysis showed that GDA-1 mRNA is abundant in SD-grown G2 pea apical buds. In long-day (LD) conditions, there was almost no detectable GDA-1 mRNA. When LD-grown G2 peas were kept in continuous darkness for 24 h, the GDA-1 mRNA content reached a level equivalent to about 50% of that in the SD samples. On the other hand, when SD-grown peas were transferred into the light for 24 h, the amount of hybridizable GDA-1 mRNA dropped to the same as that of LD-grown plants. GDA-1 expression was found to be independent of flower initiation time. GA3 application in vitro resulted in rapid accumulation of GDA-1 mRNA in LD-grown G2 pea apical buds, which is compatible with its delaying effect on apical senescence. Time-course experiments revealed that GDA-1 is induced within 15 min of GA3 application. Exogenous GA3 did not influence the expression of GDA-1 in SD-grown G2 peas. Since both photoperiod and GA induce the expression of GDA-1, we speculate that they may activate similar signal transduction pathways in G2 peas. Our work also shows that photoperiod may change the efficiency of gibberellin perception by plants. PMID:9790595

  14. Expression of human. alpha. sub 2 -macroglobulin cDNA in baby hamster kidney fibroblasts: Secretion of high levels of active. alpha. sub 2 -macroglobulin

    SciTech Connect

    Boel, E.; Mortensen, S.B. ); Kristensen, T.; Sottrup-Jensen, L. ); Petersen, C.M. )

    1990-05-01

    Human {alpha}{sub 2}-macroglobulin ({alpha}{sub 2}M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase {alpha}{sub 2}M entraps the proteinase molecule in a reaction that involves large conformational changes in {alpha}{sub 2}M. The authors describe the molecular cloning of {alpha}{sub 2}M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human {alpha}{sub 2}M with a specific enzyme-linked immunosorbent assay. Human recombinant {alpha}{sub 2}M (r{alpha}{sub 2}M), secreted and purified form isolated transfected BHK cell lines, was structurally and functionally compared to {alpha}{sub 2}M purified from human serum. The results show that r{alpha}{sub 2}M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r{alpha}{sub 2}M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference.

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

    PubMed

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

    2010-08-01

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

  16. Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

    PubMed

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Napis, Suhaimi

    2011-11-01

    An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit. PMID:21287365

  17. cDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata.

    PubMed

    Zhang, D C; Hu, Y T; Guo, H Y; Cui, S G; Su, T F; Jiang, S G

    2011-01-01

    Galectins can recognize and specifically bind to β-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters. PMID:21948759

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

    NASA Astrophysics Data System (ADS)

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

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  19. Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum.

    PubMed

    Yu, JunLong; Wang, ShiPing; Li, WenKai; Dai, Gan; Xu, ShaoRui; He, Zhuo; Peng, XianChu; Zhou, SongHua; Liu, XueQin

    2007-04-01

    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 prokaryotic 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 (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. PMID:17447029

  20. cDNA cloning, heat shock regulation and developmental expression of the hsp83 gene in the Mediterranean fruit fly Ceratitis capitata.

    PubMed

    Theodoraki, M A; Mintzas, A C

    2006-12-01

    This report presents the cDNA cloning, heat shock regulation and developmental expression of the hsp90 gene homologue of the Mediterranean fruit fly Ceratitis capitata (medfly). The isolated cDNA contained the coding region, the 3'UTR and most of the 5'UTR of the medfly hsp90 homologue, which was named Cchsp83. The deduced CcHSP83 polypeptide contained all the highly conserved amino acid segments that characterize the cytosolic members of the HSP90 family. Genomic analysis showed that the Cchsp83 gene is unique and was mapped at the 94C division of the sixth polytene chromosome. The size of the Cchsp83 mRNA was found to be approximately 2.7 kb. The predicted molecular mass of the CcHSP83 protein was 81.4 kDa, while the apparent molecular weight estimated by SDS-PAGE was approximately 90 kDa. Phylogenetic analysis based on 14 insect HSP90 amino acid sequences was consistent with the known phylogeny at low taxonomic level. The Cchsp83 gene is constitutively expressed in all stages of medfly development and is induced from a low level to several-fold by heat, depending on the developmental stage. Heat shock induction begins at 30 degrees C, reaching a maximum between 35 and 41 degrees C. Cchsp83 RNA expression is highly regulated during embryonic development; however, the temporal fluctuations in RNA levels during embryogenesis were not followed by similar fluctuations in the levels of the protein. PMID:17201776

  1. cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): Evidence for rapid evolutionary divergence of the gene

    SciTech Connect

    Li, Kehua; Christiano, A.M.; Chu, Mon Li; Uitto, J. Thomas Jefferson Univ., Philadelphia, PA ); Copeland, N.G.; Gilbert, D.J. )

    1993-06-01

    Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study the authors have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of [approximately]9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5[prime] half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7Al. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. 33 refs., 5 figs., 1 tab.

  2. Cloning

    MedlinePlus

    ... DNA Reproductive cloning, which creates 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

  3. Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

    PubMed Central

    Mao, Mao; Fu, Gang; Wu, Ji-Sheng; Zhang, Qing-Hua; Zhou, Jun; Kan, Li-Xin; Huang, Qiu-Hua; He, Kai-Li; Gu, Bai-Wei; Han, Ze-Guang; Shen, Yu; Gu, Jian; Yu, Ya-Ping; Xu, Shu-Hua; Wang, Ya-Xin; Chen, Sai-Juan; Chen, Zhu

    1998-01-01

    Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34+ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34+ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation. PMID:9653160

  4. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2. alpha. (eIF-2. alpha. ) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2. alpha. kinase

    SciTech Connect

    Chen, J.J.; Throop, M.S.; Kuo, I.; Pal, J.K.; Brodsky, M.; London, I.M. ); Gehrke, L. Harvard Medical School, Boston, MA )

    1991-09-01

    The authors have cloned the cDNA of the heme-regulated eIF-2{alpha} kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2{alpha} kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of {approx} 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2{alpha} kinase. This observation suggests that GCN2 protein kinase may be an eIF-2{alpha} kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

  5. cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

    SciTech Connect

    Kuefer, M.U.; Valentine, V.; Behm, F.G.

    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.

  6. Infectious cDNA clones of four viroids in Coleus blumei and molecular characterization of their progeny.

    PubMed

    Jiang, Dongmei; Gao, Rui; Qin, Lv; Wu, Zujian; Xie, Lianhui; Hou, Wanying; Li, Shifang

    2014-02-13

    Four viroid species infecting Coleus blumei, named Coleus blumei viroid 1-4 (CbVd-1-CbVd-4), and two tentative new viroid species, named CbVd-5 and CbVd-6, have been characterized, for two of which (CbVd-5 and CbVd-6, first reported in 2009), there is no established bioassay. Here, infectious clones were used as inoculums sources and the biological properties of CbVd-1, -3, -5 and -6 were assessed. When dimeric CbVd (+) RNAs synthesized in vitro were bioassayed, the first detection time for the four CbVds was different, ranging from 45 to 300 days. In addition, we confirmed that CbVd-5 and CbVd-6 can be transmitted via seeds. Molecular characterization of their progeny from slash-inoculated plants one year after inoculation demonstrated that the genetic diversity of CbVd populations may depend on the infected coleus plants and on the viroid genotype. PMID:24291215

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

    PubMed

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

    2016-09-01

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

  8. Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner.

    PubMed Central

    Ney, P A; Andrews, N C; Jane, S M; Safer, B; Purucker, M E; Weremowicz, S; Morton, C C; Goff, S C; Orkin, S H; Nienhuis, A W

    1993-01-01

    The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation. Images PMID:8355703

  9. [Cloning and characterization of a novel mouse short-chain dehydrogenase/reductases cDNA mHsdl2#, encoding a protein with a SDR domaid and a SCP2 domain].

    PubMed

    Dai, J; Li, P; Ji, Ch; Feng, C; Gui, M; Sun, Y; Zhang, J; Zhu, J; Dou, Ch; Gu, Sh

    2005-01-01

    The short-chain dehydrogenases/reductases (SDRs) play important roles in body's metabolism. We cloned a novel mouse SDR cDNA which encodes a deduced HSD-like protein with a conserved SDR domain and a SCP2 domain. The 1.8 kb cDNA consists of 11 exons and is mapped to mouse chromosome 4B3. The corresponding gene is widely expressed in normal mouse tissues and its expression level in liver increases after inducement with cholesterol food. The predicted mouse HSDL2 protein, which has a peroxisomal target signal, is localized in the cytoplasm of NIH 3T3 cells. PMID:16240713

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

    PubMed

    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. PMID:22132787

  11. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    PubMed Central

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

  12. The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri.

    PubMed

    Song, Linsheng; Zou, Huibin; Chang, Yaqing; Xu, Wei; Wu, Longtao

    2006-01-01

    Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. PMID:15975653

  13. Cloning and expression of a cDNA encoding epitopes shared by 15- and 60-kilodalton proteins of Cryptosporidium parvum sporozoites.

    PubMed Central

    Jenkins, M C; Fayer, R; Tilley, M; Upton, S J

    1993-01-01

    A cDNA (CP15/60) encoding epitopes of Cryptosporidium parvum 15- and 60-kDa sporozoite proteins was isolated and expressed in Escherichia coli toward the goal of developing an immunogen for producing high-titer anticryptosporidial colostrum. Antisera prepared in rats to native C. parvum 15-kDa protein and used to identify the CP15/60 bacteriophage clone recognized both 15- and 60-kDa in vitro translation products derived from sporozoite RNA. Antisera specific for recombinant CP15/60 antigen recognized native 15- and 60-kDa C. parvum sporozoite proteins by immunoblotting and identified both surface and internal antigens on C. parvum sporozoites by immunofluorescence staining. Northern (RNA) and Southern blot hybridization experiments using sporozoite RNA and DNA indicated that CP15/60 DNA is transcribed as a single 1.4-kb RNA species from a single-copy gene. Recombinant CP15/60 antigen was recognized by hyperimmune colostrum from cows immunized with C. parvum oocyst-sporozoite protein and by convalescent-phase sera from C. parvum-infected calves. Images PMID:7684726

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

    PubMed

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

    2006-05-01

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

  15. Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening.

    PubMed

    Pecker, I; Gabbay, R; Cunningham, F X; Hirschberg, J

    1996-02-01

    The cDNA which encodes lycopene cyclase, CrtL, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN) and functionally expressed in Escherichia coli. This enzyme converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8') position. Inhibition in E. coli indicated that lycopene cyclase is the target site for the inhibitor MPTA, 2-(4-methylphenoxy)tri-ethylamine hydrochloride. The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but different from that of the non-photosynthetic bacteria Erwinia. mRNA of CrtL and Pds, which encodes phytoene desaturase, was measured in leaves, flowers and ripening fruits of tomato. In contrast to genes which encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the 'breaker' stage of fruit ripening, the level of CrtL mRNA decreases at this stage. Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis. PMID:8624411

  16. Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

    PubMed Central

    Rudenskaya, Galina N; Kislitsin, Yuri A; Rebrikov, Denis V

    2004-01-01

    Background In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. Results The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa) and 226 amino acid residues (Mcalc 23.5 kDa), respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70%) and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%). The phylogenetic tree of these enzymes was constructed. Conclusions Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases. PMID:14731305

  17. Islet amyloid polypeptide (IAPP): cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus

    SciTech Connect

    Betsholtz, C.; Svensson, V.; Rorsman, F.; Wilander, E. ); Engstroem, U. ); Westermark, G.T.; Westermark, P. ); Johnson, K. )

    1989-08-01

    The authors have cloned and sequenced a human islet amyloid polypeptide (IAPP) cDNA. A secretory 89 amino acid IAPP protein precursor is predicted from which the 37 amino acid IAPP molecule is formed by amino- and carboxyterminal proteolytic processing. The IAPP peptide is 43-46% identical in amino acid sequence to the two members of the calcitonin gene-related peptide (CGRP) family. Evolutionary conserved proteolytic processing sites indicate that similar proteases are involved in the maturation of IAPP and CGRP and that the IAPP amyloid polypeptide is identical to the normal proteolytic product of the IAPP precursor. A synthetic peptide corresponding to a carboxyterminal fragment of human IAPP is shown to spontaneously form amyloid-like fibrils in vitro. Antibodies against this peptide cross-react with IAPP from species that develop amyloid in pancreatic islets in conjunction with age-related diabetes mellitus (human, cat, raccoon), but do not cross-react with IAPP from other tested species (mouse, rat, guinea pig, dog).

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

    PubMed

    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. PMID:26219580

  19. The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues

    SciTech Connect

    Shen, Z.; Chen, D.J.; Denison, K.

    1995-01-01

    The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.

  20. cDNA Cloning, Heterologous Expressions, and Functional Characterization of Malonyl-Coenzyme A:Anthocyanidin 3-O-Glucoside-6"-O-Malonyltransferase from Dahlia Flowers1

    PubMed Central

    Suzuki, Hirokazu; Nakayama, Toru; Yonekura-Sakakibara, Keiko; Fukui, Yuko; Nakamura, Noriko; Yamaguchi, Masa-atsu; Tanaka, Yoshikazu; Kusumi, Takaaki; Nishino, Tokuzo

    2002-01-01

    In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (Km, 18.8 μm) to pelargonidin 3-O-glucoside (Km, 46.7 μm) to produce pelargonidin 3-O-6"-O-malonylglucoside with a kcat value of 7.3 s−1. The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11–63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of β-glucosidase. These results

  1. An expressed sequence tag database of T-cell-enriched activated chicken splenocytes: sequence analysis of 5251 clones.

    PubMed

    Tirunagaru, V G; Sofer, L; Cui, J; Burnside, J

    2000-06-01

    The cDNA and gene sequences of many mammalian cytokines and their receptors are known. However, corresponding information on avian cytokines is limited due to the lack of cross-species activity at the functional level or strong homology at the molecular level. To improve the efficiency of identifying cytokines and novel chicken genes, a directionally cloned cDNA library from T-cell-enriched activated chicken splenocytes was constructed, and the partial sequence of 5251 clones was obtained. Sequence clustering indicates that 2357 (42%) of the clones are present as a single copy, and 2961 are distinct clones, demonstrating the high level of complexity of this library. Comparisons of the sequence data with known DNA sequences in GenBank indicate that approximately 25% of the clones match known chicken genes, 39% have similarity to known genes in other species, and 11% had no match to any sequence in the database. Several previously uncharacterized chicken cytokines and their receptors were present in our library. This collection provides a useful database for cataloging genes expressed in T cells and a valuable resource for future investigations of gene expression in avian immunology. A chicken EST Web site (http://udgenome. ags.udel. edu/chickest/chick.htm) has been created to provide access to the data, and a set of unique sequences has been deposited with GenBank (Accession Nos. AI979741-AI982511). Our new Web site (http://www. chickest.udel.edu) will be active as of March 3, 2000, and will also provide keyword-searching capabilities for BLASTX and BLASTN hits of all our clones. PMID:10860659

  2. Analysis of a Catfish Gene Resembling Interleukin-8: CDNA Cloning, Gene Structure, and Expression After Infection with Edwardsiella ictaluri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemokines are important mediators for innate immunity involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci. While almost all chemokines have been identified from mammals, only a handful of fish chemokines have been identified. Here we report molecula...

  3. Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

    PubMed

    Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

    1997-10-01

    mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

  4. cDNA cloning and mRNA expression of a FTZ-F1 homologue from the pituitary of the orange-spotted grouper, epinephelus coioides.

    PubMed

    Zhang, Weimin; Li, Xin; Zhang, Yong; Zhang, Lihong; Tian, Jing; Ma, Guangzhi

    2004-08-01

    A FTZ-F1 homologue was cloned from the pituitary cDNA library of the orange-spotted grouper. The full-length cDNA of the orange-spotted grouper FTZ-F1 spanned 1735 bp including a poly (A) tail. The open reading frame encodes a protein of 468 amino acids. Sequence analysis indicated that it had a structure typical of the orphan nuclear receptor superfamily, and the FTZ-F1 box, a characteristic of the FTZ-F1 family. Phylogenetic analysis indicated that the orange-spotted grouper FTZ-F1 was closely related to medaka FTZ-F1 and did not belong to either the SF-1/Ad4BP group or the LRH-1/FTF group. Virtual Northern Blot detected a major transcript of about 1.7 kb and a minor transcript of 2.2 kb of FTZ-F1 in the orange-spotted grouper pituitary gland. The expression of FTZ-F1 homologue gene in different tissues and during embryonic development of the orange-spotted grouper was determined using one-step RT-PCR coupled with Southern blot analysis. In addition to the pituitary gland, the orange-spotted grouper FTZ-F1 was also expressed in the hypothalamus, forebrain, heart, liver, kidney, and ovary. The stronger signal from the gel image indicated that the expression level of FTZ-F1 homologue gene was higher in the ovary of stage 3 than stage 2. During embryonic development, mRNA for the orange-spotted grouper FTZ-F1 homologue was present in newly fertilized eggs, but disappeared in embryos at 50 min post fertilization. The orange-spotted grouper FTZ-F1 homologue mRNA reappeared in embryos at 1.5 hr post fertilization. Its expression level was increased from late blastula to neurula stages. Taken together, results of the current study suggest that the orange-spotted grouper FTZ-F1 homologue exhibits characteristics indicative of both the LRH-1/FTF- and the SF-1/Ad4BP-like genes, and may also play important roles in the hypothalamus-pituitary-gonadal axis, cholesterol metabolism, and embryogenesis. PMID:15286949

  5. Rescue of infective virus from a genome-length cDNA clone of the FMDV serotype O (IND-R2/75) vaccine strain and its characterization.

    PubMed

    Rajasekhar, R; Hosamani, Madhusudan; Basagoudanavar, Suresh H; Sreenivasa, B P; Tamil Selvan, R P; Saravanan, Paramasivam; Venkataramanan, Ramamurthy

    2013-08-01

    We report here the construction and characterization of an infectious cDNA clone of the Indian vaccine strain of foot and mouth disease virus (FMDV) serotype O, IND-R2/75. Viral genome was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in five fragments and subsequently assembled sequentially in a plasmid vector to generate a complete cDNA clone, flanked by the T7 RNA polymerase promoter and poly (A) tail at 5' and 3' ends, respectively. Transfection of BHK-21 cells with the RNA transcribed from this genome-length cDNA construct allowed the recovery of infectious recombinant FMDV particles as evidenced by cytopathic effect in BHK-21 cells. Characterization of the recombinant virus revealed its similarity to the parental strain. Recombinant virus could be distinguished from the parental virus based on the presence of a unique marker sequence in the former, which was incorporated in the cDNA using a silent mutation. The virus showed no significant amino acid changes in the capsid-coding region when serially passaged up to ten times in BHK-21 cells, while retaining the marker sequence. PMID:23465777

  6. Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

    NASA Technical Reports Server (NTRS)

    Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

    1993-01-01

    An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

  7. Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

    PubMed

    Shiosaki, K; Takata, K; Omichi, K; Tomita, N; Horii, A; Ogawa, M; Matsubara, K

    1990-05-14

    A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes. PMID:2197187

  8. Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

    NASA Astrophysics Data System (ADS)

    Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

    1987-11-01

    A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

  9. Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous) cDNA libraries

    PubMed Central

    Leshkowitz, Dena; Gazit, Shirley; Reuveni, Eli; Ghanim, Murad; Czosnek, Henryk; McKenzie, Cindy; Shatters, Robert L; Brown, Judith K

    2006-01-01

    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 of eggs. Conclusion This

  10. Identification, cDNA cloning and possible roles of seed-specific rice asparaginyl endopeptidase, REP-2.

    PubMed

    Kato, Hideki; Sutoh, Keita; Minamikawa, Takao

    2003-08-01

    We previously showed that two major cysteine endopeptidases, REP-1 and REP-2, were present in germinated rice ( Oryza sativa L.) seeds, and that REP-1 was the enzyme that digests seed storage proteins. The present study shows that REP-2 is an asparaginyl endopeptidase that acts as an activator of REP-1, and we separated it into two forms, REP-2alpha (39 kDa) and REP-2beta (40 kDa), using ion-exchange chromatography and gel filtration chromatography. Although analysis of the amino terminals revealed that 10 amino acids of both forms were identical, their isoelectric points were different. SDS-PAGE/immunoblot analysis using an antiserum raised against legumain, an asparaginyl endopeptidase from jack bean, indicated that both forms were present in maturing and germinating rice seeds, and that their amounts transiently decreased in dry seeds. Northern blot analysis indicated that REP-2 mRNA was expressed in both maturing and germinating seeds. In germinating seeds, the mRNA was detected in aleurone layers but not in shoot and root tissues. Incubation of the de-embryonated seeds in 10(-6) M gibberellic acid induced the production of large amounts of REP-1, whereas REP-2beta levels declined rapidly. Southern blot analysis showed that there is one gene for REP-2 in the genome, indicating that both REP-2 enzymes are generated from a single gene. The structure of the gene was similar to that of beta-VPE and gamma-VPE isolated from Arabidopsis thaliana. PMID:12684786

  11. Characterization of Two Divergent Endo-β-1,4-Glucanase cDNA Clones Highly Expressed in the Nonclimacteric Strawberry Fruit

    PubMed Central

    Llop-Tous, Immaculada; Domínguez-Puigjaner, Eva; Palomer, Xavier; Vendrell, Miquel

    1999-01-01

    Two cDNAs clones (Cel1 and Cel2) encoding divergent endo-β-1,4-glucanases (EGases) have been isolated from a cDNA library obtained from ripe strawberry (Fragaria x ananassa Duch) fruit. The analysis of the amino acid sequence suggests that Cel1 and Cel2 EGases have different secondary and tertiary structures and that they differ in the presence of potential N-glycosylation sites. By in vitro translation we show that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kD, respectively. Phylogenetic analysis revealed that the Cel2 EGase diverged early in evolution from other plant EGases. Northern analysis showed that both EGases are highly expressed in fruit and that they have different temporal patterns of accumulation. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. In addition, the early expression of Cel2 in green fruit, well before significant softening begins, suggests that the product of this gene may also be involved in processes other than fruit softening, e.g. cell wall expansion. PMID:10198101

  12. [Cloning and analysis of cDNA encoding key enzyme gene (dxr) of the non-MVA pathway in Taxus chinensis cells].

    PubMed

    Zheng, Qing-Ping; Yu, Long-Jiang; Liu, Zhi; Li, Mo-Yi; Xiang, Fu; Yang, Qin

    2004-07-01

    Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm. PMID:15968987

  13. Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans.

    PubMed

    Kannouche, P; Mauffrey, P; Pinon-Lataillade, G; Mattei, M G; Sarasin, A; Daya-Grosjean, L; Angulo, J F

    2000-09-01

    We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15-p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. (HSA)kin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles (HSA)kin17 foci in cycling cells, indicating a link between the intranuclear distribution of (HSA)kin17 protein and the DNA damage response. PMID:10964102

  14. Androgen receptors in the bonnethead, Sphyrna tiburo: cDNA cloning and tissue-specific expression in the male reproductive tract.

    PubMed

    Tyminski, John P; Gelsleichter, James J; Motta, Philip J

    2015-12-01

    As demonstrated in past studies, androgens appear to play critical roles in regulating reproduction in male sharks. However, little is known about the cell-specific actions of androgens in these fishes. To address this, this study examined androgen targets in reproductive organs of a seasonally reproducing shark, the bonnethead (Sphyrna tiburo). A partial bonnethead AR cDNA clone was isolated and found to exhibit strong homology with known vertebrate ARs. Using RT-PCR and in situ hybridization, AR was found to be expressed in multiple cell types in the male bonnethead testis (premeiotic germ cells, Leydig-like interstitial cells, Sertoli cells, peritubular myoid cells, and mature spermatozoa) and gonadal ducts (stromal cells, luminal epithelial cells, mature spermatozoa). Furthermore, AR expression in these organs was found to vary temporally in relation to the seasonal reproductive cycle. Based on immunocytochemistry, the presence of AR protein in male bonnethead reproductive organs was largely consistent with patterns of AR gene expression with the single exception of mature spermatozoa, which exhibited consistently strong mRNA expression but only inconsistent and weak AR protein immunoreactivity. These results suggest important roles for androgens in regulating germ cell proliferation, hormone production, spermatid elongation, spermiation, and gonadal duct function in male bonnetheads. In addition, high abundance of AR mRNA in bonnethead spermatozoa suggest the potential for de novo protein synthesis following spermiation/copulation and/or a role for AR mRNA in early embryonic development, both of which have been proposed to explain the occurrence of mRNA transcripts in spermatozoa from various vertebrates. PMID:26320857

  15. Steroidogenic acute regulatory protein in eels: cDNA cloning and effects of ACTH and seawater transfer on its mRNA expression.

    PubMed

    Li, Yuan-You; Inoue, Koji; Takei, Yoshio

    2003-02-01

    Steroidogenic acute regulatory protein (StAR) is a key molecule for steroid production by translocating cholesterol from the outer to inner mitochondrial membrane. Two cDNAs of different length encoding StAR was cloned from the head kidney of the eel (Anguilla japonica). In the 3'-untranslated region (UTR) of the longer cDNA, two putative polyadenylation signals were found. The shorter one differed from the longer one solely by the lack of middle of 3'-UTR including the first polyadenylation signal. Reverse transcription-polymerase chain reaction (RT-PCR) that differentiates the two mRNAs showed that the ratio of the two was highly variable among individuals, and no preferential expression was detected between freshwater and seawater eels. The predicted protein consists of 285 amino acid residues with 64-83% identity to other StARs thus far obtained. RT-PCR analyses revealed that eel StAR mRNA was expressed abundantly in the head kidney and gonad, and faintly in the brain; but no expression was detected in the gill, heart, liver, intestine, kidney and skeletal muscle. Plasma cortisol concentration increased, but StAR mRNA content in the head kidney did not change, 3 and 24 h after transfer of freshwater eels to seawater, indicating that the transcriptional regulation of StAR may not be involved in cortisol production after seawater transfer. However, ACTH elevated both plasma cortisol and StAR mRNA levels in the head kidney 1.5 and 4.5 h after injection. Thus, the steroidogenic effect of ACTH is mediated by increased StAR production as observed in mammals. PMID:12655184

  16. cDNA cloning and molecular characterization of a defensin-like antimicrobial peptide from larvae of Protaetia brevitarsis seulensis (Kolbe).

    PubMed

    Lee, Jiae; Bang, Kyeongrin; Hwang, Sejung; Cho, Saeyoull

    2016-05-01

    We identified new defensin-like cDNA (called Psdefensin) by searching data set of high-throughput RNA sequencing (RNA-seq) expression profiling of immunized larva of white-spotted flower chafers, Protaetia brevitarsis seulensis. The length of the analyzed new defensin-like sequences were 240 base pair (bp) and encoded the deduced polypeptide of 79 amino acid residues with signal peptides (amino acids 1-20), pro-peptide region (amino acids 21-36), and mature peptide region (amino acids 37-79). The Psdefensin transcript levels were slightly up-regulated at 4 h post-infection and were highly expressed at 8 h post-infection compared to control larvae injected with sterile water. In addition, the Psdefensin did have antimicrobial activity against both Gram-negative bacteria, E. coli and Gram-positive bacteria, B. subtilis suggesting potentially pharmacologic agent. PMID:26970764

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

    PubMed

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

    1996-08-01

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

  18. 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

    SciTech Connect

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. |

    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.

  19. Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis.

    PubMed

    Hung, Chih-Liang; Chang, An-Ju; Kuo, Xhao-Kai; Sheu, Fuu

    2014-02-19

    Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway. PMID:24400969

  20. Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies ▿ †

    PubMed Central

    Gottwein, Judith M.; Scheel, Troels K. H.; Callendret, Benoit; Li, Yi-Ping; Eccleston, Heather B.; Engle, Ronald E.; Govindarajan, Sugantha; Satterfield, William; Purcell, Robert H.; Walker, Christopher M.; Bukh, Jens

    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 resulted in robust infection with peak HCV RNA titers of ∼5.5 log10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes. PMID:20200247

  1. Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex

    SciTech Connect

    Zain, S.B.; Salim, M.; Chou, W.G.; Sajdel-Sulkowska, E.M.; Majocha, R.E.; Marotta, C.A.

    1988-02-01

    To gain insight into factors associated with the excessive accumulation of ..beta..-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, the authors cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)/sup +/ RNA from AD cortices was used for the preparation of lambdagt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the ..beta..-amyloid domain and corresponded to 75% of the coding region and approx. = 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, they conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

  2. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    PubMed Central

    Liu, Xiao-Jing; Yang, Li; Luo, Feng-Ming; Wu, Hong-Bin; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis. METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted, quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR). RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation, apoptosis, and DNA damage response. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis. PMID:15162533

  3. Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species.

    PubMed

    Shimizu, Milton Massao; Melo, Geraldo Aclécio; Brombini Dos Santos, Adriana; Bottcher, Alexandra; Cesarino, Igor; Araújo, Pedro; Magalhães Silva Moura, Jullyana Cristina; Mazzafera, Paulo

    2011-09-01

    Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid. PMID:21530289

  4. Isolation and identification of the cDNA encoding the pheromone biosynthesis activating neuropeptide and additional neuropeptides in the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae).

    PubMed

    Choi, M Y; Tanaka, M; Kataoka, H; Boo, K S; Tatsuki, S

    1998-10-01

    The present study is concerned with cloning and characterizing Has-PBAN cDNA which is 756 nucleotides long, isolated from the brain and suboesophageal ganglion complex (Br-Sg) of Helicoverpa assulta adults. The 194-amino acid sequence deduced from this cDNA possessed the proteolytic endocleavage sites to generate multiple peptides. From the processing of the prepro-hormone, it can be predicted that the cDNA has a PBAN domain with 33 amino acids and four additional peptide domains: 24 amino acid-, 7 amino acid-, 18 amino acid- and 8 amino acid-long sequences, with FXPR (or K) L (X = G, T or S) amidated at their C-termini. The amino acid sequence of all five predicted peptides, including the PBAN, are identical to that of Helicoverpa zea (Raina, A.K., Jaffe, H., Kempe, T.G., Keim, P., Blacher, R.W., Fales, H.M., Riley, C.T., Klun, J.A., Ridgway, R.L., Hayes, D.K., 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244, 796-798 and Ma, P.W.K., Knipple, D.C., Roelofs, W.L., 1994. Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides. Proc. Natl. Acad. Sci., U.S.A. 91, 506-510). A single mRNA species corresponding to the size of Has-PBAN cDNA was detected from the Br-Sg of 1-3-day old female and male adults, and their expression was also at a similar level. Pheromone production was induced upon injection of female or male Br-Sg extracts or synthetic PBAN into the haemocoel of decapitated 1-3-day old female adults during the photophase when they are not supposed to produce pheromone. From these results, H. assulta adult females seem to use their own PBAN for regulating sex pheromone biosynthesis. Functions of the four other peptides ending with FXPR (or K) L in the Has-PBAN cDNA and of the male PBAN remain to be elucidated. PMID:9807222

  5. The cloning and characterization of a second brain enzyme with NAAG peptidase activity.

    PubMed

    Bzdega, Tomasz; Crowe, Samantha L; Ramadan, Epolia R; Sciarretta, Kathryn H; Olszewski, Rafal T; Ojeifo, Olumide A; Rafalski, Victoria A; Wroblewska, Barbara; Neale, Joseph H

    2004-05-01

    The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG. PMID:15086519

  6. Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA.

    PubMed

    Wongchawalit, Jintanart; Yamamoto, Takeshi; Nakai, Hiroyuki; Kim, Young-Min; Sato, Natsuko; Nishimoto, Mamoru; Okuyama, Masayuki; Mori, Haruhide; Saji, Osamu; Chanchao, Chanpen; Wongsiri, Siriwat; Surarit, Rudee; Svasti, Jisnuson; Chiba, Seiya; Kimura, Atsuo

    2006-12-01

    alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I. PMID:17151473

  7. 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.

    PubMed

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

    2016-06-01

    A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus. PMID:27234555

  8. Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase.

    PubMed

    Kojima, N; Sitthithaworn, W; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Sankaw, U

    2000-07-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs. PMID:10923851

  9. cDNA cloning of two isoforms of ornithine carbamoyltransferase from Canavalia lineata leaves and the effect of site-directed mutagenesis of the carbamoyl phosphate binding site.

    PubMed

    Lee, Y; Choi, Y A; Hwang, I D; Kim, S G; Kwon, Y M

    2001-08-01

    The immunoscreening method was used to isolate cDNAs of 1323 bp (ClOCT1) and 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) from the cDNA expression library of Canavalia lineata leaves constructed in a lambdaZAP Express vector. ClOCT1 and ClOCT2 encode 359 and 369 amino acids, respectively. The N-terminals of deduced amino acid sequences of the two cDNAs showed typical features of the transit peptide of chloroplast targeting proteins. The ornithine-binding domain (FMHCLP) and catalytic domain (HPXQ) of ClOCT1 and ClOCT2 and the carbamoyl phosphate (CP)-binding site of ClOCT1 (SMRTR) are identical to OCTs of other plant species, pea and Arabidopsis thaliana. However, the CP-binding site sequence of ClOCT2, SLRTH, has not yet been reported. Both ClOCT1 and ClOCT2 cDNAs were expressed in Escherichia coli BL21 (DE3) by using expression vector pET30a. Recombinant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity. In contrast, recombinant ClOCT2 protein showed 13 times higher canaline-dependent OCT activity than ornithine-dependent OCT activity. The two amino acids of the CP-binding site of ClOCT2 (SLRTH) were combinatorially changed to those of the CP-binding site of ClOCT1 (SMRTR) by site-directed mutagenesis. When Leu-118 of ClOCT2 was changed to Met, ornithine-dependent activity was increased significantly. It is assumed that the substrate specificity of ClOCT1 or ClOCT2 proteins partially depends on the amino acid sequence of the CP-binding site. PMID:11575720

  10. Characterization of two novel pacifastin-like peptide precursor isoforms in the desert locust (Schistocerca gregaria): cDNA cloning, functional analysis and real-time RT-PCR gene expression studies.

    PubMed

    Simonet, Gert; Breugelmans, Bert; Proost, Paul; Claeys, Ilse; Van Damme, Jozef; De Loof, Arnold; Vanden Broeck, Jozef

    2005-05-15

    In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1-3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism. PMID:15631618

  11. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    SciTech Connect

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

    1987-12-01

    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  12. Characterization and cDNA sequence of Bothriechis schlegeliil-amino acid oxidase with antibacterial activity.

    PubMed

    Vargas Muñoz, Leidy Johana; Estrada-Gomez, Sebastian; Núñez, Vitelbina; Sanz, Libia; Calvete, Juan J

    2014-08-01

    Snake venoms are complex mixtures of proteins including l-amino acid oxidase (lAAO). A lAAO (named BslAAO) with a mass of 56kDa and a theoretical Ip of 5.79, was purified from Bothriechis schlegelii venom through size-exclusion, ion exchange and affinity chromatography. The entire protein sequence of 498 amino acids, was determined from cDNA using reverse-transcribed mRNA isolated from venom gland. The enzyme showed dose-dependent inhibition of bacterial growth. BslAAO showed inhibitory effect against S. aureus with a MIC of 4μg/mL and a MBC of 8μg/mL. Against Acinetobacter baumannii, showed a MIC of 2μg/mL and MBC of 4μg/mL, No effect was observed in Escherichia coli. This antibacterial activity was inhibited by catalase, indicating that antimicrobial activity was due to H2O2 production. BslAAO did not show any cytotoxic activity toward mouse myoblast cell line C2C12 or peripheral blood mononuclear cells. The enzyme oxidated l-Leu, with a Km of 16.37μM and a Vmax of 0.39μM/min. Snake venoms lAAOs, are potential frames of different therapeutics molecules since these enzymes exhibit low MICs and MBCs and show to be harmless to human cells due to microorganisms being generally several fold more sensitive to reactive oxygen species than human tissues. PMID:24875315

  13. Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    PubMed Central

    Yang, Xueqing; Li, Xianchun; Zhang, Yalin

    2013-01-01

    Cytochrome P450 monooxygenases (CYPs or P450s) play paramount roles in detoxification of insecticides in a number of insect pests. However, little is known about the roles of P450s and their responses to insecticide exposure in the codling moth Cydia pomonella (L.), an economically important fruit pest. Here we report the characterization and expression analysis of the first P450 gene, designated as CYP9A61, from this pest. The full-length cDNA sequence of CYP9A61 is 2071 bp long and its open reading frame (ORF) encodes 538 amino acids. Sequence analysis shows that CYP9A61 shares 51%–60% identity with other known CYP9s and contains the highly conserved substrate recognition site SRS1, SRS4 and SRS5. Quantitative real-time PCR showed that CYP9A61 were 67-fold higher in the fifth instar larvae than in the first instar, and more abundant in the silk gland and fat body than other tissues. Exposure of the 3rd instar larvae to 12.5 mg L−1 of chlorpyrifos-ethyl for 60 h and 0.19 mg L−1 of lambda-cyhalothrin for 36 h resulted in 2.20-and 3.47-fold induction of CYP9A61, respectively. Exposure of the 3rd instar larvae to these two insecticides also significantly enhanced the total P450 activity. The results suggested that CYP9A61 is an insecticide-detoxifying P450. PMID:24351812

  14. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

    PubMed

    Fang, Ying; Rowland, Raymond R R; Roof, Michael; Lunney, Joan K; Christopher-Hennings, Jane; Nelson, Eric A

    2006-12-01

    The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States. PMID:16971421

  15. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

    PubMed Central

    Olsson, Per-Erik; Berg, A Håkan; von Hofsten, Jonas; Grahn, Birgitta; Hellqvist, Anna; Larsson, Anders; Karlsson, Johnny; Modig, Carina; Borg, Bertil; Thomas, Peter

    2005-01-01

    Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal. PMID:16107211

  16. cDNA cloning and gene structure of a novel water channel expressed exclusively in human kidney: Evidence for a gene cluster of aquaporins at chromosome locus 12q13

    SciTech Connect

    Ma, Tonghui; Yang, Baoxue; Verkman, A.S.

    1996-08-01

    A 1.8-kb cDNA clone (designed hKID, gene symbol AQP2L) with homology to the aquaporins was isolated from a human kidney cDNA library. The longest open reading frame of 846 bp encoded a 282-amino-acid hydrophobic protein that contained the conserved NPA motifs of MIP family members. Cell-free translation produced a nonglycosylated protein migrating at 29 kDa. Northern blot analysis revealed a 2.2-kb transcript expressed only in human kidney. PCR/Southern blot analysis of human kidney cDNA using primers flanking the hKID coding sequence revealed expression of a full-length mRNA and short transcripts with partial exon 1 and partial exon 4 deletions. Genomic Southern blot indicted a single-copy hKID gene. PCR analysis of a human/rodent somatic hybrid panel localized the hKID gene to chromosome 12. Chromosomal fluorescence in situ hybridization mapped the hKID (AQP2L) gene to chromosome locus 12q13, the same location a as the AQP-2 and MIP genes. The high sequence homology, similar genomic structure, and identical chromosomal loci of hKID, MIP, and AQP-2 suggest a MIP family gene cluster at chromosome locus 12q13. Further work is needed to establish the physiological significance of hKID. 43 refs., 6 figs.

  17. Construction of an infectious cDNA clone of foot-and-mouth disease virus type O 1 BFS 1860 and its use in the preparation of candidate vaccine.

    PubMed

    Hema, M; Chandran, D; Nagendrakumar, S B; Madhanmohan, M; Srinivasan, V A

    2009-03-01

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable,full-length cDNA clone of FMDV type O 1 BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEM R- - 7Zf(-). An 8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription.Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA,RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines. PMID:19430118

  18. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    PubMed Central

    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

  19. Channel Catfish (Ictalurus punctatus Rafinesque, 1818) CD156a (ADAM Metallopeptidase Domain 8): cDNA Clone, Characterization and Expression in Tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CD156a, also known as a disintegrin and metalloprotease domain 8 (ADAM-8), is a type 1 transmembrane glycoprotein of the ADAM family. This protein plays important roles in immune and other physiological functions. In this communication, the channel catfish CD156a cDNA was characterized and its exp...

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

    SciTech Connect

    Gronwald, R.G.K.; 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. )

    1988-05-01

    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){sup +} RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an {approx} 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.

  1. Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

    PubMed Central

    Laplante, J M; O'Rourke, F; Lu, X; Fein, A; Olsen, A; Feinstein, M B

    2000-01-01

    A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation. PMID:10794731

  2. Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

    PubMed

    Gholizadeh, A; Kohnehrouz, B B

    2009-02-01

    D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717. PMID:19267668

  3. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

    PubMed Central

    Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

    1988-01-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

  4. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation

    SciTech Connect

    Ninomiya-Tsuji, Jun ); Nomoto, Satoshi; Matsumoto, Kunihiro ); Yasuda, Hideyo ); Reed, S.I. )

    1991-10-15

    The authors have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34{sup CDC2} kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34{sup CDC2} kinase and more significantly is homologous to Xenopus Eg1 kinase, suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34{sup CDC2} kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G{sub 1} or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages.

  5. Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae).

    PubMed

    Lufrano, Daniela; Faro, Rosário; Castanheira, Pedro; Parisi, Gustavo; Veríssimo, Paula; Vairo-Cavalli, Sandra; Simões, Isaura; Faro, Carlos

    2012-09-01

    Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases. PMID:22727116

  6. Cloning and expression of buffalo active chymosin in Pichia pastoris.

    PubMed

    Vallejo, Juan Andres; Ageitos, Jose Manuel; Poza, Margarita; Villa, Tomas G

    2008-11-26

    To date, only recombinant chymosin has been obtained in its active form from supernatants of filamentous fungi, which are not as good candidates as yeasts for large-scale fermentations. Since Bos taurus chymosin was cloned and expressed, the world demand for this protease has increased to such an extent that the cheesemaking industry has been looking for novel sources of chymosin. In this sense because buffalo chymosin has properties that are more stable than those of B. taurus chymosin, it may occupy a space of its own in the chymosin market. The main objective of the present work was the production of active recombinant buffalo chymosin in the culture supernatant of Pichia pastoris . This yeast has demonstrated its usefulness as an excellent large-scale fermentation tool for the secretion of recombinant foreign proteins. RNA was extracted from the abomasum of a suckling calf water buffalo ( Bubalus arnee bubalis ). Preprochymosin, prochymosin, and chymosin DNA sequences were isolated and expressed into P. pastoris. Only the recombinant clones of P. pastoris containing the prochymosin sequence gene were able to secrete the active form of the chymosin to the culture supernatant. This paper describes for the first time the production of active recombinant chymosin in P. pastoris without the need of a previous in vitro activation. The new recombinant yeast strain could represent a novel and excellent source of rennet for the cheesemaking industry. PMID:18975968

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

    SciTech Connect

    Goedert, M.; Wischik, C.M.; Crowther, R.A.; Walker, J.E.; Klug, A. )

    1988-06-01

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

  8. The MyoD family of myogenic factors is regulated by electrical activity: isolation and characterization of a mouse Myf-5 cDNA.

    PubMed Central

    Buonanno, A; Apone, L; Morasso, M I; Beers, R; Brenner, H R; Eftimie, R

    1992-01-01

    A full-length cDNA coding for a homolog of the human Myf-5 was isolated from a BC3H-1 mouse library and characterized. The clone codes for a protein of 255 amino acids that is 89%, 88% and 68% identical to the human, bovine and Xenopus myf-5, respectively. The mouse Myf-5 cDNA (mmyf-5), as well as sequences coding for MyoD, myogenin and Mrf-4, were used to probe Northern blots to analyze the effects of innervation on the expression of the MyoD family of myogenic factors. Mouse myf-5, MyoD and myogenin mRNAs levels were found to decline in hind limb muscles of mice between embryonic day 15 (E15) and the first postnatal week, a period that coincides with innervation. In contrast, Mrf-4 transcripts increase during this period and reach steady-state levels by 1-week after birth. To distinguish if the changes in myogenic factor expression are due to a developmental program or to innervation, mRNA levels were analyzed at different times after muscle denervation. Mmyf-5 transcripts begin to accumulate 2 days postdenervation; after 1 week levels are 7-fold higher than in innervated muscle. Mrf-4, MyoD and myogenin transcripts begin to accumulate as soon as 8h after denervation, and attain levels that are 8-, 15- and 40-fold higher than found in innervated skeletal muscle, respectively. The accumulation of these three mRNAs precedes the increase of nicotinic acetylcholine receptor alpha subunit transcripts, a gene that is transcriptionally regulated by MyoD-related factors in vitro. Using extracellular electrodes to directly stimulate in situ the soleus muscle of rats, we found that 'electrical activity' per se, in absence of the nerve, represses the increases of myogenic factor mRNAs associated with denervation. Images PMID:1741288

  9. Cloning and Characterization of the Antiviral Activity of Feline Tetherin/BST-2

    PubMed Central

    Fukuma, Aiko; Abe, Masumi; Morikawa, Yuko; Miyazawa, Takayuki; Yasuda, Jiro

    2011-01-01

    Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection. PMID:21479233

  10. Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2.

    PubMed

    Chen, Lei-Lei; Liu, Li-Jun; Shi, Mei; Song, Xiao-Yan; Zheng, Chang-Ying; Chen, Xiu-Lan; Zhang, Yu-Zhong

    2009-10-01

    Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60-65 degrees C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59-69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma. PMID:19702879

  11. Cloning and heterologous expression of SS10, a subtilisin-like protease displaying antifungal activity from Trichoderma harzianum.

    PubMed

    Yan, Liu; Qian, Yang

    2009-01-01

    Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene (SS10) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5' and 3' rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL(-1)) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 degrees C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma. PMID:19025577

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

    PubMed

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

    2014-10-01

    Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. PMID:25038276

  13. Structural defect linked to nonrandom mutations in the matrix gene of Biden strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes

    SciTech Connect

    Ayata, M.; Hirano, A.; Wong, T.C.

    1989-03-01

    Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 M/sub r/ protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general.

  14. Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

    PubMed Central

    DeJong, J; Bernstein, R; Roeder, R G

    1995-01-01

    The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7724559

  15. Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.

    PubMed Central

    Hemmi, S; Peghini, P; Metzler, M; Merlin, G; Dembic, Z; Aguet, M

    1989-01-01

    A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction. Images PMID:2532365

  16. Molecular cloning, expression and bioactivity of B cell activating factor (BAFF) in African ostrich.

    PubMed

    Yang, Keli; Xiao, Ke; Huang, Haibo; Lu, Shun; Zhong, Juming; Ansari, Abdur Rahman; Khaliq, Haseeb; Song, Hui; Liu, Huazhen; Peng, Kemei

    2015-09-01

    B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds. PMID:26256697

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

    PubMed

    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. PMID:17286044

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

    PubMed

    Shanthi, S; Vaseeharan, B

    2012-03-20

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

  19. Molecular cloning, cDNA analysis, and localization of a monomer of the N-acetylglucosamine-specific receptor of the thyroid, NAGR1, to chromosome 19p13. 3-13. 2

    SciTech Connect

    Blanck, O.; Perrin, C.; Mziaut, H.; Miquelis, R. ); Darbon, H. ); Mattei, M.G. )

    1994-05-01

    The authors have proposed that the GlcNAc thyroid receptor triggers selective recycling of immature GlcNAc-bearing thyroglobulin molecules through the Golgi back to the apical membrane for further processing until maturation is achieved. This process, which is called [open quotes]receptor-mediated exocytosis,[close quotes] prevents lysosomal degradation of thyroid prohormones. In the present study, the authors report cloning of the cDNA encoding the (or one of the) monomer(s) constituting the human GlcNAc thyroid receptor. This novel gene, called NAGR1, was assigned by in situ hybridization to subbands p13.3-p13.2 of chromosome 19. Northern blot analysis showed that the mRNA encoding NAGR1 was present as a single transcript of 2.1 kb in the thyroid, but not in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The deduced amino acid sequence comprised a 51-kDa type I membrane protein with a single spanning region and a short intracytoplasmic domain. Sequence analysis showed that NAGR1 is a glycine-, tryptophan-, and methionine-rich protein with no cysteine residues or glycosylation site. No sequence homology with any known cDNA or protein was noted. The extracellular domain is composed of 420 amino acids and contains a region of 204 residues showing 15 repeats of 4 amino acids, each 1 having an acidic amino acid presumably involved in calcium coordination. The intracellular domain contained what appeared to be a tyrosine internalization signal. The usefulness of this clone in glycobiology, cell biology, and thyroid pathology studies is discussed. 53 refs., 6 figs.

  20. Molecular cloning, recombinant expression and antibacterial activity analysis of hepcidin from Simensis crocodile (Crocodylus siamensis).

    PubMed

    Hao, Juan; Li, Yan-Wei; Xie, Ming-Quan; Li, An-Xing

    2012-01-01

    Hepcidin, a cysteine-rich cationic antibacterial peptide, plays an important role in human defense against pathogen infection. However, its role in reptile immune response and whether it is involved in antibacterial immune have not yet been proven. In order to study the antibacterial activity of Crocodylus siamensis hepcidin (Cshepc), a common reptile which lives in topic region of Southeast Asia, a cDNA sequence of Cshepc was cloned, which included an open reading frame (ORF) of 300 bp encoding a 99 amino acid preprohepcidin. Cshepc has eight cysteines formed four conserved disulfide bridges, similarly to that of human's. Sequence analysis showed that Cshepc mature peptide was more conserved than that of preprohepcidin. Tissue expression analysis indicated that Cshepc transcripts were highly expressed in the liver, muscle and heart of C. siamensis. Recombinant expressed hepcidin could significantly inhibit the growth of the Gram-negative bacteria Escherichia coli and Aeromonas sobria as well as the Gram-positive bacterium Staphylococcus aureus, and Bacillus subtilis in vitro, suggesting that Cshepc, like human hepcidin could play a role in the antibacterial function in hosts innate immune response. PMID:22967859

  1. Isolation, cDNA cloning, and characterization of an 18-kDa hemagglutinin and amebocyte aggregation factor from Limulus polyphemus.

    PubMed

    Fujii, N; Minetti, C A; Nakhasi, H L; Chen, S W; Barbehenn, E; Nunes, P H; Nguyen, N Y

    1992-11-01

    An 18-kDa hemagglutinin which possesses the property of inducing both aggregation of amebocytes and agglutination of erythrocytes has been isolated from Limulus polyphemus amebocytes and purified by ion exchange chromatography. This nonglycosylated, single chain polypeptide with an M(r) of 18,506 and isoelectric point of 8.3 is stored exclusively in the large secretory granules of amebocytes. Based on the partial N-terminal amino acid sequence of 63 residues, DNA probes have been synthesized for screening a pBR322 cDNA library constructed from Limulus amebocytes. The cDNA coding for this protein reveals the presence of a 19-residue signal peptide preceding the 153-residue open reading frame. Northern blot analysis indicates the presence of a single mRNA species. The primary structure derived from the corresponding cDNA sequence reveals an internal homology consisting of two consensus sequences, Val-Asn-Asp/Ser-Trp-Asp and Glu-Asp-Arg-Arg-Trp. The formation of 5 disulfide bonds between 10 half-cysteines divides the molecule into three looped domains each containing the Glu-Asp-Arg-Arg-Trp repeating unit. One of the novel features of this protein is that it shares 37% identity with a 22-kDa mammalian extracellular matrix protein isolated from fetal bovine skin (Neame, P.J., Choi, H.U., and Rosenberg, L.C. (1989) J. Biol. Chem. 264, 5474-5479). The two proteins exhibit a similar pattern of looped domains, each domain containing a homologous consensus sequence (i.e. Glu-Asp-Arg-Arg-Trp). The overall structure of both proteins seems to be highly related, with the exception of an N-terminal tyrosine-rich region present only in the mammalian extracellular matrix protein. The functional properties of the two proteins are similar in that the Limulus 18-kDa protein agglutinates horse erythrocytes and aggregates Limulus amebocytes, and the mammalian 22-kDa protein is an effective adhesion promoter for dermal fibroblasts. On the basis of these unique properties, the newly

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

    SciTech Connect

    Yu, L.M.

    1988-01-01

    The chloroplast ATP synthase was partially purified from the green alga Chlamydomonas reinhardtii by extracting membranes with deoxycholate and KCl, followed by centrifugation and ammonium sulfate fractionation of the supernatant. The enzyme assay involved the reconstitution of such fractions with bacteriorhodopsin and soybean phospholipids to form vesicles capable of light-dependent ({sup 32}P)-phosphate esterification. A cDNA for the gamma subunit from Chlamydomonas was isolated, expressed in vitro and sequenced. It contains the entire coding region for the gamma subunit precursor. A 35 amino acid long transit peptide resides at the NH{sub 2}-terminus of a 323 amino acid long mature peptide that is 77% similar to the spinach gamma subunit. Six cysteines were found; three were conserved in Chlamydomonas and spinach.

  3. Characterization of a human glycoprotein with a potential role in sperm-egg fusion: cDNA cloning, immunohistochemical localization, and chromosomal assignment of the gene (AEGL1)

    SciTech Connect

    Hayashi, Masaru; Fujimoto, Seiichiro; Takano, Hiroko

    1996-03-05

    Acidic epididymal glycoprotein (AEG), thus far identified only in rodents, is one of the sperm surface proteins involved in the fusion of the sperm and egg plasma membranes. In the present study, we describe the isolation and characterization of cDNA encoding a human glycoprotein related to AEG. Although this protein, designated ARP (AEG-related protein), is not the ortholog of rodent AEG, it resembles AEG in that it is an epididymal secretory glycoprotein that binds to the postacrosomal region of the sperm head. The fact that no AEG mRNA can be detected in the human epididymis suggests that ARP might be the functional counterpart of rodent AEG. The gene encoding ARP (AEGL1) was mapped by fluorescence in situ hybridization to 6p21.1-p21.2. This result indicates that AEGL1 and the mouse gene for AEG are located in the chromosomal segments with conserved syntenies. 43 refs., 6 figs.

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

    PubMed

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

    1999-01-18

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

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

    SciTech Connect

    Chen, Haiming; Morris, M.A.; Rossier, C.

    1995-08-10

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

  6. Cloning and stable expression of a new member of the human liver phenol/bilirubin: UDP-glucuronosyltransferase cDNA family.

    PubMed Central

    Wooster, R; Sutherland, L; Ebner, T; Clarke, D; Da Cruz e Silva, O; Burchell, B

    1991-01-01

    A new human liver UDP-glucuronosyltransferase (HlugP4) has been cloned and expressed in cell culture. The expressed enzyme has a molecular mass of 56 kDa and preferentially catalysed the glucuronidation of halogenated and bulky alkyl phenols. The C-terminal half of the sequence (246 amino acids) is 96% identical with the same portion of HlugP1, whereas the N-terminal half of the deduced protein sequences are only 38% identical. These results suggest that the two isoenzymes may be derived from the same gene by differential splicing of the gene product. Images Fig. 2. PMID:1910331

  7. Effects of chlorpyrifos on the transcription of CYP3A cDNA, activity of acetylcholinesterase, and oxidative stress response of goldfish (Carassius auratus).

    PubMed

    Ma, Junguo; Liu, Yang; Niu, Daichun; Li, Xiaoyu

    2015-04-01

    Chlorpyrifos (CPF) is the widely used organophosphate pesticide in agriculture throughout the world. It has been found that CPF is relatively safe to human but highly toxic to fish. In this study, acute toxicity of CPF on goldfish was determined and then the transcription of goldfish cytochrome P450 (CYP) 3A was evaluated after 96 h of CPF exposure at concentrations of 15.3 [1/10 50% lethal concentration (LC50 )] or 51 μg L(-1) (1/3 LC50 ) of CPF. Meanwhile, the enzymatic activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), and catalase (CAT), total antioxidant activity (T-AOC), and the contents of malondialdehyde (MDA) in the liver or brain of goldfish were also determined. The results of acute toxicity testing showed that the 96-h LC50 of CPF to the goldfish was 153 μg L(-1) . Moreover, a length sequence of 1243 bp CYP3A cDNA encoding for 413 amino acids from goldfish liver was cloned. Polymerase chain reaction results reveal that CPF exposure downregulates CYP 3A transcription in goldfish liver, suggesting that goldfish CYP 3A may be not involved in CPF bioactivation. Finally, the results of biochemical assays indicate that 96 h of CPF exposure remarkably inhibits AChE activity in fish liver or brain, alters hepatic antioxidant enzyme activities, decreases brain T-AOC, and causes lipid peroxidation in fish liver. These results suggest that oxidative stress might be involved in CPF toxicity on goldfish. PMID:24190793

  8. Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

    PubMed Central

    Mendez, Ernesto; Ruggli, Nicolas; Collett, Marc S.; Rice, Charles M.

    1998-01-01

    Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>105 PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity. PMID:9573238

  9. Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS

    SciTech Connect

    Onodera, Osamu; Oyake, Mutsuo; Takano, Hiroki

    1995-11-01

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by genetic anticipation and variable combinations of symptoms including myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a gene on the short arm of chromosome 12. We determined the consensus DRPLA cDNA sequence containing the complete coding region for 1,185 amino acids. The CAG repeat, which is expanded in DRPLA, is located 1,462 bp downstream from the putative methionine initiation codon and encodes a poly-glutamine tract. Although poly-serine and proline tracts exist near the CAG repeats, these poly-serine or proline tracts did not show any polymorphisms, which is in strong contrast to the high heterogeneity in the length of the CAG repeat. Northern blot analysis revealed a 4.7-kb transcript that is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. Reverse transcription-PCR analysis revealed that the expanded alleles are transcribed to levels comparable to those of normal alleles. These results indicate that there is no difference in transcriptional efficiency between expanded and normal alleles. Furthermore, mRNA from cerebellar hemispheres of DRPLA patients showed smaller sizes of CAG repeats compared with other regions of the brain, which reflects somatic mosaicism of the expanded alleles of the DRPLA gene. 49 refs., 6 figs.

  10. SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river.

    PubMed

    Bigot, Aurélie; Vasseur, Paule; Rodius, François

    2010-02-01

    The cDNA sequences encoding manganese superoxide dismutase (Mn-SOD) and catalase (CAT) were isolated in the freshwater bivalve Unio tumidus by reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers. Quantitative real-time PCR approach was used to evaluate the mRNA expression patterns of SOD, CAT, selenium-dependent glutathione peroxidase (Se-GPx), pi class glutathione S-transferase (pi-GST) and metallothionein (MT), in the digestive gland of Unio tumidus transplanted from a control site to four stations in the Moselle River (M1-M4), for periods of 8 and 21 days. These sites were chosen upstream and downstream of populated areas. Chemical analysis performed on sediments from the Moselle river sites did not show high levels of pollutants. Decrease of SOD, CAT, Se-GPx and MT mRNA levels were observed at M3 site after a 21-day exposure compared to control site. These results suggest inefficiency of antioxidant systems affected by cytotoxic mechanisms and confirm an environmental perturbation. Organisms transplanted at M4 site showed a strong increase of biomarkers transcription levels after 21 days of exposure. These inductions could correspond to an adaptive response to an altered environment. Our results showed that biological approaches using multibiomarkers appear as essential tools complementary to measurement of contaminants, to detect environmental degradations. PMID:19784772

  11. Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides*

    PubMed Central

    Engels, Benedikt; Heinig, Uwe; Grothe, Torsten; Stadler, Marc; Jennewein, Stefan

    2011-01-01

    Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6–7 double bond into the 7–8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns. PMID:21148562

  12. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus

    PubMed Central

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78–4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  13. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus.

    PubMed

    Niu, Rongli; Chen, Xiang

    2016-01-01

    Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78-4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. PMID:27471730

  14. Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

    PubMed Central

    Beall, Anne; Yount, Boyd; Lin, Chun-Ming; Hou, Yixuan; Wang, Qiuhong; Saif, Linda

    2016-01-01

    ABSTRACT Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. PMID:26733065

  15. Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

    PubMed

    Weis, J J; Fearon, D T; Klickstein, L B; Wong, W W; Richards, S A; de Bruyn Kops, A; Smith, J A; Weis, J H

    1986-08-01

    Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2. PMID:3016712

  16. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin.

    PubMed Central

    Bobak, D A; Nightingale, M S; Murtagh, J J; Price, S R; Moss, J; Vaughan, M

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFs also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs. Images PMID:2474826

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

    SciTech Connect

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A.

    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.

  18. CLONING AND EXPRESSION OF RABBIT INTERLEUKIN-15

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA co...

  19. Cloning and Expression of Yak Active Chymosin in Pichia pastoris.

    PubMed

    Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

    2016-09-01

    Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

  20. Cloning and Expression of Yak Active Chymosin in Pichia pastoris

    PubMed Central

    Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

    2016-01-01

    Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

  1. Grazing-Activated Production of Dimethyl Sulfide (DMS) by two clones of Emiliania huxleyi

    NASA Technical Reports Server (NTRS)

    Wolfe, Gordon V.; Steinke, Michael

    1996-01-01

    Emiliania huxleyi clones CCMP 370 and CCMP 373 produced similar amounts of dimethylsulfoniopropionate (DMSP) during axenic exponential growth, averaging 109 mM internal DMSP. Both clones had detectable DMSP lyase activity, as measured by production of dimethyl sulfide (DMS) during in vitro assays of crude cell preparations, but activities and conditions differed considerably between clones. Clone 373 had high activity; clone 370 had low activity and required chloride. For both strains, enzyme activity per cell was constant during exponential growth, but little DMS was produced by healthy cells. Rather, DMS production was activated when cells were subjected to physical or chemical stresses that caused cell lysis. We propose that DMSP lyase and DMSP are segregated within these cells and re-action only under conditions that result in cell stress or damage. Such activation occurs during microzooplankton grazing. When these clones were grazed by the dinoflagellate Oxyrrhis marina, DMS was produced; ungrazed cells, as well as those exposed to grazer exudates and associated bacteria, generated no DMS. Grazing of clone 373 produced much more DMS than grazing of clone 370, consistent with their relative in vitro DMSP lyase activities. DMS was only generated when cells were actually being grazed, indicating that ingested cells were responsible for the DMS formation. We suggest that even low levels of grazing can greatly accelerate DMS production.

  2. Molecular cloning, tissue distribution and ontogenetic expression of the amino acid transporter b(0,+) cDNA in the small intestine of Tibetan suckling piglets.

    PubMed

    Wang, Wence; Gu, Wanting; Tang, Xiangfang; Geng, Meimei; Fan, M; Li, Tiejun; Chu, Wuying; Shi, Changyou; Huang, Ruilin; Zhang, Hongfu; Yin, Yulong

    2009-09-01

    The small intestine is the main absorption place of peptides and free amino acids in mammals. The amino acid transporter system b(0,+) mediates apical uptake of basic amino acids, especially lysine, arginine and cysteine. The aim of the current study was to clone Tibetan porcine amino acid transporter b(0,+)AT (SLC7A9) for comparing the sequences of Tibetan and common (Sus scrofa) pigs, and investigating the tissue distribution and ontogenetic expression in the small intestine of Tibetan suckling piglets. The Tibetan porcine SLC7A9 gene was first cloned from the porcine small intestine and found to encode the amino acid transporter b(0,+)AT. The entire open reading frame (ORF) of the SLC7A9 is 1464 bp and codes for 487 amino acid residues, with a higher degree of sequence similarity with common pig (99.59%) and horse counterparts (91.2%) than with monkey (89.5%) or human (88.7%). The deduced protein has 12 putative transmembrane domains. In this study, SLC7A9 mRNA was detected in brain, kidney, duodenum, jejunum, ileum, heart, liver, lung and muscle from Tibetan pigs at 7 and 21 days by PCR. We also investigated the age-dependent expression of b(0,+)AT in Tibetan suckling piglets in duodenum, anterior jejunum, posterior jejunum, ileum and kidney from day 1 to 35. The abundance of SLC7A9 mRNA in duodenum and jejunum was highest and lowest, respectively. Expression patterns were similar in duodenum and anterior jejunum, where the mRNA level was decreased before the suckling period and increased until day 35. Posterior jejunum expression was increasing steadily with age, except on day 7. The ileum has the highest expression at day 14 and became steady after day 28. The mRNA abundance in the kidney is opposite to duodenum, increasing until day 14 and reducing thereafter. Our results showed the pattern of b(0,+)AT expressed in small intestine of Tibetan pig and lay the foundation for in depth investigations of the regulation of b(0,+)AT in vivo. PMID:19501668

  3. Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp.

    PubMed

    Yu, E M; Liu, B H; Wang, G J; Yu, D G; Xie, J; Xia, Y; Gong, W B; Wang, H H; Li, Z F; Wei, N

    2014-08-01

    Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean-fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059-bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α-helixes, eight β-sheets, 16 regions of triple helical repeats, 21 low-complexity regions, 10 function domains and two zinc-binding sites; however, no calcium-binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi-quantitative RT-PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real-time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean-fed grass carp. PMID:24127725

  4. cDNA cloning and transcript distribution of two novel members of the neuroparsin peptide family in a hemipteran insect (Nezara viridula) and a decapod crustacean (Jasus lalandii).

    PubMed

    Marco, Heather G; Anders, Lance; Gäde, Gerd

    2014-03-01

    Two novel neuroparsin (NP) precursor cDNAs were cloned: one from the corpora cardiaca of an insect, the green stink bug Nezara viridula, and the other from the X-organ of a decapod crustacean, the spiny lobster Jasus lalandii. The translated NP precursor consists of 106 amino acid residues in N. viridula and 103 amino acid residues in J. lalandii, with 14 and 12 cysteine residues, respectively, in conserved positions when aligned with known NPs. Reverse transcriptase PCR shows that in both arthropods, NP is expressed in some neural tissues: corpora cardiaca, sub-esophageal ganglion and brain of N. viridula; X-organ, brain, sub-esophageal and thoracic ganglion in J. lalandii. Additionally, NP is also expressed in non-neural tissues, such as fat body, leg muscle, flight muscle, reproductive organs and antennae in N. viridula, and heart and ovary in J. lalandii. There are no major differences in the NP transcript expression in mature and immature stink bugs, and also no difference between male and female stink bugs. PMID:24512948

  5. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

    PubMed

    Handley-Gearhart, P M; Stephen, A G; Trausch-Azar, J S; Ciechanover, A; Schwartz, A L

    1994-12-30

    The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1

  6. Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily

    SciTech Connect

    Kozma, S.C.; Ferrari, S. Bassand, P.; Siegmann, M.; Thomas, G. ); Totty, N. )

    1990-10-01

    Recently the authors reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A){sup {sup plus}} mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.

  7. Large-scale cDNA transfection screening for genes related to cancer development and progression

    PubMed Central

    Wan, Dafang; Gong, Yi; Qin, Wenxin; Zhang, Pingping; Li, Jinjun; Wei, Lin; Zhou, Xiaomei; Li, Hongnian; Qiu, Xiaokun; Zhong, Fei; He, Liping; Yu, Jian; Yao, Genfu; Jiang, Huiqiu; Qian, Lianfang; Yu, Ye; Shu, Huiqun; Chen, Xianlian; Xu, Huili; Guo, Minglei; Pan, Zhimei; Chen, Yan; Ge, Chao; Yang, Shengli; Gu, Jianren

    2004-01-01

    A large-scale assay was performed by transfecting 29,910 individual cDNA clones derived from human placenta, fetus, and normal liver tissues into human hepatoma cells and 22,926 cDNA clones into mouse NIH 3T3 cells. Based on the results of colony formation in hepatoma cells and foci formation in NIH 3T3 cells, 3,806 cDNA species (8,237 clones) were found to possess the ability of either stimulating or inhibiting cell growth. Among them, 2,836 (6,958 clones) were known genes, 372 (384 clones) were previously unrecognized genes, and 598 (895 clones) were unigenes of uncharacterized structure and function. A comprehensive analysis of the genes and the potential mechanisms for their involvement in the regulation of cell growth is provided. The genes were classified into four categories: I, genes related to the basic cellular mechanism for growth and survival; II, genes related to the cellular microenvironment; III, genes related to host-cell systemic regulation; and IV, genes of miscellaneous function. The extensive growth-regulatory activity of genes with such highly diversified functions suggests that cancer may be related to multiple levels of cellular and systemic controls. The present assay provides a direct genomewide functional screening method. It offers a better understanding of the basic machinery of oncogenesis, including previously undescribed systemic regulatory mechanisms, and also provides a tool for gene discovery with potential clinical applications. PMID:15498874

  8. Cloning and sequence analysis of the coding sequence of β-actin cDNA from the Chinese alligator and suitable internal reference primers from the β-actin gene.

    PubMed

    Zhu, H N; Zhang, S Z; Zhou, Y K; Wang, C L; Wu, X B

    2015-01-01

    β-Actin is an essential component of the cytoskeleton and is stably expressed in various tissues of animals, thus, it is commonly used as an internal reference for gene expression studies. In this study, a 1731-bp fragment of β-actin cDNA from Alligator sinensis was obtained using the homology cloning technique. Sequence analysis showed that this fragment contained the complete coding sequence of the β-actin gene (1128 bp), encoding 375 amino acids. The amino acid sequence of β-actin is highly conserved and its nucleotide sequence is slightly variable. Multiple alignment analyses showed that the nucleotide sequence of the β-actin gene from A. sinensis is very similar to sequences from birds, with 94-95% identity. Ten pairs of primers with different product sizes and different annealing temperatures were screened by PCR amplification, agarose gel electrophoresis, and DNA sequencing, and could be used as internal reference primers in gene expression studies. This study expands our knowledge of β-actin gene phylogenetic evolution and provides a basis for quantitative gene expression studies in A. sinensis. PMID:26505364

  9. Isolation, cDNA Cloning, and Structure-based Functional Characterization of Oryctin, a Hemolymph Protein from the Coconut Rhinoceros Beetle, Oryctes rhinoceros, as a Novel Serine Protease Inhibitor*

    PubMed Central

    Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

    2010-01-01

    We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant 13C,15N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with Ki values of 3.9 × 10−10 m, 6.2 × 10−10 m, 1.4 × 10−9 m, and 1.2 × 10−8 m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections. PMID:20630859

  10. 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

    SciTech Connect

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. ); Mattei, M.G. ); Seldin, M.F. ); Riviere, M.; Szpirer, J. )

    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.

  11. Cloning of a neoteleost (Oreochromis mossambicus) pro-opiomelanocortin (POMC) cDNA reveals a deletion of the gamma-melanotropin region and most of the joining peptide region: implications for POMC processing.

    PubMed

    Lee, J; Danielson, P; Sollars, C; Alrubaian, J; Balm, P; Dores, R M

    1999-12-01

    A signature feature of tetrapod pro-opiomelanocortin (POMC) is the presence of three melantropin (MSH) coding regions (alpha-MSH, beta-MSH, gamma-MSH). The MSH duplication events occurred early during the radiation of the jawed vertebrates well over 400 million years ago. However, in at least one order of modern bony fish (subdivision Teleostei; order Salmoniformes; i.e. salmon and trout) the gamma-MSH sequence has been deleted from POMC. To determine whether the gamma-MSH deletion has occurred in other teleost orders, a POMC cDNA was cloned from the pituitary of the neoteleost Oreochromis mossambicus (order Perciformes). In O. mossambicus POMC, the deletion is more extensive and includes the gamma-MSH sequence and most of the joining peptide region. Because the salmoniform and perciform teleosts do not share a direct common ancestor, the gamma-MSH deletion event must have occurred early in the evolution of the neoteleost fishes. The post-translational processing of O. mossambicus POMC occurs despite the fact that the proteolytic recognition sequence, (R/K)-Xn-(R/K) where n can be 0, 2, 4, or 6, a common feature in mammalian neuropeptide and polypeptide hormone precursors, is not present at several cleavage sites in O. mossambicus POMC. These observations would indicate that either the prohormone convertases in teleost fish use distinct recognition sequences or vertebrate prohormone convertases are capable of recognizing a greater number of primary sequence motifs around proteolytic cleavage sites. PMID:10698113

  12. Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

    PubMed Central

    2004-01-01

    Cystathionine γ-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by λ genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice. PMID:15038791

  13. Molecular cloning and characterization of duck interleukin-17

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Cloning of murine ferrochelatase.

    PubMed Central

    Brenner, D A; Frasier, F

    1991-01-01

    Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The activity of ferrochelatase is deficient in the inherited disease protoporphyria. In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe. The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences. Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined. The ferrochelatase protein and mRNAs have different relative concentrations in different tissues. The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria. Images PMID:1704134

  15. Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

    PubMed Central

    Ralph, Steven G; Chun, Hye Jung E; Cooper, Dawn; Kirkpatrick, Robert; Kolosova, Natalia; Gunter, Lee; Tuskan, Gerald A; Douglas, Carl J; Holt, Robert A; Jones, Steven JM; Marra, Marco A; Bohlmann, Jörg

    2008-01-01

    Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were

  16. Identification, cloning and regulation of cDNA encoding aldo-keto reductase 1B7 in the adrenal gland of two Saharan rodents Meriones libycus (Libyan jird) and Gerbillus gerbillus (gerbil).

    PubMed

    Mataoui-Mazari, Houria; Amirat, Zaïna; Khammar, Farida; Martinez, Antoine

    2011-12-01

    Aldo-Keto Reductase 1B7 (AKR1B7) is a mouse aldose reductase-like protein with two major sites of expression, the vas deferens and the adrenal cortex. In the adrenal cortex, Akr1b7 is an adrenocorticotropin (ACTH)-responsive-gene whose product scavenges harmful byproducts of steroidogenesis and limits stress response through the biosynthesis of prostaglandin F2α. The purpose of the present study was to explore the possible expression of AKR1B7 in the adrenal glands of two saharan rodents, Libyan jird and Lesser Egyptian gerbil. Western blot analyses demonstrated that a protein related to murine/rat AKR1B7 was highly expressed in adrenals and absent from vas deferens of both saharan species. Based on conserved sequences between mouse and rat, full length cDNA were cloned and sequenced in both species while hormonal regulation and tissue localization were explored in Libyan jird. Both cDNA encoded the expected 316 amino acids protein typical of AKR1B subfamily and contained the highly conserved catalytic tetrad consisting in Asp-44, Tyr-49, Lys-78 and His-111 residues. The deduced proteins shared higher identities with aldose reductase-like, i.e. AKR1B7 (86-94%), AKR1B8 and AKR1B10 (83-86%) than with aldose reductase group, i.e. AKR1B1 and AKR1B3 (70%). Phylogenetic analysis showed that the Libyan jird and gerbil enzymes were more closely related to murine and rat AKR1B7 than to the other AKR1B members. Northern blot analyses of total RNA from Libyan jird adrenals showed a single mRNA transcript of 1.4 kb whose expression was dependent on circulating ACTH levels. In conclusion, we demonstrate here that adrenal glands of Libyan jird and gerbil express both an ortholog of the murine/rat Akr1b7 gene and that ACTH-responsiveness is at least conserved in Libyan jird. PMID:21963864

  17. [cDNA cloning, expression and determination of substrate specificity of mice selenocysteine-containing protein SelV (Selenoprotein V)].

    PubMed

    Varlamova, E G; Novoselov, S V; Novoselov, V I

    2015-01-01

    To date various bioinformatics tools allowed to identify 25 selenocysteine-containing mammalian proteins. The name of these proteins assumes that they contain the amino acid selenocysteine (Sec). Functionally characterized selenocysteine-containing proteins are oxidoreductases with various functions, including glutathione peroxidases, thioredoxin reductases, deiodinases etc. However, the functions of more than half of identified proteins are still unclear, and mammalian selenoprotein SeIV is among them. We studied the selV in all stages of postnatal development with the maximum level of mRNA expression during puberty, whereas in adult mice (8-18 months) we observed a gradual decrease of expression. In order to get closer to the functional role of Selenoprotein V, we have carried out experiments on the substrate specificity and enzymatic activity measurement of this selenocysteine-containing protein. It was shown that SelV posseses glutathionperoxidase and thioredoxinreductase activities. PMID:26510596

  18. Molecular cloning and mRNA distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide in the lungfish.

    PubMed

    Lee, L T O; Tam, J K V; Chan, D W; Chow, B K C

    2009-04-01

    In this article, we report the isolation of a full-length cDNA clone encoding pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) from lungfish Protopterus dolloi. When comparing the deduced amino acid sequences, the lungfish PACAP was found to be highly conserved with other vertebrates; however, the PRP shares only lower levels of sequence identity with known PRP sequences. Consistently in phylogenetic analysis, the lungfish PRP, similar to sturgeon PRP, fails to cluster with other PRPs. In addition to the full-length clone, another cDNA encoding a short precursor that lacks the first 32 amino acids of the PRP was also isolated. Interestingly, similar isoforms were also identified in several nonmammalian vertebrates, and it was suggested that exon skipping of PRP/PACAP transcripts was a mechanism that regulated the expression ratio of PACAP to PRP in nonmammalian vertebrates. By real-time PCR, both long and short PRP/PACAP transcripts were found almost exclusively in the brain, and the short isoform is the more abundant transcript (3.7 times more), indicating that PACAP is the major product produced in lungfish brain. The expression patterns of lungfish and previously studied frog PRP/PACAP suggest that the PRP/PACAP gene in the tetrapod lineage may first express in the central nervous system; in the process of evolution, the functions of these peptides diversified and were later found in other tissues. PMID:19456341

  19. A comparative study of 2',3'-cyclic-nucleotide 3'-phosphodiesterase in vertebrates: cDNA cloning and amino acid sequences for chicken and bullfrog enzymes.

    PubMed

    Kasama-Yoshida, H; Tohyama, Y; Kurihara, T; Sakuma, M; Kojima, H; Tamai, Y

    1997-10-01

    In mammalian brain, two 2',3'-cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) isoforms, CNP1 and CNP2, are translated, respectively, from the two mRNAs, which have been transcribed and processed by alternative use of the two transcription start points and by differential splicing. In the present study, the cDNAs encoding chicken CNP2 and bullfrog CNP1, respectively, were isolated, and the amino acid sequences of chicken CNP2 and bullfrog CNP1 were deduced. Western blot analysis showed that chicken brain contains a major CNP2-type protein together with a minor unidentified isoform, and bullfrog brain contains only a CNP1-type protein. All available amino acid sequences of vertebrate 2',3'-cyclic-nucleotide 3'-phosphodiesterases were aligned and compared. Three conserved motif sequences were noted: (a) an ATP-binding site near the amino terminus, (b) an isoprenylation site at the carboxyl terminus, and (c) a probable catalytic site resembling the active site of beta-ketoacyl synthase (EC 2.3.1.41). The second and the third motifs are conserved also in goldfish RICH (regeneration-induced 2',3'-cyclic-nucleotide 3'-phosphodiesterase homologue), which has been shown recently to have 2',3'-cyclic-nucleotide 3'-phosphodiesterase activity. The third motif (probably catalytic site) was assigned for the first time in the present report. PMID:9326261

  20. 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

    SciTech Connect

    Gafvels, M.E.; Strauss, J.F. III ); Caird, M.; Patterson, D. ); Britt, D.; Jackson, C.L. )

    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.

  1. Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

    SciTech Connect

    Chen, Kun; Sun, Guoxun; Lv, Zhiyuan; Wang, Chen; Jiang, Xueyuan; Li, Donghai; Zhang, Chenyu

    2010-10-01

    Research highlights: {yields} Invertebrates, for example amphioxus, do express uncoupling proteins. {yields} Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. {yields} UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

  2. Cloning, expression and characterization of a gene encoding mitogen activated protein kinase 2 (MPK2) from Tetrahymena thermophila.

    PubMed

    Arslanyolu, Muhittin; Yıldız, Mehmet Taha

    2014-08-01

    Environmental effects and mitogens determine cell phenotype in eukaryotes mainly through MAPK pathways. However, MAPK signaling pathways in T. thermophila have not been studied comprehensively. This study aims to express recombinant MPK2, a MAPK from T. thermophila, in E. coli to characterize its kinase activity. MPK2 was cloned by RT-PCR using degenerate oligonucleotide primers and RACE method. The full-length cDNA of the MPK2 gene is 1705bp that includes 1281bp ORF coding for a putative protein of 426 amino acids having a mass of 50.2kDa. The putative MPK2 protein contains all eleven conserved subdomains that are characteristics of serine/threonine protein kinases, and a TDY motif, which is a putative dual phosphorylation site common in Protista. MPK2 displays highest 48% overall identity to human ERK5 (MAPK7). The expression vector pGEX4T-1-MPK2 was constructed by inserting the coding region of MPK2 cDNA into pGEX4T-1 after introducing the nine point mutations, and then transformed into E. coli BL21(DE3). Autophosphorylation of 76kDa GST-MPK2 at tyrosine residues was confirmed not only by Western blot using anti-phosphotyrosine monoclonal antibody but also by in vitro kinase assay. GST-MPK2 was also able to phosphorylate the artificial substrate myelin basic protein. This study concludes that the free-living unicellular protist T. thermophila MPK2 has commonly conserved MAPK enzyme features, possibly involved in the regulation of cell survival responding to abiotic or biotic stressors, and the production and movement of haploid gametic nuclei between pairs during conjugation. PMID:24858074

  3. Cloning and expression analysis of recombination activating genes (RAG1/2) in red snapper (Lutjanus sanguineus).

    PubMed

    Zhang, X L; Lu, Y S; Jian, J C; Wu, Z H

    2012-04-01

    Recombination activating genes (RAG1 and RAG2), involved in the V(D)J recombination of immunoglobulin and T-cell receptor genes play a crucial role in the adaptive immune response in vertebrates. The expression of these genes was required for the proper development and maturity of lymphocytes so that they can be used as useful markers to evaluate the development of lymphoid organ. In this paper, the cDNA of RAG1 and RAG2 in red snapper, Lutjanus sanguineus were cloned by homological cloning and rapid amplification of cDNA ends (RACE) methods. Results showed the full length of RAG1 cDNA was 3944 bp, containing a 5' untranslated region (UTR) of 200 bp, a 3'-UTR of 561 bp and an open reading frame of 3183 bp encoding 1060 amino acids. Three important structural motifs, a RING/U-box domain, a RING/FYVE/PHD-type domain and a RAG Nonamer-binding domain were detected in the deduced amino acid sequence of RAG1 by InterProScan analysis. The full length of RAG2 cDNA was 2200 bp, consisting of a 141 bp 5'-UTR, a 457 bp 3'-UTR and an open reading frame of 1602 bp encoding 533 amino acids. Two important structural motifs, a Galactose oxidase/kelch, beta-propeller domain and a kelch-type beta-propeller domain were detected in the deduced amino acid sequence of RAG2 by InterProScan analysis. BLAST analysis revealed that the RAG1 and RAG2 in red snapper shared a high homology with other known RAG1 and RAG2 genes, while the greatest degree of identity was observed with Hippoglossus hippoglossus RAG1 at 82% and Takifugu rubripes RAG2 at 87%, respectively. The differential expressions of RAG1 and RAG2 in various tissues of red snapper were analyzed by fluorescent quantitative real-time PCR. The overall expression pattern of the two genes was quite similar. In healthy red snappers, the RAGs transcripts were mainly detected in thymus, following head kidney, spleen, intestine, liver and brain. After vaccinated with inactivated Vibrio alginolyticus 48 h later, the RAGs m

  4. Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

    PubMed Central

    Miltenburg, A M; Van Laar, J M; De Kuiper, P; Daha, M R; Breedveld, F C

    1990-01-01

    A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function. PMID:2148285

  5. Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity.

    PubMed

    Mori, Tsukasa; Hiraka, Ikuei; Kurata, Youichi; Kawachi, Hiroko; Mano, Nobuhiro; Devlin, Robert H; Nagoya, Hiroyuki; Araki, Kazuo

    2007-03-01

    Growth hormone (GH) transgenic amago salmon (Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F(2) and F(3) generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Delta6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F(2) and F(3) salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F(2) and F(3) fish were completely

  6. Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity.

    PubMed

    Sun, X L; Jayaram, H N; Gharehbaghi, K; Li, Q J; Xiao, X; Antony, A C

    1999-02-15

    Cervical carcinoma is an AIDS-defining illness. The expression of folate receptors (FRs) in cervical carcinoma (HeLa-IU1) cells was modulated by stable transduction of FR cDNA encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). Although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) FCS, [methyl-3H]thymidine incorporation into DNA was significantly increased in sense cells in 10% serum; therefore, the basis for this discrepancy was investigated. The activity of thymidine kinase (TK) was subsequently directly correlated with the extent of FR expression in single cell-derived clones of transduced cells. This elevated TK activity was not a result of recruitment of the salvage pathway based on the presence of adequate dTTP pools, normal thymidylate synthase (TS) activity, persistence of increased thymidine incorporation despite the exogenous provision of excess 5,10-methylene-tetrahydrofolate, and documentation of adequate folates in sense cells. The increase in TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as demonstrated by augmented phosphorylation of 3'-azido-3'-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT. Conversely, sense cells were highly resistant to methotrexate, but this was reversed by the addition of AZT. The direct correlation of FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression. PMID:10029088

  7. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Namhai Chua; Kush, A.

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids.

  8. beta 1-4N-acetylgalactosaminyltransferase can synthesize both asialoglycosphingolipid GM2 and glycosphingolipid GM2 in vitro and in vivo: isolation and characterization of a beta 1-4N-acetylgalactosaminyltransferase cDNA clone from rat ascites hepatoma cell line AH7974F.

    PubMed Central

    Hidari, J K; Ichikawa, S; Furukawa, K; Yamasaki, M; Hirabayashi, Y

    1994-01-01

    We have cloned a cDNA encoding beta 1-4N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, M., and Kojima, K. (1979) Biochim. Biophys. Acta 572, 113-120]. The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in lambda gt10 with a probe. The probe was obtained from AH7974F cDNA by PCR using primers with the nucleotide sequence of the human GalNAc-T cDNA. The amino acid sequence deduced from the nucleotide sequence of pGNA56 exhibited 88% similarity to the human GalNAc-T sequence. The enzyme was a typical type II membrane protein, which consisted of a short N-terminal residue, a transmembrane region, and a long C-terminal residue, including the catalytic domain. The substrate specificity of rat GalNAc-T was determined using homogenates from cells into which the cDNA clone was transfected. The enzyme catalysed not only the formation of GM2 and GD2 from GM3 and GD3 respectively, but also asialo-GM2 from CDH. It also acted on GSL substrates, including GM1b, sialylparagloboside and GD1 alpha. On the other hand, the enzyme did not transfer GalNAc to soluble substrates such as glycoproteins and oligosaccharide. The GSL compositional and immunocytochemical analyses of stable transfectants obtained by transfection of the cDNA showed simultaneous expression of asialo-GM2 and GM2 on the plasma membrane. Therefore, we concluded that the formation of asialo-GM2, GM2 and GD2 was catalysed by the single GalNAc-T. Northern-blot hybridization showed that the GalNAc-T mRNA was strongly expressed in rat brain, testis, and spleen. The gene was also expressed in rat normal liver to a lesser extent. We found the GSLs in asialo- and alpha-pathways such as asialo-GM1 and GD1 alpha in the rat tissues by using a sensitive t.l.c.-immunostaining method. These observations also

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  11. Cloning, sequence, and expression of bovine interleukin 2.

    PubMed Central

    Cerretti, D P; McKereghan, K; Larsen, A; Cantrell, M A; Anderson, D; Gillis, S; Cosman, D; Baker, P E

    1986-01-01

    Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system. Images PMID:3517854

  12. Liganded Thyroid Hormone Receptor Inhibits Phorbol 12-O-Tetradecanoate-13-Acetate-Induced Enhancer Activity via Firefly Luciferase cDNA

    PubMed Central

    Misawa, Hiroko; Sasaki, Shigekazu; Matsushita, Akio; Ohba, Kenji; Iwaki, Hiroyuki; Matsunaga, Hideyuki; Suzuki, Shingo; Ishizuka, Keiko; Oki, Yutaka; Nakamura, Hirotoshi

    2012-01-01

    Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity

  13. Identification of HUGT1 as a potential BiP activator and a cellular target for improvement of recombinant protein production using a cDNA screening system.

    PubMed

    Ku, Sebastian Chih Yuan; Lwa, Teng Rhui; Giam, Maybelline; Yap, Miranda Gek Sim; Chao, Sheng-Hao

    2009-05-31

    The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon gamma, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells. PMID:19466607

  14. Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.

    PubMed Central

    Waldo, G L; Paterson, A; Boyer, J L; Nicholas, R A; Harden, T K

    1996-01-01

    A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by alpha-subunits of the Gq family and by G-protein beta gamma-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-beta family, with the highest identity (71%) shared with PLC-beta 2 and lesser identities observed with PLC-beta 1 (49%), PLC-beta 3 (46%) and PLC-beta 4 (37%). The largest differences in sequence between the turkey PLC-beta and other PLC-beta isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-beta 2, which differ in their regulation by G-protein alpha-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in alpha q activation of these proteins. Recombinant turkey PLC-beta was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-beta. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-beta in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with G alpha 11 or G-protein beta gamma-subunits. The turkey PLC-beta was more sensitive to activation by G alpha 11 than was PLC-beta 2, and was more sensitive to activation by beta gamma-subunits than either PLC

  15. Prophenoloxidase from Pieris rapae: gene cloning, activity, and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata *

    PubMed Central

    Zhu, Jia-ying; Yang, Pu; Wu, Guo-xing

    2011-01-01

    Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis,