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Sample records for deoxycytidine kinase cdna

  1. Enhanced activity of deoxycytidine kinase after pulsed low dose rate and single dose gamma irradiation

    NARCIS (Netherlands)

    Sigmond, J.; Haveman, J.; Kreder, N. Castro; Loves, W. J.; van Bree, C.; Franken, N. A.; Peters, G. J.

    2006-01-01

    In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for

  2. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

    Science.gov (United States)

    Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

    2016-04-12

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

  3. The apoptotic effects of toosendanin are partially mediated by activation of deoxycytidine kinase in HL-60 cells.

    Science.gov (United States)

    Ju, Jianming; Qi, Zhichao; Cai, Xueting; Cao, Peng; Huang, Yan; Wang, Shuzhen; Liu, Nan; Chen, Yijun

    2012-01-01

    Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

  4. The apoptotic effects of toosendanin are partially mediated by activation of deoxycytidine kinase in HL-60 cells.

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    Jianming Ju

    Full Text Available Triterpenoid toosendanin (TSN exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK. Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

  5. Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

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    Sujatha P Koduvayur

    Full Text Available Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK, to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3 indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells.

  6. Nonenantioselectivity Property of Human Deoxycytidine Kinase Explained by Structures of the Enzyme in Complex with [subscript L]- and [subscript D]-Nucleosides

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    Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred; Lavie, Arnon (UIC); (MXPL-G)

    2008-07-31

    Biological molecules are predominantly enantioselective. Yet currently two nucleoside analogue prodrugs (3TC and FTC) with opposite chirality compared to physiological nucleosides are clinically approved for the treatment of HIV infections. These prodrugs require conversion to their triphosphorylated forms to achieve pharmacological activity. The first step in the activation of these agents is catalyzed by human deoxycytidine kinase (dCK). This enzyme possesses the ability to phosphorylate nucleosides of the unnatural L-chirality. To understand the molecular basis for the nonenantioselectivity of dCK, we solved the crystal structures of the enzyme in complex with the L-enantiomer and of its physiological substrate deoxycytidine and with the L-nucleoside analogue FTC. These were compared to a structure solved with D-dC. Our results highlight structural adjustments imposed on the L-nucleosides and properties of the enzyme endowing it with the ability to phosphorylate substrates with nonphysiological chirality. This work reveals the molecular basis for the activation of L-nucleosides by dCK.

  7. Role of Genetic Polymorphisms of Deoxycytidine Kinase and Cytidine Deaminase to Predict Risk of Death in Children with Acute Myeloid Leukemia

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    Aurora Medina-Sanson

    2015-01-01

    Full Text Available Cytarabine is one of the most effective antineoplastic agents among those used for the treatment of acute myeloid leukemia. However, some patients develop resistance and/or severe side effects to the drug, which may interfere with the efficacy of the treatment. The polymorphisms of some Ara-C metabolizing enzymes seem to affect outcome and toxicity in AML patients receiving cytarabine. We conducted this study in a cohort of Mexican pediatric patients with AML to investigate whether the polymorphisms of the deoxycytidine kinase and cytidine deaminase enzymes are implicated in clinical response and toxicity. Bone marrow and/or peripheral blood samples obtained at diagnosis from 27 previously untreated pediatric patients with de novo AML were processed for genotyping and in vitro chemosensitivity assay, and we analyzed the impact of genotypes and in vitro sensitivity on disease outcome and toxicity. In the multivariate Cox regression analysis, we found that age at diagnosis, wild-type genotype of the CDA A79C polymorphism, and wild-type genotype of the dCK C360G polymorphism were the most significant prognostic factors for predicting the risk of death.

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

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    Jennifer C. C. Neale

    2004-01-01

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

  9. Deoxycytidine transport and metabolism in choroid plexus

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    Spector, R.; Huntoon, S.

    1983-05-01

    In vitro, the transport into and release of (/sup 3/H)deoxycytidine from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. By use of the ability of nitrobenzylthioinosine (NBTI) to inhibit deoxycytidine efflux from choroid plexus, the transport of 1 microM (/sup 3/H)deoxycytidine into choroid plexus at 37 degrees C was measured. Deoxycytidine was transported into choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production, but not intracellular binding or metabolism. The Michaelis-Menten constant (KT) for the active transport of deoxycytidine into choroid plexus was 15 microM. The active transport system for deoxycytidine was inhibited by naturally occurring nucleosides and deoxynucleosides, but not by 1 mM probenecid and 2-deoxyribose or 100 microM cytosine and cytosine arabinoside. With less than 1 microM (/sup 3/H)deoxycytidine in the medium, the choroid plexus accumulated (/sup 3/H)deoxycytidine against a concentration gradient. However, approximately 50% of the (/sup 3/H)deoxycytidine was phosphorylated to (/sup 3/H)deoxycytidine nucleotides at a low extracellular (/sup 3/H)deoxycytidine concentration (6 nM) in 15-min incubations. This accumulation process depended, in part, on saturable intracellular phosphorylation. These studies provide further evidence that the choroid plexus contains an active nucleoside transport system of low specificity for deoxynucleosides and ribonucleosides, and a separate, saturable efflux system for deoxynucleosides which is very sensitive to inhibition by NBTI.

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

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

    2006-09-01

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

  11. Stable transfection of protein kinase C alpha cDNA in hormone-dependent breast cancer cell lines

    OpenAIRE

    Tonetti, D A; Chisamore, M J; Grdina, W; Schurz, H; Jordan, V C

    2000-01-01

    An inverse relationship between protein kinase C (PKC) activity and oestrogen receptor (ER) expression in human breast cell lines and tumours has been firmly established over the past 10 years. To determine whether specific alterations in PKC expression accompany hormone-independence, we examined the expression of PKC isozymes in the hormone-independent human breast cancer cell clones MCF-7 5C and T47D:C42 compared with their hormone-dependent counterparts, MCF-7 A4, MCF-7 WS8 and T47D:A18 re...

  12. Complementation of a yeast cell cycle mutant by an alfalfa cDNA encoding a protein kinase homologous to p34cdc2.

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    Hirt, H; Páy, A; Györgyey, J; Bakó, L; Németh, K; Bögre, L; Schweyen, R J; Heberle-Bors, E; Dudits, D

    1991-03-01

    The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.

  13. The effect of 2'-fluoro-2'-deoxycytidine on herpes virus growth.

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    Wohlrab, F; Jamieson, A T; Hay, J; Mengel, R; Guschlbauer, W

    1985-03-20

    The effect of 2'-fluoro-2'-deoxycytidine (dCfl) on the growth of certain viruses of the herpes type was investigated. It is shown that the compound has considerable anti-viral activity against HSV-I, HSV-II, pseudorabies virus and equine abortion virus. It has an effect comparable to that of araC and is more efficient than br5dC, but less so than acyclovir. Experiments with thymidine kinase-negative strains of HSV-I indicated that dCfl was phosphorylated by the viral kinase, and its Km appears to be low and close to that of thymidine. Density gradient centrifugation enabled us to show that dCfl was incorporated into cellular and viral DNA and RNA. The cytotoxic activity of dCfl appears to be about 10-times smaller than that of araC. Removal of the nucleoside analog, washing and replacement with deoxycytidine reversed this effect, indicating rather a cytostatic than cytotoxic effect.

  14. A multisubstrate deoxyribonucleoside kinase from plants

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Knecht, Wolfgang

    2008-01-01

    and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene...

  15. Identification of four genomic loci highly related to casein-kinase-2-alpha cDNA and characterization of a casein kinase-2-alpha pseudogene within the mouse genome

    DEFF Research Database (Denmark)

    Boldyreff, B; Wehr, K; Hecht, R

    1992-01-01

    Using the coding region of the human CK-2 alpha cDNA as a probe for screening a genomic mouse library, positive clones representing four different genomic loci were isolated. Partial DNA sequences of these loci encompassing the first 120 nucleotides of the putative coding region are reported. One...

  16. Quantification of 5-methyl-2'-deoxycytidine in the DNA.

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    Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

    2015-01-01

    Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

  17. Origin of increased deoxycytidine excretion into urine of rats bearing Yoshida ascites sarcoma

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    Shimizu, M.; Fujimura, S.

    1984-06-01

    The metabolism of deoxycytidine (dCyd) and dCyd nucleotides in Yoshida ascites sarcoma (YS) cells and the host rat liver was investigated with reference to the increased excretion of urinary dCyd. Incorporation of (/sup 14/C)orotic acid into the livers of rats at the fifth day after the transplantation of YS cells, was 2 times higher than that into the normal rat livers. After the injection of (/sup 14/C)orotic acid, the ratio of the specific radioactivity of cytidylate to uridylate moieties of the host liver RNA was measured and found to be higher than that of normal rat liver RNA and to be similar to that of YS cell RNA. When (/sup 14/C)orotic acid was injected into rats followed by the transplantation of YS cells, the radioactivities present in the livers disappeared more rapidly than those in the control rat livers. The activities of pyrimidine de novo synthesis enzymes, such as cytidine triphosphate synthetase and cytidine diphosphate reductase, in YS were higher than those in both rat ascites hepatoma AH 7974 and Walker 256 carcinosarcoma, the transplantations of which did not induce increased excretion of dCyd into urine of the hosts. The activities of dCyd kinase and dCyd deaminase in YS cells were lower than those in the other two tumors investigated. The activities of cytidine triphosphate synthetase and cytidine diphosphate reductase in the livers of YS-bearing rats were elevated compared with those in the livers of rat ascites hepatoma AH 7974- or Walker 256 carcinosarcoma-bearing rats and normal rats, while the activities of dCyd kinase, 5'-nucleotidase, and dCyd deaminase were similar between normal rat livers and tumor-bearing rat livers.

  18. cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I)

    DEFF Research Database (Denmark)

    Saris, Chris J M; Kristensen, Torsten; D’Eustachio, Peter

    1987-01-01

    We have isolated and sequenced cDNA clones of bovine nd murine pl 1 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p l l mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5”untranslated region of 73 nucleotides in bovine p l l m...

  19. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin......, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions....

  20. Gene expression profiling of the synergy of 5-aza-2′-deoxycytidine and paclitaxel against renal cell carcinoma

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    Han Tiandong

    2012-09-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is one of the most common kidney cancers and is highly resistant to chemotherapy. We previously demonstrated that 5-aza-2′-deoxycytidine (DAC could significantly increase the susceptibility of renal cell carcinoma (RCC cells to paclitaxel (PTX treatment in vitro, and showed the synergy of DAC and PTX against RCC. The purpose of this study is to investigated the gene transcriptional alteration and investigate possible molecular mechanism and pathways implicated in the synergy of DAC and PTX against RCC. Methods cDNA microarray was performed and coupled with real-time PCR to identify critical genes in the synergistic mechanism of both agents against RCC cells. Various patterns of gene expression were observed by cluster analysis. IPA software was used to analyze possible biological pathways and to explore the inter-relationships between interesting network genes. Results We found that lymphoid enhancer-binding factor 1 (LEF1, transforming growth factor β-induced (TGFBI, C-X-C motif ligand 5 (CXCL5 and myelocytomatosis viral related oncogene (c-myc may play a pivotal role in the synergy of DAC and PTX. The PI3K/Akt pathway and other pathways associated with cyclins, DNA replication and cell cycle/mitotic regulation were also associated with the synergy of DAC and PTX against RCC. Conclusion The activation of PI3K/Akt-LEF1/β-catenin pathway could be suppressed synergistically by two agents and that PI3K/Akt-LEF1/β-catenin pathway is participated in the synergy of two agents.

  1. Normalizing cDNA libraries.

    Science.gov (United States)

    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A

    2010-04-01

    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  2. DEAMINATION OF DEOXYCYTIDINE AND 5-METHYLDEOXYCYTIDINE IN DEVELOPING ANTHERS OF LILIUM LONGIFLORUM (VAR. CROFT)

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    Hotta, Yasuo; Stern, Herbert

    1961-01-01

    A study has been made of developmental variations in anthers of Lilium longiflorum with respect to the deamination of deoxycytidine and 5-methyldeoxycytidine. Although a periodicity of enzymatic activities surrounding the mitotic cycle has been found, the over-all evidence is against the significance of such periodicity to chromosome duplication. On the basis of evidence obtained a distinction has been drawn between enzyme systems functioning in a broad supporting role and those immediately tied to morphogenetic events. PMID:13716116

  3. Hypomethylating agent 5-aza-2'-deoxycytidine (DAC) ameliorates multiple sclerosis in mouse models

    DEFF Research Database (Denmark)

    Mangano, Katia; Fagone, Paolo; Bendtzen, Klaus

    2014-01-01

    Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) in two ....... Finally, DAC treatment increased the percentage of circulating regulatory T cells by inducing Foxp3 expression via demethylation of a CpG island in Foxp3....

  4. cDNA library preparation.

    Science.gov (United States)

    Kooiker, Maarten; Xue, Gang-Ping

    2014-01-01

    The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5' and 3' end of the cDNA. The 3' adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5' adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.

  5. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  6. Global demethylation of rat chondrosarcoma cells after treatment with 5-aza-2'-deoxycytidine results in increased tumorigenicity.

    Directory of Open Access Journals (Sweden)

    Christopher A Hamm

    Full Text Available Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.

  7. Fingerprinting DNA oxidation processes: IR characterization of the 5-methyl-2'-deoxycytidine radical cation.

    Science.gov (United States)

    Bucher, Dominik B; Pilles, Bert M; Pfaffeneder, Toni; Carell, Thomas; Zinth, Wolfgang

    2014-02-24

    Methylated cytidine plays an important role as an epigenetic signal in gene regulation. Its oxidation products are assumed to be involved in active demethylation processes but also in damaging DNA. Here, we report the photochemical production of the 5-methyl-2'-deoxycytidine radical cation via a two-photon ionization process. The radical cation is detected by time-resolved IR spectroscopy and identified by band assignment using density functional theory calculations. Two final oxidation products are characterized with liquid chromatography coupled to mass spectrometry. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2 '-deoxyuridine and 5-ethynyl-2 '- deoxycytidine

    Czech Academy of Sciences Publication Activity Database

    Ligasová, A.; Liboska, Radek; Friedecký, D.; Mičová, K.; Adam, T.; Oždian, T.; Rosenberg, Ivan; Koberna, K.

    2016-01-01

    Roč. 6, č. 1 (2016), č. článku 150172. ISSN 2046-2441 R&D Projects: GA MZd NV15-31604A Institutional support: RVO:61388963 Keywords : cytidine deaminase * dCMP deaminase * 5-ethynyl-2 '-deoxyuridine * 5-ethynyl-2 '-deoxycytidine * DNA replication Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.481, year: 2016 http://rsob.royalsocietypublishing.org/content/6/1/150172

  9. Toward a cDNA map of the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.; Chen, X.N. [Cedars-Sinai Research Institute, Los Angeles, CA (United States); Adams, M.D.; Venter, J.C. [Institute for Genomic Research, Gaithersburg, MD (United States)

    1995-09-20

    Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosomes band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for the rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to 41 cDNAs with an average insert size of < 2 kb to single human chromosome bands. The results provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular single-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphotase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (205 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases. 16 refs., 1 fig., 3 tabs.

  10. Tritiated thymidine and deoxycytidine suicide of mouse hemopoietic colony forming cells (CFC)

    International Nuclear Information System (INIS)

    Uyeki, E.M.; Wierzba, K.; Bisel, T.U.

    1981-01-01

    Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1-100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd. (author)

  11. Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

    Directory of Open Access Journals (Sweden)

    Yogini P. Bhavsar

    2011-01-01

    Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  12. Molecular cloning and characterization of a novel human kinase ...

    Indian Academy of Sciences (India)

    throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the ...

  13. Novel expression patterns of phosphatidylinositol 3-hydroxy kinase in nodulated Medicago spp. plants

    NARCIS (Netherlands)

    Hernández, L.E.; Escobar, C.; Drobak, B.K.; Bisseling, T.; Brewin, N.J.

    2004-01-01

    A cDNA clone encoding a phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been characterized from Medicago truncatula, Mtpi3k, that is highly homologous to their counterparts from soybean (over 84%). The results suggest the presence of at least two genes coding PtdIns 3-kinases in M. truncatula.

  14. Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

    International Nuclear Information System (INIS)

    Szopa, J.

    1995-01-01

    The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

  15. Labelling of the thymidine and deoxyctidine bases of DNA by (2-14C) deoxycytidine in cultured L1210 cells

    International Nuclear Information System (INIS)

    Karle, J.M.; Hoeraut, R.M.; Cysyk, R.L.

    1983-01-01

    Exposure of cultured L1210 cells to (2- 14 C) deoxycytidine and analysis of radioactivity incorporated into DNA-pyrimidines revealed that 2.7-5.5-fold more radioactivity is incorporated into DNA-thymine than into cytosine bases. Thus, the pathway involving deamination of deoxycytidylate to deoxyuridylate and methylation to thymidylate is highly favoured over successive phosphorlation to dCTP. Several modified and endogenous pyrimidines altered the labelling of DNA-thymine and DNA-cytosine with (2- 14 C)-deoxycytidine. 3-deazauridine at 0.1 mM caused a 56% increase in the labelling of DNA-thymine. Both thymidine and 3-deazauridine (>=10 μM) increased the specific activity to DNA-cytosine by 4-fold. Cytosine arabinoside (ara-C) (>= 10 μM) reduced the labelling of both DNA-cytosine and DNA-thymine. Excess cytidine (0.1 mM) reduced the labelling of DNA-cytosine by 40%. Tetrahydrouridine at concentrations up to 1 mM had no effect. (author)

  16. HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA: methodological optimization for chestnut and other woody species.

    Science.gov (United States)

    Hasbún, Rodrigo; Valledor, Luís; Rodríguez, José L; Santamaria, Estrella; Ríos, Darcy; Sanchez, Manuel; Cañal, María J; Rodríguez, Roberto

    2008-01-01

    Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2'-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2'-deoxycytidine.

  17. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...

  18. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposite...

  19. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...... subunit and 71% identity to the alpha' subunit of human casein kinase 2....

  1. An Escherichia coli strain deficient for both exonuclease 5 and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Estevenon, A.M.; Kooistra, J.; Sicard, N.

    1995-01-01

    An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity

  2. Development of an HIV-based cDNA expression cloning system.

    Science.gov (United States)

    van Maanen, Marc; Tidwell, Jennie K; Donehower, Lawrence A; Sutton, Richard E

    2003-07-01

    Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

  3. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  4. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length ...

  5. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    317. 2.4 cDNA sequencing and analysis. The nucleotide sequence of the cloned H. fossilis GH. cDNA was determined by Sanger's dideoxy chain termi- nation method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All other computational analysis of the GH cDNA was done using.

  6. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  7. Expression, purification and kinase activity analysis of maize ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... Kinase activity is essential for a protein kinase to perform its biological function. In previous study we have cloned a novel plant SnRK2 subfamily gene from maize and named it as ZmSPK1. In this study the. cDNA of ZmSPK1 with dHA-His6 tag was amplified by PCR and was subcloned into the yeast.

  8. 5-Aza-2'-deoxycytidine protects against emphysema in mice via suppressing p16Ink4a expression in lung tissue

    Directory of Open Access Journals (Sweden)

    He ZH

    2017-10-01

    Full Text Available Zhi-Hui He,1 Yan Chen,2 Ping Chen,2 Sheng-Dong He,2 Hui-Hui Zeng,2 Ji-Ru Ye,2 Da Liu,2 Jun Cao3 1Intensive Care Unit, 2Department of Respiratory Medicine, Second Xiangya Hospital, Central South University, Changsha, 3Department of Respiratory Medicine, Hunan Provincial People’s Hospital, Changsha, China Background: There is a growing realization that COPD, or at least emphysema, involves several processes presenting in aging and cellular senescence. Endothelial progenitor cells (EPCs contribute to neovascularization and play an important role in the development of COPD. The gene for p16Ink4a is a major dominant senescence one. The aim of the present study was to observe changes in lung function, histomorphology of lung tissue, and expression of p16Ink4a in lung tissue and bone marrow-derived EPCs in emphysematous mice induced by cigarette-smoke extract (CSE, and further to search for a potential candidate agent protecting against emphysema induced by CSE. Materials and methods: An animal emphysema model was induced by intraperitoneal injection of CSE. 5-Aza-2'-deoxycytidine (5-Aza-CdR was administered to the emphysematous mice. Lung function and histomorphology of lung tissue were measured. The p16Ink4a protein and mRNA in EPCs and lung tissues were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction, respectively. Results: CSE induced emphysema with increased p16Ink4a expression in lung tissue and bone marrow-derived EPCs. 5-Aza-CdR partly protected against emphysema, especially in the lung-morphology profile, and partly protest against the overexpression of p16Ink4a in EPCs and lung tissue induced by CSE. Conclusion: 5-Aza-CdR partly protected against emphysema in mice via suppressing p16Ink4a expression in EPCs and lung tissue. Keywords: 5-Aza-2'-deoxycytidine, cigarette smoke, emphysema, endothelial progenitor cells, p16Ink4a

  9. Alpha anomer of 5-aza-2'-deoxycytidine down-regulates hTERT mRNA expression in human leukemia HL-60 cells

    Czech Academy of Sciences Publication Activity Database

    Hájek, Miroslav; Votruba, Ivan; Holý, Antonín; Krečmerová, Marcela; Tloušťová, Eva

    2008-01-01

    Roč. 75, č. 4 (2008), s. 965-972 ISSN 0006-2952 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : alpha-5-aza-2'-deoxycytidine * DNA hypomethylation * hTERT * c- myc Subject RIV: CC - Organic Chemistry Impact factor: 4.838, year: 2008

  10. Identification of 3,N(4)-etheno-5-methyl-2'-deoxycytidine in human DNA: a new modified nucleoside which may perturb genome methylation.

    Science.gov (United States)

    Nair, Jagadeesan; Godschalk, Roger W; Nair, Urmila; Owen, Robert W; Hull, William E; Bartsch, Helmut

    2012-01-13

    Methylation of cytidine at dCpdG sequences regulates gene expression and is altered in many chronic inflammatory diseases. Inflammation generates lipid peroxidation (LPO) products which can react with deoxycytidine, deoxyadenosine, and deoxyguanosine in DNA to form pro-mutagenic exocyclic etheno-nucleoside residues. Since 5-methyl-2'-deoxycytidine (5mdC) residues exhibit increased nucleophilicity at N3, they should be even better targets for LPO products. We synthesized and characterized 3,N(4)-etheno-5-methyl-2'-deoxycytidine-3'-phosphate and showed that LPO products can indeed form the corresponding etheno-5mdC (ε5mdC) lesion in DNA in vitro. Our newly developed (32)P-postlabeling method was subsequently used to detect ε5mdC lesions in DNA from human white blood cells, lung, and liver at concentrations 4-10 times higher than that observed for etheno adducts on nonmethylated cytidine. Our new detection method can now be used to explore the hypothesis that this DNA lesion perturbs the DNA methylation status.

  11. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... ... Journal of Genetics; Volume 95; Issue 4. Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. CHENCUI HUANG KUN YU HUIYANG HUANG HAIHUI YE. RESEARCH ARTICLE Volume 95 Issue 4 December 2016 pp ...

  12. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    CHENCUI HUANG

    Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. CHENCUI HUANG1, KUN YU1, HUIYANG HUANG1,2 and HAIHUI YE1,2∗. 1College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, People's Republic of China.

  13. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain. [Huang C., Yu K., Huang H. and Ye H. 2016 Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. J. Genet.

  14. Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X.Q. [Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong University, Jinan (China); Department of Oral and Maxillofacial Surgery, The First People' s Hospital of Jining, Shandong (China); Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China); Huang, S.Y. [Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China); Zhang, D.S. [Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong University, Jinan (China); Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China); Zhang, S.Z.; Li, W.G.; Chen, Z.W.; Wu, H.W. [Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Affiliated to Shandong University, Jinan (China)

    2014-12-12

    Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

  15. Cloning and sequence analysis of lily and tobacco guanylate kinases.

    Science.gov (United States)

    Kumar, V

    2000-03-01

    Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phosphoryl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74-84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.

  16. Kinases and Cancer.

    Science.gov (United States)

    Cicenas, Jonas; Zalyte, Egle; Bairoch, Amos; Gaudet, Pascale

    2018-03-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...].

  17. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  18. The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    J. Zych

    2013-05-01

    Full Text Available Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs derived from bone marrow (BM-MSCs and adipose tissue (ADSCs using the chromatin-modifying agents trichostatin A (TSA, a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC, a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

  19. Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome.

    Directory of Open Access Journals (Sweden)

    Xiangning Qiu

    2010-09-01

    Full Text Available The hypomethylating agent 5-Azacytidine (5-Aza-CR is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS. Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT. While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA, which may potentially affect the activities of the drugs in patients.A hematopoietic (HL-60; acute myeloid leukemia and a solid (T24; transitional cell carcinoma cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2 and lasting (day 8 effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells.Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

  20. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  1. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin. cDNA cloned ...

  2. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  3. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

    Directory of Open Access Journals (Sweden)

    Stear Michael

    2003-03-01

    Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

  4. [Construction of chicken embryo fibroblasts cDNA expression library].

    Science.gov (United States)

    Liu, Wei; Gao, Yu-long; Gao, Hong-lei; Wang, Xiao-mei; Xu, Xiu-hong

    2007-06-01

    Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1 x 10(6) cfu/mL, total clones of 1.2 x 10(7) cfu and an average insertion size of about 2243 bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5 x 10(5) cfu/mL, total clones of 5.5 x 10(6) cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

  5. [The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

    Science.gov (United States)

    Liang, X; Gong, Y; Liu, Q; Li, J; Chen, B; Guo, C

    2001-02-01

    To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes. Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers. A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed. The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

  6. Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer

    International Nuclear Information System (INIS)

    Lemaire, Maryse; Chabot, Guy G; Raynal, Noël JM; Momparler, Louise F; Hurtubise, Annie; Bernstein, Mark L; Momparler, Richard L

    2008-01-01

    The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established. Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay. Increasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 μM). We have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is

  7. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  8. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

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

    1987-01-01

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

  9. Molecular cloning and characterization of a cDNA encoding ...

    African Journals Online (AJOL)

    enoh

    2012-03-29

    Nanjing) co., Ltd. The nucleotide sequences of these primers are as follows: ..... Ebizuka Y (2000). Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase. Biol. Pharm. Bull. 23(2):231-234.

  10. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  11. Screening of cDNA libraries on glass slide microarrays.

    Science.gov (United States)

    Berger, Dave K; Crampton, Bridget G; Hein, Ingo; Vos, Wiesner

    2007-01-01

    A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.

  12. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  13. Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray.

    Science.gov (United States)

    Chung, Eun Jung; Sung, Young Kwan; Farooq, Mohammad; Kim, Younghee; Im, Sanguk; Tak, Won Young; Hwang, Yoon Jin; Kim, Yang Il; Han, Hyung Soo; Kim, Jung-Chul; Kim, Moon Kyu

    2002-12-31

    We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.

  14. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  15. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  16. [Cold induced cDNA library construction of highland barley (Hordeum vulgare L. var. nudum Hk. f.) using suppression subtractive hybridization technology].

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

    Cold-induced genes of highland barley (Hordeum vulgare L. var. nudum Hk. f.) were studied using suppression subtractive hybridization (SSH) technique. The cDNA from the materials treated with 4 degrees C was used as "tester", and that from the materials growing in green house (20+/-2 degrees C) as "driver". A subtractive library of highland barley including 640 cDNA clones was constructed in this study. Enzyme digestion of 32 clones chosen randomly from the library indicated that 87.5% of them contained inserts. The cDNA inserts of 16 clones were sequenced. Blast search analyses showed that these cDNAs were homologies to genes encoding the following proteins: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1,5-bisphosphate carboxylase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein, and sugar transporter-like protein. These results indicated that the cDNA clones in the library were related to cold-induced genes, and suggested that the cold-tolerant mechanism of highland barley might be a complicated, interactive system involving multiple approaches and genes. Construction of subtractive cDNA library provided an advantage for further studies to isolate and clone cold-induced genes in highland barley.

  17. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

    Science.gov (United States)

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

    The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

  18. MgSlt2, a cellular integrity MAP kinase of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

    NARCIS (Netherlands)

    Mehrabi, R.; Lee, van der T.A.J.; Waalwijk, C.; Kema, G.H.J.

    2006-01-01

    Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1,242-bp open reading frame, and encodes a

  19. Equitoxic Doses of 5-Azacytidine and 5-Aza-2 ′ Deoxycytidine Induce Diverse Immediate and Overlapping Heritable Changes in the Transcriptome

    DEFF Research Database (Denmark)

    Qiu, X.; Hother, C.; Ralfkiær, U. M.

    2010-01-01

    influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. METHODOLOGY/PRINCIPAL FINDINGS: A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell......BACKGROUND: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical...... trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may...

  20. Gemcitabine, Pyrrologemcitabine, and 2'-Fluoro-2'-Deoxycytidines: Synthesis, Physical Properties, and Impact of Sugar Fluorination on Silver Ion Mediated Base Pairing.

    Science.gov (United States)

    Guo, Xiurong; Leonard, Peter; Ingale, Sachin A; Seela, Frank

    2017-12-14

    The stability of silver-mediated "dC-dC" base pairs relies not only on the structure of the nucleobase, but is also sensitive to structural modification of the sugar moiety. 2'-Fluorinated 2'-deoxycytidines with fluorine atoms in the arabino (up) and ribo (down) configuration as well as with geminal fluorine substitution (anticancer drug gemcitabine) and the novel fluorescent phenylpyrrolo-gemcitabine ( ph PyrGem) have been synthesized. All the nucleosides display the recognition face of naturally occurring 2'-deoxycytidine. The nucleosides were converted into phosphoramidites and incorporated into 12-mer oligonucleotides by solid-phase synthesis. The addition of silver ions to DNA duplexes with a fluorine-modified "dC-dC" pair near the central position led to significant duplex stabilization. The increase in stability was higher for duplexes with fluorinated sugar residues than for those with an unchanged 2'-deoxyribose moiety. Similar observations were made for "dC-dT" pairs and to a minor extent for "dC-dA" pairs. The increase in silver ion mediated base-pair stability was reversed by annulation of a pyrrole ring to the cytosine moiety, as shown for 2'-fluorinated ph PyrGem in comparison with phenylpyrrolo-dC ( ph PyrdC). This phenomenon results from stereoelectronic effects induced by fluoro substitution, which are transmitted from the sugar moiety to the silver ion mediated base pairs. The extent of the effect depends on the number of fluorine substituents, their configuration, and the structure of the nucleobase. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. cDNA library Table - KAIKOcDNA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us KAIKOcDNA... cDNA library Table Data detail Data name cDNA library Table DOI 10.18908/lsdba.nbd...c00951-005 Description of data contents List of Bombyx mori cDNA libraries. Data file File name: kaiko_cdna_...iption Registered library name Registered name of the partial cDNA library Library synonym Another name for cDNA... Download License Update History of This Database Site Policy | Contact Us cDNA library Table - KAIKOcDNA | LSDB Archive ...

  2. Study on construction of cDNA library of the treated changliver cell and quality analysis

    OpenAIRE

    Juntang, Lin; Pramanik, Jogenananda; Congrui, Wang; Huiyong, Zhang; Huigen, Feng; Baosheng, Yang; Yuchang, Li; Cunshuan, Xu

    2004-01-01

    The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5′ end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 1010 pfu/ml and the averag...

  3. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    DEFF Research Database (Denmark)

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A

    1999-01-01

    We have isolated and characterized the genomic clone of maize casein kinase 2 (CK2) alpha subunit using the previously described alphaCK2-1 cDNA clone as a probe. The genomic clone is 7.5 kb long and contains 10 exons, separated by 9 introns of different size, two larger than 1.5 kb and the other...

  4. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

    Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li; Lei, YuYang; Dan, WeiChao

    2017-06-01

    The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.

  5. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  6. cDNA libraries for virus-induced gene silencing.

    Science.gov (United States)

    Todd, Andrea T; Liu, Enwu; Page, Jonathan E

    2010-01-01

    Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

  7. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  8. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    Science.gov (United States)

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

  9. The catalytic domain of acanthamoeba myosin I heavy chain kinase. II. Expression of active catalytic domain and sequence homology to p21-activated kinase (PAK).

    Science.gov (United States)

    Brzeska, H; Szczepanowska, J; Hoey, J; Korn, E D

    1996-10-25

    Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by autophosphorylation. Activation by phospholipids is inhibited by Ca2+-calmodulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049-27055), we identified the catalytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase. In this paper, we report the cloning and sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specificity similar to that of native kinase and resistance to trypsin similar to that of fully phosphorylated MIHC kinase. MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of protein kinase A and similarly low sequence identity to the catalytic domains of protein kinase C- and calmodulin-dependent kinases, but 50% sequence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regulated by small GTP-binding proteins. The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserved Tyr and Ser/Thr residues in the region corresponding to the P+1 loop of protein kinase A. A synthetic peptide corresponding to this region of MIHC kinase is phosphorylated by both the expressed catalytic domain and native MIHC kinase.

  10. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    cell embryo and the expression was monitored continuously. The expression shown here is in developing embryo and freshly hatched fish. The intensity of green colour indicate the strong expression of EGFP in all the tissues of the embryo/fry. The expression of EGPF indicates the co-expression of catfish GH cDNA and the ...

  11. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 96; Issue 2. cDNA cloning, structural analysis, SNP detection and tissue ... Abstract. Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the ...

  12. Cloning and characterization of cDNA encoding xyloglucan ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... construction and restructuring of xyloglucan cross-links, thereby controlling the mechanical properties of cell wall. We cloned complete cDNA of an ..... are marked by horizontal lines. The conserved cysteine residues (amino acids 220, 229, 274 and 288 in P. glaucum) are marked by vertical blue arrows.

  13. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  14. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    [Naicy T., Venkatachalapathy T., Aravindakshan T., Raghavan K. C., Mini M. and Shyama K. 2017 cDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India. J. Genet. 96, xx–xx]. Introduction. Insulin-like growth factor 1 (IGF1), an important ...

  15. Construction of yeast surface-displayed cDNA libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    Using yeast display, heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall. Yeast display can be used to screen large expressed protein libraries for proteins or protein fragments with specific binding properties. Recently, yeast surface-displayed cDNA libraries have been constructed and used to identify proteins that bind to various target molecules such as peptides, small molecules, and antibodies. Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Coupled with fluorescence-activated cell sorting, yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface-displayed cDNA libraries using preexisting yeast two-hybrid cDNA libraries as a starting point.

  16. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  17. Selective Phosphorylation of South and North-Cytidine and Adenosine Methanocarba-Nucleosides by Human Nucleoside and Nucleotide Kinases Correlates with Their Growth Inhibitory Effects on Cultured Cells.

    Science.gov (United States)

    Sjuvarsson, Elena; Marquez, Victor E; Eriksson, Staffan

    2015-01-01

    Here bicyclo[3.1.0]hexane locked deoxycytidine (S-MCdC, N-MCdC), and deoxyadenosine analogs (S-MCdA and N-MCdA) were examined as substrates for purified preparations of human deoxynucleoside kinases: dCK, dGK, TK2, TK1, the ribonucleoside kinase UCK2, two NMP kinases (CMPK1, TMPK) and a NDP kinase. dCK can be important for the first step of phosphorylation of S-MCdC in cells, but S-MCdCMP was not a substrate for CMPK1, TMPK, or NDPK. dCK and dGK had a preference for the S-MCdA whereas N-MCdA was not a substrate for dCK, TK1, UCK2, TK2, dGK nucleoside kinases. The cell growth experiments suggested that N-MCdC and S-MCdA could be activated in cells by cellular kinases so that a triphosphate metabolite was formed. List of abbreviations: ddC, 2', 3'-didioxycytosine, Zalcitabine; 3TC, β-L-(-)-2',3'-dideoxy-3'-thiacytidine, Lamivudine; CdA, 2-cloro-2'-deoxyadenosine, Cladribine; AraA, 9-β-D-arabinofuranosyladenine; hCNT 1-3, human Concentrative Nucleoside Transporter type 1, 2 and 3; hENT 1-4, human Equilibrative Nucleoside Transporter type 1, 2, 3, and 4.

  18. Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    2001-01-01

    A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x 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 isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase 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 isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

  19. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  20. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  1. Isolation and characterization of a Coffea canephora ERF-like c-DNA

    African Journals Online (AJOL)

    A cDNA corresponding to an ERF gene has been isolated from a Coffea canephora fruit cDNA library. The cDNA was 1,317 nucleotides long and has an open reading frame of 987 bp. The predicted polypeptide showed a great similitude with equivalent proteins from others plant species. The binding domain shows 98.3% ...

  2. Molecular cloning of growth hormone encoding cDNA of Indian

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  3. cDNA cloning and characterization of a mannose-binding lectin

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  4. cDNA library information - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available (C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to const...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library construction

  5. [CDNA cloning of human leptin and its expression].

    Science.gov (United States)

    Jia, Zhen-Yu; Fu, Xiao-Min; Jin, Ai-Hua; Cao, Jiang

    2003-07-01

    To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.

  6. Construction of cDNA library of Pyrocystis lunula (Pyrophyta)

    Science.gov (United States)

    Sui, Zhenghong; Kowallik, Klaus V.

    2004-10-01

    Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20µgg-1 net cells, and the band intensity ratio of 28S/18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.

  7. Construction of cDNA libraries in vaccinia virus.

    Science.gov (United States)

    Smith, Ernest S; Shi, Shuying; Zauderer, Maurice

    2004-01-01

    Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and useful properties as a library vector. Most importantly, because poxviruses are packaged into fully infectious particles in the cell cytoplasm, specific recombinants can be readily recovered even from a very small number of selected cells. Moreover, in contrast to libraries constructed in retrovirus or plasmid-based vectors, recombinant vaccinia virus can be efficiently recovered even from cells that have been induced to undergo apoptosis or cessation of cell growth. In the past, the major obstacle in this application to poxviruses has been the low frequency with which recombinants can be generated. The most commonly used method to construct recombinant poxvirus is homologous recombination. The frequency of recombinants derived in this manner is of the order of 0.1%, sufficient to recover a recombinant of a purified DNA clone in a transfer plasmid, but far too low to permit construction of a representative cDNA library. We have developed a method that generates nearly 100% recombinant vaccinia viruses at good titer. We have termed this method trimolecular recombination. cDNA libraries of as many as 107 or more independent viral recombinants can be constructed by trimolecular recombination. For the first time, large, diverse, and representative cDNA libraries can be screened in a vaccinia virus-based expression vector.

  8. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    E-mail: naicy@kvasu.ac.in. and conception rate ... transformed into DH5α strain of Escherichia coli and the clones harbouring ... Primer pairs for caprine IGF1 and GAPDH were designed using Primer3 software (table 1). RTq-PCR was conducted in a 25 μL reaction volume containing 50 ng of cDNA and 2× Max- ima SYBR ...

  9. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    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 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  10. cDNA library generation from ribonucleoprotein particles.

    Science.gov (United States)

    Rederstorff, Mathieu; Hüttenhofer, Alexander

    2011-02-01

    Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

  11. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    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 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  12. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  13. cDNA cloning and mRNA expression of cat and dog Cdkal1

    Directory of Open Access Journals (Sweden)

    Sako T

    2012-08-01

    Full Text Available Ichiro Yamamoto, Shingo Ishikawa, Li Gebin, Hiroshi Takemitsu, Megumi Fujiwara, Nobuko Mori, Yutaka Hatano, Tomoko Suzuki, Akihiro Mori, Nobuhiro Nakao, Koh Kawasumi, Toshinori Sako, Toshiro AraiLaboratory of Veterinary Biochemistry, Nippon Veterinary and Life Science University, Tokyo, JapanAbstract: The cyclin-dependent kinase 5 regulatory subunit–associated protein 1–like 1 (CDKAL1 gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabetes in humans. In this study, we performed complementary DNA cloning for Cdkal1 in the cat and dog and characterized the tissue expression profiles of its messenger RNA. Cat and dog Cdkal1 complementary DNA encoded 576 and 578 amino acids, showing very high sequence homology to mammalian CDKAL1 (>88.4%. Real-time polymerase chain reaction analyses revealed that Cdkal1 messenger RNA is highly expressed in smooth muscle and that tissue distribution of Cdkal1 is similar in cats and dogs. Genotyping analysis of single-nucleotide polymorphism for cat Cdkal1 revealed that obese cats had different tendencies from normal cats. These findings suggest that the cat and dog Cdkal1 gene is highly conserved among mammals and that cat Cdkal1 may be a candidate marker for genetic diagnosis of obesity.Keywords: cat, dog, Cdkal1, obese, cDNA cloning, Q-PCR

  14. [cDNA library construction from panicle meristem of finger millet].

    Science.gov (United States)

    Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

    2014-01-01

    The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

  15. [Construction and identification of the expression library of album pollen allergens cDNA].

    Science.gov (United States)

    Zhang, Jie; Sun, Xiu-zhen; Yan, Hong; Zhang, Ni; Feng, Xiang-li

    2011-05-01

    To construct and identify the express library of album pollen allergens cDNA. Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

  16. Assignment of the murine protein kinase gene DLK to chromosome 15 in the vicinity of the bt/Koa locus by genetic linkage analysis

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Toshio; Yanagisawa, Masahiro; Matsubara, Nobumichi [Tokyo Univ. (Japan)] [and others

    1997-03-01

    We have cloned protein kinase genes from murine primordial germ cell-derived EG cells by a PCR-based strategy using degenerate primers corresponding to the conserved sequences in the catalytic domain of protein kinases. One of these clones, designated Gek2 (germ cell kinase 2), was used as a probe for screening of a mouse brain cDNA library and obtained clones contained an entire coding sequence. Comparison of the sequence of Gek2 with those in databases revealed that it was identical to a previously reported protein kinase gene, DLK. 8 refs., 1 fig.

  17. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

    Science.gov (United States)

    Klar, Agnes S; Gopinadh, Jakka; Kleber, Sascha; Wadle, Andreas; Renner, Christoph

    2015-01-01

    NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC). We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels. These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

  18. DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

    Science.gov (United States)

    Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

    2012-03-01

    Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

    Directory of Open Access Journals (Sweden)

    Agnes S Klar

    Full Text Available NY-ESO-1 belongs to the cancer/testis antigen (CTA family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC.We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165 peptide specific chimeric antigen receptor (CAR CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

  20. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  1. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  2. Construction and characterization of a normalized cDNA library.

    OpenAIRE

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-01-01

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each...

  3. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  4. [Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

    Science.gov (United States)

    Zhang, Zhe; Wu, Xiang-Yu; Feng, Lu; Huang, Shang-Ke; Luo, Min-Na; Shao, Shan; Zhao, Xin-Han

    2016-09-01

    To construct a cDNA phage expression library for human esophageal cancer cells. After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked with Eco R1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×10⁶ pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

  5. Characterization of a cDNA encoding cottonseed catalase.

    Science.gov (United States)

    Ni, W; Turley, R B; Trelease, R N

    1990-06-21

    A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

  6. Infectious Maize rayado fino virus from Cloned cDNA.

    Science.gov (United States)

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

    2015-06-01

    A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

  7. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    Science.gov (United States)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  8. Liganded thyroid hormone receptor inhibits phorbol 12-O-tetradecanoate-13-acetate-induced enhancer activity via firefly luciferase cDNA.

    Directory of Open Access Journals (Sweden)

    Hiroko Misawa

    Full Text Available 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

  9. Preparation of full-length cDNA libraries: focus on metazoans.

    Science.gov (United States)

    Harada, Masako; Hayashizaki, Yoshihide

    2009-01-01

    Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

  10. Solid-phase cDNA library construction, a versatile approach.

    OpenAIRE

    Roeder, T

    1998-01-01

    A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for m...

  11. [Software development in data analysis and mining for cDNA microarray].

    Science.gov (United States)

    Wu, Bin; Wang, Jianguo; Wang, Miqu

    2007-12-01

    Data analysis and mining is a key issue to microarray technology and is usually implemented through software development. This paper summarizes the state-of-art software development in cDNA microarray data analysis and mining. The updated software developments are discussed in three stages: data inquisition from cDNA microarray tests, statistical treatment of cDNA data and data mining from gene network.

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

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

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

  13. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

  14. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    Directory of Open Access Journals (Sweden)

    Kim Kyu-Tae

    2010-07-01

    Full Text Available Abstract Background DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC and Trichostatin A (TSA respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Method Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC. Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Results Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS. In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. Conclusion These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically

  15. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    International Nuclear Information System (INIS)

    Mossman, David; Kim, Kyu-Tae; Scott, Rodney J

    2010-01-01

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of

  16. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  17. Retrospective natural history of thymidine kinase 2 deficiency.

    Science.gov (United States)

    Garone, Caterina; Taylor, Robert W; Nascimento, Andrés; Poulton, Joanna; Fratter, Carl; Domínguez-González, Cristina; Evans, Julie C; Loos, Mariana; Isohanni, Pirjo; Suomalainen, Anu; Ram, Dipak; Hughes, M Imelda; McFarland, Robert; Barca, Emanuele; Lopez Gomez, Carlos; Jayawant, Sandeep; Thomas, Neil D; Manzur, Adnan Y; Kleinsteuber, Karin; Martin, Miguel A; Kerr, Timothy; Gorman, Grainne S; Sommerville, Ewen W; Chinnery, Patrick F; Hofer, Monika; Karch, Christoph; Ralph, Jeffrey; Cámara, Yolanda; Madruga-Garrido, Marcos; Domínguez-Carral, Jana; Ortez, Carlos; Emperador, Sonia; Montoya, Julio; Chakrapani, Anupam; Kriger, Joshua F; Schoenaker, Robert; Levin, Bruce; Thompson, John L P; Long, Yuelin; Rahman, Shamima; Donati, Maria Alice; DiMauro, Salvatore; Hirano, Michio

    2018-03-30

    Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. The study was conducted by 42 investigators across 31 academic medical centres. We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  18. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  19. Construction and analysis of a cDNA library from yellow-fruit ginseng

    African Journals Online (AJOL)

    The total RNA was isolated from yellow-fruit ginseng (Panax ginseng C.A. Meyer) leaf tissue. A cDNA library of panax ginseng leaves was constructed by using pDNR-LIB vector according to the SMART cDNA library construction kit protocol. We obtained 378 high quality sequences (GenBank accession number: ...

  20. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ile URL: ftp://ftp.biosciencedbc.jp/archive/rpd/LATEST/rpd_cdna.zip File size: 15 KB Simple search URL http:...age About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA table - RPD | LSDB Archive ...

  1. [Construction of cDNA library of Magnaporthe grisea with magnetic bead].

    Science.gov (United States)

    Feng, Xu; Xiaoli, Wu; Dewen, Qiu

    2008-06-01

    We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3'terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. The cDNA library constructed had a high titer of 8.9 x 10(6) cfu/mL, and contained a total clones of 8.9 x 10(7) cfu, with an average inserts size of about 1380 bp. Constructing cDNA library with magnetic bead was a highly efficient method using only small amount of experimental materials within a short period.

  2. Development of a simple and powerful method, cDNA AFLP-SSPAG ...

    African Journals Online (AJOL)

    Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on polyacylamide gels. The cDNA was then reamplified, cloned and fragments were sequenced. Sequenced clones were used as probes in northern dot blot analyses and library screening. Full-length cDNA was cloned from a library ...

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  4. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB ...

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

    International Nuclear Information System (INIS)

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

    1989-01-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K 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

  6. Cloning and sequence analysis of H. contortus HC58cDNA gene ...

    African Journals Online (AJOL)

    Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the cathepsin B like proteases, suggesting that HC58cDNA was a member of the papain family. Keywords:Haemonchus contortus, HC58cDNA, cathepsin B like protease, papain family. Kenya Veterinarian Vol.

  7. [A novel vector for construction of a cDNA library].

    Science.gov (United States)

    Fedchenko, V I; Kaloshin, A A; Medvedev, A E

    2010-01-01

    A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

  8. Formation of fused-ring 2'-deoxycytidine adducts from 1-chloro-3-buten-2-one, an in vitro 1,3-butadiene metabolite, under in vitro physiological conditions.

    Science.gov (United States)

    Sun, Liang; Pelah, Avishay; Zhang, Dong-Ping; Zhong, Yu-Fang; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu; Elfarra, Adnan A

    2013-10-21

    1-Chloro-3-buten-2-one (CBO) is a potential metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO is a bifunctional alkylating agent that readily reacts with glutathione (GSH) to form mono-GSH and di-GSH adducts. Recently, CBO and its precursor 1-chloro-2-hydroxy-3-butene (CHB) were found to be cytotoxic and genotoxic in human liver cells in culture with CBO being approximately 100-fold more potent than CHB. In the present study, CBO was shown to react readily with 2'-deoxycytidine (dC) under in vitro physiological conditions (pH 7.4, 37 °C) to form four dC adducts with the CBO moieties forming fused rings with the N3 and N(4) atoms of dC. The four products were structurally characterized as 2-hydroxy-2-hydroxymethyl-7-(2-deoxy-β-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-1 and dC-2, a pair of diastereomers), 4-chloromethyl-4-hydroxy-7-(2-deoxy-β-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-3), and 2-chloromethyl-2-hydroxy-7-(2-deoxy-β-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-4). Interestingly, dC-1 and dC-2 were stable under our experimental conditions (pH 7.4, 37 °C, and 6 h) and existed in equilibrium as indicated by HPLC analysis, whereas dC-3 and dC-4 were labile with the half-lives being 3.0 ± 0.36 and 1.7 ± 0.06 h, respectively. Decomposition of dC-4 produced both dC-1 and dC-2, whereas acid hydrolysis of dC-1/dC-2 and dC-4 in 1 M HCl at 100 °C for 30 min yielded the deribosylated adducts dC-1H/dC-2H and dC-4H, respectively. Because fused-ring dC adducts of other chemicals are mutagenic, the characterized CBO-dC adducts could be mutagenic and play a role in the cytotoxicity and genotoxicity of CBO and its precursors, CHB and BD. The CBO-dC adducts may also be used as standards to characterize CBO-DNA adducts and to develop potential biomarkers for CBO formation in vivo.

  9. Construction and characterization of a normalized cDNA library.

    Science.gov (United States)

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-09-27

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

  10. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  11. A plant homologue to mammalian brain 14-3-3 protein and protein kinase C inhibitor.

    Science.gov (United States)

    Hirsch, S; Aitken, A; Bertsch, U; Soll, J

    1992-01-20

    We have isolated cDNA clones of Spinacea oleracea L. and Oenothera hookeri of 930 and 1017 base pairs, respectively. The open reading frame deduced from the Oenothera sequence codes for a protein of a calculated molecular mass of 29,200. The primary amino acid sequence exhibits a very high degree (88%) of homology to the 14-3-3 protein from bovine brain, and protein kinase C inhibitor from sheep brain. Subsequently the plant protein was partially purified from leaf extract. The partially purified plant protein inhibited protein kinase C from sheep brain in a heterologous assay system. The active fraction consisted of 5-6 different polypeptides of similar molecular size. One of these proteins crossreacted with a peptide-specific antibody against protein kinase C inhibitor protein from sheep brain.

  12. Osmotic Stress Induces the Expression of VvMAP Kinase Gene in Grapevine (Vitis vinifera L.

    Directory of Open Access Journals (Sweden)

    Samia Daldoul

    2012-01-01

    Full Text Available Abiotic stress adversely affects the growth of grapevine plants. In order to study the early expression changes of genes particularly involved in signal transduction upon salt and drought stresses in grapevines, ESTs derived from a suppressive subtractive hybridization approach (SSH were selected for expression studies. We were particularly interested in the expression behaviour of the MAP kinase cDNA clone identified by differential screening of the salt-stressed SSH libraries. Interestingly, VvMAP kinase transcript showed a differential expression towards salt and drought treatment in the salt tolerant cultivar Razegui. The upregulation of this transcript was confirmed by RNA blot analysis. Our results revealed that the VvMAP kinase gene could be classified as an osmotic stress responsive gene as its expression was induced by salinity and drought. Furthermore, our study provides the basis for future research on the diverse signaling pathways mediated by MAPKs in grapevine.

  13. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  14. Human Leukocyte Antigen-G Is Frequently Expressed in a Multicentric Study on Glioblastoma and May Be Induced in Vitro by Combined 5-aza-2'-deoxycytidine and Interferon-γ Treatments

    DEFF Research Database (Denmark)

    Wastowski, Isabela J; Simões, Renata T; Yaghi, Layale

    2012-01-01

    -G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2'-deoxycytidine treatment and enhanced......Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly...... expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA...

  15. [Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

    Science.gov (United States)

    Qin, Sheng-fang; Tan, Wei-dong; Chen, You-nan; Ding, Yang; Li, Sheng-fu; Li, Hong-xia; Wang, Li; Yang, Rong; Lu, Yan-rong

    2007-06-01

    To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

  16. [cDNA libraries construction and screening in gene expression profiling of disease resistance in wheat].

    Science.gov (United States)

    Luo, Meng; Kong, Xiu-Ying; Liu, Yue; Zhou, Rong-Hua; Jia, Ji-Zeng

    2002-09-01

    A wheat line, Bai Nong 3217/Mardler BC5F4 with resistance to powdery mildew, was used to construct a conventional cDNA library and a suppression subtractive hybridization (SSH) cDNA library from wheat leaves inoculated by Erysiphe graminis DC. Three hundred and eighty-seven non-redundant ESTs from the conventional cDNA library and 760 ESTs from the SSH cDNA library were obtained, and the ESTs similarity analysis using BLASTn and BLASTx were conducted by comparing these ESTs with sequences in GenBank. The results showed that the redundancy of some kinds of genes such as photosynthesis related genes and ribosome related genes was higher in the conventional cDNA library but the varieties and quantities of disease resistance genes were less than those in the SSH cDNA library. The SSH cDNA library was found to have obvious advantages in gene expression profiling of disease resistance such as simple library construction procedure, rich specific DRR (disease-resistance-related) genes and decreased sequencing amount. To acquire genes that were involved in the powdery mildew resistance of wheat, hybridization with high-density dots membranes was used to screen the two libraries. The result showed that the method was relatively simple in operation, and the membranes could be used for many times. But some problems also existed with this screening method. For instance, a large amount of mRNA and radioactive isotope were needed and the hybridization procedure must be repeated several times to obtain stable hybridization results. About 54.1% function-known ESTs in the SSH cDNA library were identified to be DRR genes by screening. There were 247 clones of the SSH cDNA library that had positive signal in the repeated hybridizations with the pathogen uninfected probe. The identified DRR genes distributed in the whole procedure of powdery mildew resistance, but mainly focused on the SAR (systemic of acquired resistance).

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

    International Nuclear Information System (INIS)

    Spicer, E.K.; Horton, R.; Bloem, L.

    1987-01-01

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

  18. Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

    OpenAIRE

    Goddard, J M; Caput, D; Williams, S R; Martin, D W

    1983-01-01

    We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled c...

  19. Generation of cDNA expression libraries enriched for in-frame sequences

    OpenAIRE

    Davis, Claytus A.; Benzer, Seymour

    1997-01-01

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore deve...

  20. Generation of full-length cDNA libraries: focus on plants.

    Science.gov (United States)

    Seki, Motoaki; Kamiya, Asako; Carninci, Piero; Hayashizaki, Yoshihide; Shinozaki, Kazuo

    2009-01-01

    Full-length cDNAs are essential for the correct annotation of transcriptional units and gene products from genomic sequence data and for functional analysis of the genes. Full-length cDNA libraries are very important resources for isolation of the full-length cDNAs. The biotinylated cap trapper method using the trehalose-thermostabilized reverse transcriptase has been developed and has become an efficient method for construction of high-content full-length cDNA libraries. We have constructed full-length cDNA libraries from various plants and animals using this method. The protocol of the method is described in this chapter.

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

    Science.gov (United States)

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

    1994-01-01

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

  2. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

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

  3. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  4. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76...... in human. Although additional minor bands of 1.5 and 3.3 kb were found in Northern blots, RT-PCR (reverse transcription polymerase chain reaction) analysis failed to provide evidence that these minor bands are products of the tetranectin gene. Finally, the genetic map location for this gene, Tna...

  5. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    length τ-crystallin cDNA from crocodilian lens and α-enolase from other tissues. ... human (Acc. No. NM_001428). The sequences were used to construct a phylogenetic tree depicting gene lineage, using the clustering program DNAML.

  6. Construction and characterization of a cDNA expression library from the endangered Hu sheep.

    Science.gov (United States)

    Hu, P-F; Li, X-C; Liu, H-K; Guan, W-J; Ma, Y-H

    2014-10-31

    Hu sheep is one of the most important species in China; it is also listed as one of the 78 nationally protected domestic animals by the Chinese government in 2000. The construction of cDNA expression library of Hu sheep is of great significance for protecting individual genomes, generating transgenic sheep, and conducting clinical research using cDNA from Hu sheep. In this study, the total RNA from the ear tissue of Hu sheep was extracted, and a cDNA expression library was constructed using the SMART(TM) technique. The titer of amplified cDNA library was 1.09 x 10(10) PFU/mL, the rate of recombination was above 91.6%, and the average size of fragments was 1.1 kb. This study has an important significance for the preservation of Hu sheep resources at the genome level.

  7. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  8. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available ing/transcriptional initiation regionss About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA - ASTRA | LSDB Archive ...

  9. Construction of primary and subtracted cDNA libraries from early embryos.

    Science.gov (United States)

    Rothstein, J L; Johnson, D; Jessee, J; Skowronski, J; DeLoia, J A; Solter, D; Knowles, B B

    1993-01-01

    By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification

  10. Assignment of casein kinase 2 alpha sequences to two different human chromosomes

    DEFF Research Database (Denmark)

    Boldyreff, B; Klett, C; Göttert, E

    1992-01-01

    Human casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter...... and one on 20p13. The existence of two separate chromosomal loci suggests that CK-2 alpha is a member of a gene family. Only the locus on chromosome 11 was confirmed by somatic cell hybrid analysis. The analysis was based on the presence of a CK-2-alpha-specific 20-kb fragment. However, the CK-2 alpha c...

  11. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    International Nuclear Information System (INIS)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

    2003-01-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  12. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  13. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  14. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

    OpenAIRE

    Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

    1999-01-01

    A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

  15. Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

    Science.gov (United States)

    Long, Jian-Er; He, Li-Qiang; Cai, Xia; Ren, Zhao-Rui; Huang, Shu-Zhen; Zeng, Yi-Tao

    2006-11-01

    To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  17. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 ...

  18. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  19. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

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

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ 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 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  20. Trehalose as a good candidate for enriching full-length cDNAs in cDNA library construction.

    Science.gov (United States)

    Chen, Lei; Cao, Lixue; Zhou, Longhai; Jing, Yudong; Chen, Zuozhou; Deng, Cheng; Shen, Yu; Chen, Liangbiao

    2007-01-10

    It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

  1. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Science.gov (United States)

    Yoon, Ju-Yeon; Cho, In-Sook; Choi, Gug-Seoun; Choi, Seung-Kook

    2014-01-01

    Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants. PMID:25288987

  2. Construction of a T7 Human Lung Cancer cDNA Library

    Directory of Open Access Journals (Sweden)

    Wentao YUE

    2008-10-01

    Full Text Available Background and objective Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Results Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106 pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5×1010 pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24 in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24. Insert range from 300 bp to 1 500 bp. Conclusion Two phage display cDNA library from NSCLC were constructed.

  3. [Construction of suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus].

    Science.gov (United States)

    Wu, Jia-hong; Zhao, Tong-yan; Dong, Yan-de

    2006-08-01

    To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Total RNA was extracted from the deltamethrin-resistant (R-lab) and -sensitive (S-lab) isolates, mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%, with a length of cDNA fragments ranged from 150bp to 750bp. The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

  4. [Construction of the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei].

    Science.gov (United States)

    Bei, Zhu-chun; Wang, Jing-yan

    2004-06-01

    To construct the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei using suppression subtractive hybridization PCR (SSH PCR). Total RNA was extracted from the artemisinin-sensitive (NS) and artemisinin-resistant (AR) strains of Plasmodium berghei K173. The cDNA synthesis followed the protocol of super SMART cDNA synthesis kit. Taking the NS as driver, AR as tester and reverse, two subtractions were performed by SSH PCR. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries of NS-AR and AR-NS contained 395 and 506 positive clones respectively. The PCR results of 108 clones picked randomly from each library showed 100 and 104 positive inserts contained in the plasmids respectively, and distributing in 250-2000 bp. The successful construction of the subtracted cDNA libraries related to artemisinin-resistance of P. berghei enable us to identify the different expressed genes involved in the resistance mechanism.

  5. [cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

    Science.gov (United States)

    Xu, Lei; Wang, Ling-ling; Liu, Shuo; Ling, Yuan; Ma, Lie; Wang, Qun; Zhang, Li-jiao; He, Xiao-yu; Zhao, Ming-jing; Wang, Xiao-ge

    2012-03-01

    To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

  6. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  7. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  8. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  9. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  10. Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

    Science.gov (United States)

    Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

    2017-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8 + T cell responses in vivo . As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4 + and CD8 + T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4 + T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4 + and CD8 + T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4 + T cells responding to Ag85B- and ESAT-6

  11. Tv-RIO1 – an atypical protein kinase from the parasitic nematode Trichostrongylus vitrinus

    Directory of Open Access Journals (Sweden)

    Sternberg Paul W

    2008-09-01

    Full Text Available Abstract Background Protein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs and atypical protein kinases (aPKs; RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite. Results A full-length cDNA (Tv-rio-1 encoding a RIO1 protein kinase (Tv-RIO1 was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida. The uninterrupted open reading frame (ORF of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3, and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1. Conclusion This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes.

  12. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  13. [Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

    Science.gov (United States)

    Sun, Yi; Jia, Ren-chu; Liu, Jin-ming; Yuan, Chun-xiu; Shi, Yao-jun; Lu, Ke; Fu, Zhi-qiang; Sun, Huan; Cai, You-min; Lin, Jiao-jiao

    2007-10-01

    To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

  14. [Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

    Science.gov (United States)

    Chai, Hui-ping; Liu, Guang-yuan; Zhang, Lin; Gong, Zhen-li; Xie, Jun-ren; Tian, Zhan-cheng; Wang, Lu; Jia, Ning

    2009-02-28

    To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

  15. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Science.gov (United States)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  16. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, L.A.; Zhang, H. Nasir, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1994-09-01

    Mucopolysaccharidosis I (MPS I) are a group of disorders caused by deficiency of the lysosomal enzyme alpha-L-iduronidase. The characterization of the human gene and the identification of mutations underlying MPS I in humans has led to the delineation of the molecular basis of this disorder. Model systems are now needed for the evaluation and development of therapeutics for this disorder. Both canine and feline models for MPS type I have been described but only the canine gene has been isolated and characterized. We report here the cloning and expression of the murine alpha-L-iduronidase cDNA. The murine cDNA was obtained by screening a mouse liver cDNA library with a probe from the human cDNA. The full length murine cDNA is 3120 base pairs in length and thus is considerably larger than both the human and canine transcripts. The increase in size is due to a 1.2 kb 3{prime} untranslated region in the murine cDNA that contains a CA dinucleotide repeat. Within the coding region the murine cDNA shows sequences. At the protein level the murine protein shows 77% similarity with the human protein and 75% similarity with the canine protein. There are significant differences in both the start and stop sites with the murine protein 9 amino acids shorter at both the N terminal signal peptide region and the C terminus. Expression of the murine cDNA in COS-1 cells resulted in a 20 fold increase in intracellular alpha-L-iduronidase activity as well as the detection of considerable enzyme activity in the culture medium. Comparison of the reported missense mutations underlying MPS I in humans (A75T, H82P, R89Q, L218P, P533R, Q310X, T366P) has shown conservation of these amino acid residues in the murine protein. The isolation of the murine iduronidase cDNA will now allow for the development of a murine model for MPS I.

  17. Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

    Full Text Available Abstract Background Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance, that calculates the relative abundance of genes in cDNA libraries. Results DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach. The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone. Conclusions The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with

  18. Kinetic and photochemical studies and alteration of ultraviolet sensitivity of Escherichia coli thymidine kinase of halogenated allosteric regulators and substrate analogues

    International Nuclear Information System (INIS)

    Chen, M.S.; Prusoff, W.H.

    1977-01-01

    The effect of various halogenated and nonhalogenated allosteric effectors on the sensitivity of Escherichia coli thymidine kinase to ultraviolet radiations (uv,253.7 nm) was investigated. All naturally occurring dNTPs convert the monomeric form of the enzyme into the dimeric form which is less sensitive to uv inactivation. Whereas 5-iodo-2'-deoxycytidine triphosphate (IdCTP) and 5-iodo-2' deoxyuridine triphosphate (IdUTP) enhance the uv inactivation of the enzyme, 5-bromo-2'-deoxyuridine triphosphate and 5-bromo-2'-deoxycytidine triphosphate exert a protective effect similar to that produced by the corresponding naturally occurring effectors, dTTP and dCTP. The enhanced uv inactivation by IdUTP is prevented totally by dTTP, but only partially by dCTP or dThd, whereas the enhanced sensitization by IdCTP is prevented almost totally by dCTP, partially by dTTP, and not at all by dThd. The uv sensitization of thymidine kinase by IdCTP appears to be at the regulatory site since a maximum saturation effect is observed, and the concentration required to exert a 50% maximal uv sensitization is similar to its K/sub m/ for enhancement of catalytic activity. When the enzyme was irradiated in the presence of either [2- 14 C]IdUTP or [2- 14 C]IdUrd, zone sedimentation analysis in sucrose density gradients showed the sedimentation coefficient of the radioactive labeled proteins to be the same, 3.8 S. Hence uv irradiation of the effector-induced dimer resulted in not only dissociation to the monomer, but also complete loss of catalytic activity. The substitution of an azido group for the 5'-OH group of 5-iodo-, 5-bromo-, 5-chloro-, or 5-fluorodeoxyuridine greatly decreased their affinity for thymidine kinase, and in addition the kinetics of inhibition changed from a competitive to a noncompetitive pattern. The presence of the azido moiety in the 5' position of the halogenated nucleosides did not enhance the rate of uv inactivation of the enzyme

  19. Generation of cDNA expression libraries enriched for in-frame sequences.

    Science.gov (United States)

    Davis, C A; Benzer, S

    1997-03-18

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

  20. [Construction of a yeast two-hybrid cDNA library from the human testis].

    Science.gov (United States)

    Zheng, Ying; Zhang, Lu-Ping; Jia, Xiao-Qin; Wang, Hai-Yan

    2012-04-01

    To construct a human testis cDNA library for yeast two-hybrid screening. Human normal testis mRNA was purified from total RNA, and ds cDNA was synthesized and amplified using primers SMART III and CDS III oligo (dT) as the base of recombination. The purified PCR products and linearized plasmid pGADT7-Rec were co-transformed into the competent yeast Y187 and recombined by yeast homologous recombinase in the yeast cells to form an active cyclic plasmid. All the clones growing on the SD/-Leu plates were harvested to constitute a human testis cDNA library. We constructed a human testis cDNA library with high multiplication and adequate capacity, from which 2.0 x 10(6) recombinants were obtained. The amplified PCR fragments were between 0.3 kb and 4.0 kb in length. The yeast two-hybrid cDNA library of human testis was successfully constructed by the Clontech SMART method, which has prepared a ground for further studies on the molecular mechanism of spermatogenesis.

  1. [Construction of cDNA expression library of salivary gland from Boophilus microplus].

    Science.gov (United States)

    Tian, Zhan-Cheng; Liu, Guang-Yuan; Xie, Jun-Ren; Gong, Zhen-Li

    2008-10-30

    Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo (dT)-primed cDNA with added directional EcoR I/Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/Hind III arms of the lambda SCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.38x10(6) PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2x10(9) PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.

  2. Construction of equalized short hairpin RNA library from human brain cDNA.

    Science.gov (United States)

    Xu, Lei; Li, Jingqi; Liu, Li; Lu, Lixia; Gao, Jingxia; Li, Xueli

    2007-02-20

    Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.

  3. Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

    Science.gov (United States)

    Jayasekara, Walimuni Samantha Nilanthi; Yonezawa, Tomohiro; Ishida, Maho; Yamanouchi, Keitaro; Nishihara, Masugi

    2004-06-01

    20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

  4. [Combining SSH and cDNA microarray for identification of lung cancer related genes].

    Science.gov (United States)

    Fan, Baoxing; Zhang, Kaitai; Da, Jiping; Xie, Ling; Wang, Shengqi; Wu, Dechang

    2003-04-20

    To screen and identify differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and some other kinds of tumor tissues using suppression subtractive hybridization (SSH) and cDNA Microarray. One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries which came from BEP2D cell lines during three different malignant transformed phases. Then the clones were hybridizated with cDNA probes which extracted from 15 cases of lung cancer tissues, 5 cases of paracancerous pulmonary tissues and 24 cases of other 8 kinds of tumor tissues respectively. Twenty-six cDNAs were obtained which expressed higher in lung cancer tissues than that in paracancerous pulmonary tissues. Thirty-one cDNAs expressed remarkably higher in paracancerous tissues than those in cancer tissues. Compared with other 8 kinds of tumors, paracancerous tissues had 63 overexpressed cDNAs and lung cancer tissues had 87 overexpressed cDNAs. The combination of SSH and cDNA microarray is rapid and effective for screening and identification of differentially expressed genes in different samples. It may be potentially useful for diagnosis of lung cancer to further study the differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and other tumor tissues.

  5. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  6. Overexpression of Populus trichocarpa Mitogen-Activated Protein Kinase Kinase4 Enhances Salt Tolerance in Tobacco

    Directory of Open Access Journals (Sweden)

    Chengjun Yang

    2017-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK is one of the factors of cascade reactions affecting responses to signal pathway of environmental stimuli. Throughout the life of plants, MAPK family members participate in signal transduction pathways and regulate various intracellular physiological and metabolic reactions. To gain insights into regulatory function of MAPK kinase (MAPKK in Populus trichocarpa under salt stress, we obtained full-length cDNA of PtMAPKK4 and analyzed different expression levels of PtMAPKK4 gene in leaves, stems, and root organs. The relationship between PtMAPKK4 and salt stress was studied by detecting expression characteristics of mRNA under 150 mM NaCl stress using real-time quantitative polymerase chain reaction. The results showed that expression of PtMAPKK4 increased under salt (NaCl stress in leaves but initially reduced and then increased in roots. Thus, salt stress failed to induce PtMAPKK4 expression in stems. PtMAPKK4 possibly participates in regulation of plant growth and metabolism, thereby improving its salt tolerance. We used Saccharomyces cerevisiae strain INVScI to verify subcellular localization of PtMAPKK4 kinase. The yeast strains containing pYES2-PtMAPKK4-GFP plasmid expressed GFP fusion proteins under the induction of d-galactose, and the products were located in nucleus. These results were consistent with network prediction and confirmed location of PtMAPKK4 enzyme in the nucleus. We tested NaCl tolerance in transgenic tobacco lines overexpressing PtMAPKK4 under the control of 35S promoter at germination stage to detect salt tolerance function of PtMAPKK4. Compared withK326 (a wild-type tobacco, lines overexpressing PtMAPKK4 showed a certain degree of improvement in tolerance, germination, and growth. NaCl inhibited growth of overexpressed line and K326 at the seedling stage. However, statistical analysis showed longer root length, higher fresh weight, and lower MDA content in transgenic lines in

  7. Isolation of a full-length mitotic cyclin cDNA clone CycIIIMs from Medicago sativa: chromosomal mapping and expression.

    Science.gov (United States)

    Savouré, A; Fehér, A; Kaló, P; Petrovics, G; Csanádi, G; Szécsi, J; Kiss, G; Brown, S; Kondorosi, A; Kondorosi, E

    1995-03-01

    Cyclins in association with the protein kinase p34cdc2 and related cyclin-dependent protein kinases (cdks) are key regulatory elements in controlling the cell division cycle. Here, we describe the identification and characterization of a full-length cDNA clone of alfalfa mitotic cyclin, termed CycIIIMs. Computer analysis of known plant cyclin gene sequences revealed that this cyclin belongs to the same structural group as the other known partial alfalfa cyclin sequences. Genetic segregation analysis based on DNA-DNA hybridization data showed that the CycIIIMs gene(s) locates in a single chromosomal region on linkage group 5 of the alfalfa genetic map between RFLP markers UO89A and CG13. The assignment of this cyclin to the mitotic cyclin class was based on its cDNA-derived sequence and its differential expression during G2/M cell cycle phase transition of a partially synchronized alfalfa cell culture. Sequence analysis indicated common motifs with both the A- and B-types of mitotic cyclins similarly to the newly described B3-type of animal cyclins.

  8. Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\\ normalization and subtraction of cDNA libraries.

    Science.gov (United States)

    Soares, Marcelo Bento; de Fatima Bonaldo, Maria; Hackett, Jeremiah D; Bhattacharya, Debashish

    2009-01-01

    Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

  9. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

    Science.gov (United States)

    Alba, Rob; Fei, Zhangjun; Payton, Paxton; Liu, Yang; Moore, Shanna L; Debbie, Paul; Cohn, Jonathan; D'Ascenzo, Mark; Gordon, Jeffrey S; Rose, Jocelyn K C; Martin, Gregory; Tanksley, Steven D; Bouzayen, Mondher; Jahn, Molly M; Giovannoni, Jim

    2004-09-01

    Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

  10. Construction and analysis of full-length and normalized cDNA libraries from citrus.

    Science.gov (United States)

    Marques, M Carmen; Perez-Amador, Miguel A

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional analysis of the corresponding genes enabling manipulation of their expression and the generation of a variety of tagged versions of the native protein. The development of full-length cDNA sequences has the power to improve the quality of genome annotation, as well as provide tools for functional characterization of genes.

  11. Design and Screening of M13 Phage Display cDNA Libraries

    Directory of Open Access Journals (Sweden)

    Yuliya Georgieva

    2011-02-01

    Full Text Available The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS will be presented.

  12. Genetic profile of Egyptian hepatocellular-carcinoma associated with hepatitis C virus Genotype 4 by 15 K cDNA microarray: Preliminary study

    Directory of Open Access Journals (Sweden)

    Mansour Tarek

    2008-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory. Findings We studied gene expression profile of 17 Egyptian HCC patients associated with HCV genotype-4 infection by c-DNA microarray. Out of the 15,660 studied genes, 446 were differentially expressed; 180 of them were up regulated and 134 were down regulated. Seventeen genes out of the 180 up-regulated genes are involved in 28 different pathways. Protein phosphatase 3 (PPP3R1 is involved in 10 different pathways followed by fibroblast growth factor receptor 1 (FGFR1, Cas-Br-M ecotropic retroviral transforming sequence b (CBLB, spleen tyrosine kinase (SYK involved in three pathways; bone morphogenetic protein 8a (BMP8A, laminin alpha 3 (LAMA3, cell division cycle 23 (CDC23 involved in 2 pathways and NOTCH4 which regulate Notch signaling pathway. On the other hand, 25 out of the 134 down-regulated genes are involved in 20 different pathways. Integrin alpha V alpha polypeptide antigen CD51 (ITGVA is involved in 4 pathways followed by lymphotoxin alpha (TNF superfamily, member 1 (LTA involved in 3 pathways and alpha-2-macroglobulin (A2M, phosphorylase kinase alpha 2-liver (PHKA2 and MAGI1 membrane associated guanylate kinase 1 (MAGI1 involved in 2 pathways. In addition, 22 genes showed significantly differential expression between HCC cases with cirrhosis and without cirrhosis. Confirmation analysis was performed on subsets of these genes by RT-PCR, including some up-regulated genes such as CDK4, Bax, NOTCH4 and some down-regulated genes such as ISGF3G, TNF, and VISA. Conclusion This is the first preliminary study on gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a new gate for prognostic and diagnostic markers for HCC associated

  13. [Screening of specifically expressed genes in amphioxus neurula by construction of a subtractive cDNA library].

    Science.gov (United States)

    Zhang, Lei; Yang, Yong-Jie; Zhang, Yan-Jun

    2010-12-01

    To screen specifically expressed genes in the development of nerve, muscle, and body axis of amphioxus, Branchiostoma belcheri tsingtauenese. A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA. The total RNA was extracted from the 12-hour neurula and 6-hour gastrula, then reverse transcribed into cDNA. The 12-hour neurula cDNA was designated as the experimental group (the tester) and the 6-hour gastrula cDNA as the control group (the driver). The differentially expressed sequences were exponentially amplified using suppression PCR. Background was subtracted and differentially expressed sequences were further enriched. The PCR products were ligated to the T Vector. After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells, the cDNA library was constructed successfully. Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained. SSH is an effective method for searching differentially expressed genes. The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.

  14. Gene therapy for bladder pain with gene gun particle encoding pro-opiomelanocortin cDNA.

    Science.gov (United States)

    Chuang, Yao-Chi; Chou, A-K; Wu, P-C; Chiang, Po-Hui; Yu, T-J; Yang, L-C; Yoshimura, Naoki; Chancellor, Michael B

    2003-11-01

    Interstitial cystitis is a bladder hypersensitivity disease associated with bladder pain that has been a major challenge to understand and treat. We hypothesized that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study we developed a gene gun method for the transfer of POMC cDNA in vivo and investigated its therapeutic effect on acetic acid induced bladder hyperactivity in rats. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered into the bladder wall of adult female rats by direct injection or the gene gun. Three days after gene therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. The intercontraction interval was decreased after intravesical instillation of acetic acid (73.1% or 68.1% decrease) in 2 control groups treated with saline or the gene gun without POMC cDNA, respectively. However, rats that received POMC cDNA via the gene gun showed a significantly decreased response (intercontraction interval 35% decreased) to acetic acid instillation, whereas this antinociceptive effect was not detected in the plasmid POMC cDNA direct injection group. This effect induced by POMC gene gun treatment was reversed by intramuscular naloxone (1 mg/kg), an opioid antagonist. Increased endorphin immunoreactivity with anti-endorphin antibodies was observed in the bladder of gene gun treated animals. The POMC gene can be transferred in the bladder using the gene gun and increased bladder expression of endorphin can suppress nociceptive responses induced by bladder irritation. Thus, POMC gene gun delivery may be useful for the treatment of interstitial cystitis and other types of visceral pain.

  15. Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2008-10-01

    Full Text Available Abstract Background cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. Results With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation was written in Perl. cDNA-AFLP protocols described in the literatur and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Conclusion Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each

  16. [Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

    2005-03-01

    To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

  17. Display of a maize cDNA library on baculovirus infected insect cells.

    Science.gov (United States)

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-08-12

    Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  18. Cloning, sequencing, and expression of cDNA for human β-glucuronidase

    International Nuclear Information System (INIS)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-01-01

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-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 2 -terminal amino acid sequence determined for human spleen β-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 β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-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

  19. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 micro...

  20. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

  1. Construction of cDNA libraries: focus on protists and fungi.

    Science.gov (United States)

    Rodríguez-Ezpeleta, Naiara; Teijeiro, Shona; Forget, Lise; Burger, Gertraud; Lang, B Franz

    2009-01-01

    Sequencing of cDNA libraries is an efficient and inexpensive approach to analyze the protein-coding portion of a genome. It is frequently used for surveying the genomes of poorly studied eukaryotes, and is particularly useful for species that are not easily amenable to genome sequencing, because they are nonaxenic and/or difficult to cultivate. In this chapter, we describe protocols that have been applied successfully to construct and normalize a variety of cDNA libraries from many different species of free-living protists and fungi, and that require only small quantities of cell material.

  2. Genomic organization of Bruton`s tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Rohrer, J.; Conley, M.E. [Univ. of Tennessee, Memphis, TN (United States)

    1994-09-01

    Bruton`s tyrosine kinase (Btk), is a nonreceptor tyrosine kinase that has been identified as the defective gene in X-linked agammaglobulinemia (XLA). XLA patients have profound hypogammaglobulinemia and markedly reduced numbers of B cells while their T cell and phagocyte numbers remain normal. To determine the genomic organization of Btk, intron/exon borders were identified by sequencing cosmid DNA using cDNA primers. Nineteen exons spanning 37 kb of genomic DNA were identified. All the intron/exon splice junctions followed the GT/AG rule. The translational ATG start codon was in exon 2 which was 6 kb downstream of exon 1. Exon 19, 519 bp in length and 3.8 kb distal to exon 18, was the largest exon and included the 450 bp of the 3{prime} untranslated region. Exons 6 through 18 formed the largest cluster of exons with no intron being longer than 1550 bp. There was no apparent correlation between the exon boundaries of Btk and the functional domains of the protein or the exon boundaries of src, the nonreceptor protein tyrosine kinase prototype. The region 500 bp upstream of the presumed transcriptional start site was sequenced and found to have a G+C content of 52%. No TATA-type promoter elements in the -20 bp to -30 bp region were identified. However, at position -48 bp, a TGTGAA motif was found that bears some similarity to the TATA box. This sequence was preceded by a perfect inverted CCAAT box at position -90 bp. Three retinoic acid binding sites were also identified at positions -50 bp, -83 bp and -197 bp. Defining the genomic structure of Btk will permit us to identify regulatory elements in this gene and to identify mutations in genomic DNA of patients with XLA.

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

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  4. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of τ-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the ...

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  6. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  7. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  8. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... 2Ningbo City College of Vocational Technology, Ningbo, 315100 People's Republic of China. 3National Marine Environmental Monitoring Center. Dalian, 116023, People's ... The SMART cDNA library of S. constricta was constructed by our laboratory. Random sequencing of the library using T3 primer.

  9. cDNA, genomic sequence cloning and analysis of the ribosomal ...

    African Journals Online (AJOL)

    Ribosomal protein L37A (RPL37A) is a component of 60S large ribosomal subunit encoded by the RPL37A gene, which belongs to the family of ribosomal L37AE proteins, located in the cytoplasm. The complementary deoxyribonucleic acid (cDNA) and the genomic sequence of RPL37A were cloned successfully from giant ...

  10. Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library.

    Science.gov (United States)

    Li, S; Chen, Y

    2001-02-01

    To construct a lambda gt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 micrograms) was submitted to reverse transcription using random hexamers [pd(N6)]. The double-strand blunt-ended cDNAs were ligated with an EcoRI/Notl adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector lambda gt11 EcoRI arms. After being packaged in vitro, lambda gt11 was put to an infectious bacteria Echinococcus coli (E. coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments. The recombinant ratio was nearly 100% and approximately 1 x 10(6) clones could be derived from this lambda gt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.

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

    DEFF Research Database (Denmark)

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

    1988-01-01

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

  12. cDNA sequence of the long mRNA for human glutamine synthase

    NARCIS (Netherlands)

    van den Hoff, M. J.; Geerts, W. J.; Das, A. T.; Moorman, A. F.; Lamers, W. H.

    1991-01-01

    Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp

  13. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  14. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

    NARCIS (Netherlands)

    Akkerdaas, Jaap H.; Schocker, Frauke; Vieths, Stefan; Versteeg, Serge; Zuidmeer, Laurian; Hefle, Sue L.; Aalberse, Rob C.; Richter, Klaus; Ferreira, Fatima; van Ree, Ronald

    2006-01-01

    The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA

  15. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

    Science.gov (United States)

    Cartolano, Maria; Huettel, Bruno; Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

  16. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... Complete cDNA sequence of ScATPase and its deduced amino acid sequence. Nucleotides were numbered from the first base at the 5'end. The canonical polyadenylation signal-sequence was italic and underlined. The asterisk indicated the stop codon. The domain for ATP synthase C was underlined.

  17. Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin

    2013-01-01

    of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR...

  18. Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence

    NARCIS (Netherlands)

    Mechelen, J.R. van; Smits, M.; Douma, A.C.; Rouster, J.; Cameron-Mills, V.; Heidekamp, F.; Valk, B.E.

    1995-01-01

    A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5' untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3' untranslated region of 142 nucleotides. The molecular mass of the encoded

  19. cDNA cloning and expression analysis of two distinct Sox8 genes in ...

    Indian Academy of Sciences (India)

    2010-08-06

    Aug 6, 2010 ... cDNA cloning and expression analysis of two distinct Sox8 genes in. Paramisgurnus dabryanus (Cypriniformes). XIAOHUA XIA, JIE ZHAO, QIYAN DU and ZHONGJIE CHANG. ∗. Molecular and Genetic Laboratory, College of Life Sciences, Henan Normal University, 46 East of Construction Road,. Xinxiang ...

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

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The. cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown- ...

  1. Construction and characterization of a cDNA library from human ...

    African Journals Online (AJOL)

    The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain ...

  2. First-strand cDNA synthesis primed with oligo(dT)

    International Nuclear Information System (INIS)

    Krug, M.S.; Berger, S.L.

    1987-01-01

    The quality of a cDNA library depends on the integrity of the messenger RNA and the fidelity with which it can be reverse transcribed. RNA cannot be cloned directly; in a reaction catalyzed by reverse transcriptase, the RNA, together with a suitable primer and a supply of deoxyribonucleoside triphosphates (dNTPs), must be converted to a double-stranded molecule. The product contains a complementary strand (first, antisense, or minus-strand cDNA) that is hybridized to what remains of the original RNA template. Such DNA-RNA hybrids can be cloned albeit often with lower efficiency than their double-stranded DNA counterparts. Usually the hybrid molecules are treated as intermediates in a scheme aimed at replacing the fragmented RNA with continuous DNA to form a double-stranded cDNA molecule. From this brief summary of cDNA cloning, it should be obvious that, regardless of the strategy, reverse transcriptase does and how it does it in vitro is discussed

  3. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  4. cDNA cloning and primary structure analysis of invariant chain in ...

    African Journals Online (AJOL)

    cDNA cloning and primary structure analysis of invariant chain in Chinese Pengze crucian carp. X Liu, W Yu, J Li, F Chen, S Liu, C Wu, J Xu. Abstract. Invariant chain (Ii) plays an important role in MHC class II molecules assembly and exogenous peptide presentation in vertebrates. Although mammalian Ii has been ...

  5. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    Unknown

    Evans and Long 1921) and the human growth hormone (GH) encoding cDNA was per- haps the first to be isolated and characterized (Li and. Evans 1944). GH, chorionic somatomamotropin (placental lactogen) and prolactin (PRL) are all a family of ...

  6. Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

    Science.gov (United States)

    Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

    1998-08-01

    Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders. Copyright 1998 Academic Press.

  7. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma.

    Science.gov (United States)

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-11-01

    To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database. Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used. Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed. Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.

  8. New Approaches to Attenuated Hepatitis a Vaccine Development: Cloning and Sequencing of Cell-Culture Adapted Viral cDNA.

    Science.gov (United States)

    1987-10-13

    Insert frag- ments from p16 cDNA clones were subcloned into the phage vector Ml3mp8 or Ml3mpl9 qnd subjected to rapid sequencing using the...2), and selected cDNA insert fragments were subcloned into M13 vectors for sequencing. The sequence of the complete genome was determined, with over

  9. Construction and Screening of an Expression cDNA Library from the Triactinomyxon Spores of Myxobolus cerebralis, the causative agent of Salmonid Whirling Diseases

    OpenAIRE

    Soliman, Hatem Mohamed Touhan

    2005-01-01

    The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract...

  10. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    . The catalytic alpha subunits are distantly related to the CMGC subfamily of kinases, such as the Cdk kinases. There are some peculiarities associated with protein kinase CK2, which are not found with most other protein kinases: (i) the enzyme is constitutively active, (ii) it can use ATP and GTP and (iii...

  11. A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants1

    Science.gov (United States)

    Chico, José Manuel; Raíces, Marcela; Téllez-Iñón, María Teresa; Ulloa, Rita María

    2002-01-01

    A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks. PMID:11788771

  12. KLONING cDNA HORMON PERTUMBUHAN DARI IKAN GURAME (Osphronemus gouramy

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2016-11-01

    Full Text Available Penelitian mengenai kloning cDNA pengkode hormon pertumbuhan ikan gurame telah dilakukan. Tujuan dari penelitian ini adalah untuk memperoleh sekuens DNA komplemen hormon pertumbuhan sebagai langkah awal dalam rangka pengembangan teknologi rekayasa genetik ikan gurame. Empat buah kelenjar hifopisa ikan gurame digunakan sebagai bahan bakunya dan dilakukan proses ekstraksi RNA total dari kelenjar hipofisa, dilanjutkan dengan sintesis cDNA, amplifikasi PCR, purifikasi fragmen DNA dari gel, ligasi produk PCR dengan vektor kloning, transformasi dan inkubasi bakteri, seleksi koloni bakteri putih, isolasi plasmid, dan sekuensing. Hasil sekuensing menunjukkan bahwa panjang produk amplifikasi PCR adalah 843 bp yang menyandikan 204 asam amino residu dan mengandung sekuens-sekuens yang konserf untuk gen hormon pertumbuhan (GH. Analisis homologi menunjukkan kesamaan sekuens hasil isolasi antara 52,4%--97,6% dengan gen GH ikan lainnya, dengan persentase homologi tertinggi adalah dengan ikan sepat. Dengan demikian dapat disimpulkan bahwa sekuens hasil isolasi merupakan sekuens gen GH. Dari hasil analisis sekuens terlihat bahwa gen GH ikan gurame secara evolusi adalah konserf. Research on cDNA cloning encoded the gouramy growth hormone was conducted. The aim of the research was to get complementary DNA, cDNA, sequences of growth hormone as an initial step to develop genetic engineering of gouramy fish. Four pituitary glands of the gouramy were taken and then processed with total RNA extraction, and continued with cDNA synthesis, PCR amplification, DNA fragment purification from the gel, PCR product legation with cloning vector, transformation and incubation of bacteria, white colony bacteria selection, plasmid isolation and sequencing analysis. Sequencing result showed that the amplified PCR product length had 834 bp, encoding 204 amino acid residue and contained conserve sequence for GH (growth hormone gen. Homolog analysis showed sequence similarity of

  13. [Construction and immunoscreening of cDNA library of Babesia orientalis].

    Science.gov (United States)

    Liu, Qin; Zhou, Dan-Na; Zhou, Yan-Qin; Zhang, Ying; He, Lan; Yao, Bao-An; Zhao, Jun-Long

    2009-06-01

    To construct a cDNA library for Babesia orientalis and screen immunologically positive clones. Total RNA of B. orientalis in red blood cells from an infected calf was isolated. cDNA was synthesized by reverse transcriptase, amplified by PCR and ligated into lambdaTriplEx2 vector. The recombined vectors were packaged and the unamplified cDNA library was constructed. The cDNA library was then amplified and immunologically screened with rabbit anti-B. orientalis serum. The recombinant lambdaTriplEx2 of positive clones were converted to the corresponding recombinant pTriplEx2. The inserted fragments were identified by PCR amplification. The plasmids were sequenced and compared against GenBank database by Blast. The titer of the unamplified library was 2.0 x 10(6) pfu/ml. The inserted fragment length of the library ranged from 500 to 3,000 bp, and the recombination efficiency accounted for 98.8%. The titer of the amplified library was 5.8 x 10(8) pfu/ml. Three positive clones were selected by serum immunological screening and named B04, B05, and B41, respectively. The inserted fragments of the B04, B05 and B41 were about 1,300 bp, 1,000 bp, and 2,400 bp, respectively. Sequence analysis revealed that the 3 clones contained open reading frames. Blast results showed that they were highly homologous to the nuclear movement protein gene, the hypothetical protein gene and the heat shock protein 70 (HSP70) gene, respectively. The deduced amino acid sequences of B04, B05 and B41 contained 310, 192 and 647 amino acid residues, with Mr of 34,000, 21,000, and 70,700, respectively. A qualified cDNA library of B. orientalis has been constructed and three positive clones of B. orientalis discovered.

  14. Screening a cDNA Library for Protein–Protein Interactions Directly in Planta[W

    Science.gov (United States)

    Lee, Lan-Ying; Wu, Fu-Hui; Hsu, Chen-Tran; Shen, Shu-Chen; Yeh, Hsuan-Yu; Liao, De-Chih; Fang, Mei-Jane; Liu, Nien-Tze; Yen, Yu-Chen; Dokládal, Ladislav; Sýkorová, Eva; Gelvin, Stanton B.; Lin, Choun-Sea

    2012-01-01

    Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein– or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ∼2 × 105 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species. PMID:22623495

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

    Science.gov (United States)

    Eißmann, Moritz; Schwamb, Bettina; Melzer, Inga Maria; Moser, Julia; Siele, Dagmar; Köhl, Ulrike; Rieker, Ralf Joachim; Wachter, David Lukas; Agaimy, Abbas; Herpel, Esther; Baumgarten, Peter; Mittelbronn, Michel; Rakel, Stefanie; Kögel, Donat; Böhm, Stefanie; Gutschner, Tony; Diederichs, Sven; Zörnig, Martin

    2013-01-01

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

  16. Citron kinase - renaissance of a neglected mitotic kinase.

    Science.gov (United States)

    D'Avino, Pier Paolo

    2017-05-15

    Cell division controls the faithful segregation of genomic and cytoplasmic materials between the two nascent daughter cells. Members of the Aurora, Polo and cyclin-dependent (Cdk) kinase families are known to regulate multiple events throughout cell division, whereas another kinase, citron kinase (CIT-K), for a long time has been considered to function solely during cytokinesis, the last phase of cell division. CIT-K was originally proposed to regulate the ingression of the cleavage furrow that forms at the equatorial cortex of the dividing cell after chromosome segregation. However, studies in the last decade have clarified that this kinase is, instead, required for the organization of the midbody in late cytokinesis, and also revealed novel functions of CIT-K earlier in mitosis and in DNA damage control. Moreover, CIT-K mutations have recently been linked to the development of human microcephaly, and CIT-K has been identified as a potential target in cancer therapy. In this Commentary, I describe and re-evaluate the functions and regulation of CIT-K during cell division and its involvement in human disease. Finally, I offer my perspectives on the open questions and future challenges that are necessary to address, in order to fully understand this important and yet unjustly neglected mitotic kinase. © 2017. Published by The Company of Biologists Ltd.

  17. The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2'-deoxycytidine.

    Science.gov (United States)

    Alleman, Wade G; Tabios, Ray L; Chandramouli, Gadisetti V R; Aprelikova, Olga N; Torres-Cabala, Carlos; Mendoza, Arnulfo; Rogers, Craig; Rodgers, Craig; Sopko, Nikolai A; Linehan, W Marston; Vasselli, James R

    2004-10-15

    Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models. Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis. 5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment. Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.

  18. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  19. Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus).

    Science.gov (United States)

    Shao, Zhan-tao; Cong, Xiao; Yuan, Jin-duo; Yang, Gui-wen; Chen, Ying; Pan, Jie; An, Li-guo

    2009-09-01

    In this paper, a cDNA expression library from head kidney of Japanese sea bass (Lateolabrax japonicus) was constructed for the first time. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase and the double-stranded cDNA was digested by Xho I enzyme. Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bps were ligated into the lambdaZAPExpress vector. The recombinant DNA was packaged in vitro with Gigapack III gold packaging extract. The titers of the primary and amplified library were 1.0 x 10(5) and 5.0 x 10(9) pfu/ml, respectively. To characterize the constructed cDNA library, 15 phage plaques were selected randomly to test the inserted fragments. The results showed that the inserts were mostly longer than 500 bps. To test the utility, the library was screened with primers designed for three immune-related genes of, Myxovirus resistant (Mx), tumor necrosis factor-alpha (TNF-alpha) and Toll-like receptor (TLR). Results of Blastn and alignment showed that they are members of Mx, TNF-alpha and TLR gene families, respectively, which meets our anticipates for this cDNA library as an immune-related one. These results confirmed that the cDNA library constructed will provide a useful tool for gene cloning and expression analysis in immune system of Japanese sea bass.

  20. [Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages].

    Science.gov (United States)

    Zhang, Z X; Zhang, F D; Tang, W H; Pi, Y J; Zheng, Y L

    2005-01-01

    Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.

  1. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  2. [Cloning and sequence analysis of Eg95 cDNA from different stages of Echinococcus granulosus in Xinjiang].

    Science.gov (United States)

    Lin, Ren-yong; Ding, Jian-bing; Wen, Hao; Zhang, Wen-bao; Li, Jun; Lu, Xiao-mei

    2003-01-01

    To study expression and sequence differences of Echinococcus granulosus 95(Eg95) antigen cDNA from different stages of protoscolex, oncosphere and adult worm of E. granulosus from Xinjiang Uighur Aut. Reg. In accordance with the sequence of Eg95 antigen cDNA, the primers of Eg95 were designed. Eg95 antigen cDNAs were amplified by PCR from protoscolex, oncosphere and adult worm cDNA libraries of E. granulosus, respectively and were cloned into pUCm-T plasmid, and sequenced. The sequences were analyzed by DNAman and GenBank/BLAST biosoftware. PCR results showed that Eg95 antigen cDNA was amplified from three stages of E. granulosus cDNA libraries. Sequencing analysis indicated that the Eg95 cDNA length was 402 bp, same as the reported data in GenBank. The Eg95 antigen cDNA was expressed in the different life-cycle stages of E. granulosus in Xinjiang and there was no nucleic acid sequence difference of Eg95 antigen among the protoscolex, oncosphere and adult worm of E. granulosus.

  3. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies.......cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  4. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Rodriguez-del Valle Nuri

    2007-11-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  6. cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen

    NARCIS (Netherlands)

    Vrtala, S.; Sperr, W. R.; Reimitzer, I.; van Ree, R.; Laffer, S.; Müller, W. D.; Valent, P.; Lechner, K.; Rumpold, H.; Kraft, D.

    1993-01-01

    We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found

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

    Science.gov (United States)

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

    1990-09-25

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-05-01

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

  9. [Construction and identification of a full-length cDNA library from Spirometra erinaceieuropaei].

    Science.gov (United States)

    Lv, Gang; Lu, Ya-Jun; Fan, Zhi-Gang; Shi, Da-Zhong; Gan, Xiu-Feng; Zhong, Sai-Feng

    2010-10-30

    The full-length pBluescript II SK cDNA library of adult Spirometra erinaceieuropaei was constructed by using the SMART method. Data showed that 95.5% of the library was recombinant and the titer of the library was 1.06 x 10(6). The average insert size of the library was about 1.4 kb. Forty-eight randomly selected clones were sequenced. A set of 36 effective expressed sequence tags (ESTs) with the average size of 674 bp was obtained after excluding clones shorter than 450 bp. The unigenes occupied 58.3% of the 36 ESTs. The rate of full-length cDNAs were 57.7% (15/26). The high-quality of full-length cDNA library could be used for large scale EST sequencing.

  10. Construction and analysis of full-length cDNA library of Cryptosporidium parvum.

    Science.gov (United States)

    Yamagishi, Junya; Wakaguri, Hiroyuki; Sugano, Sumio; Kawano, Suguru; Fujisaki, Kozo; Sugimoto, Chihiro; Watanabe, Junichi; Suzuki, Yutaka; Kimata, Isao; Xuan, Xuenan

    2011-06-01

    A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  11. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

    the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. BIOLOGICAL SIGNIFICANCE: Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes......UNLABELLED: Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity...... of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  12. Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida.

    Science.gov (United States)

    Dong, Guo-Qing; Yuan, Xiao-Ling; Shan, Ya-Jun; Zhao, Zhen-Hu; Chen, Jia-Pei; Cong, Yu-Wen

    2004-04-01

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

  13. Consistent errors in first strand cDNA due to random hexamer mispriming.

    Directory of Open Access Journals (Sweden)

    Thomas P van Gurp

    Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

  14. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Soumen Naskar

    2012-01-01

    Full Text Available In the present study, water buffalo MHC (Bubu-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

  15. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that l5-nucleotide-long random...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...

  16. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

    Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

    2017-01-01

    The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

  17. Attenuation and cell culture adaptation of hepatitis A virus (HAV): a genetic analysis with HAV cDNA.

    OpenAIRE

    Cohen, J I; Rosenblum, B; Feinstone, S M; Ticehurst, J; Purcell, R H

    1989-01-01

    RNA transcripts of hepatitis A virus (HAV) HM-175 cDNA from attenuated, cell culture-adapted HAV were infectious in cell culture. A full-length HAV cDNA from wild-type HAV (propagated in marmosets in vivo) was constructed. Chimeric cDNAs that contained portions of both wild-type and attenuated genomes were produced. Oligonucleotide-directed mutagenesis was used to engineer a point mutation into the VP1 gene of attenuated HAV cDNA, so that the sequence of this capsid protein would be identical...

  18. Identification of differential genes by suppression subtractive hybridization: I. Preparation of subtracted cDNA or genomic DNA library.

    Science.gov (United States)

    Rebrikov, Denis V

    2008-07-01

    INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

  19. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    Prakash

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ~8.2 kb PCR product was amplified ...

  20. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

    OpenAIRE

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-01-01

    Background: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results: We report here a ...

  1. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  2. cDNA cloning and expression analysis of a mannose-binding lectin ...

    Indian Academy of Sciences (India)

    Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using ...

  3. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... detection of phosphorylated tyrosines by phosphoprotomics studies in bacteria. Other pioneering studies conducted in recent years, such as the first structures of BY-kinases and biochemical and phyiological studies of new BY-kinase substrates significantly furthered our understanding of these enzymes...

  4. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    International Nuclear Information System (INIS)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-01-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis

  5. Screening for candidate genes related to breast cancer with cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Yu-Juan Xiang

    2015-06-01

    Full Text Available Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quantitatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Keywords: Breast neoplasms, Candidate genes, Microarray

  6. A novel method using edge detection for signal extraction from cDNA microarray image analysis.

    Science.gov (United States)

    Kim, J H; Kim, H Y; Lee, Y S

    2001-06-30

    Gene expression analyses by probes of hybridization from mRNA to cDNA targets arrayed on membranes or activated glass surfaces have revolutionized the way of profiling mega level gene expression. The main remaining problems however are sensitivity of detection, reproducibility and data processing. During processing of microarray images, especially irregularities of spot position and shape could generate significant errors: small regions of signal spots can be mis-included into background area and vice versa. Here we report a novel method to eliminate such obstacles by sensing their edges. Application of edge detection technology on separating spots from the background decreases the probability of the errors and gives more accurate information about the states of spots such as the pixel number, degree of fragmentation, width and height of spot, and circumference of spot. Such information can be used for the quality control of cDNA microarray experiments and filtering of low quality spots. We analyzed the cDNA microarray image that contains 10,368 genes using edge detection and compared the result with that of conventional method which draws circle around the spot.

  7. Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis.

    Science.gov (United States)

    Yang, Liping; Meng, Zining; Liu, Yun; Zhang, Yong; Liu, Xiaochun; Lu, Danqi; Huang, Junhai; Lin, Haoran

    2010-01-15

    The Chinese giant salamander (Andrias davidianus) is one of the largest and 'living fossil' species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.

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

    International Nuclear Information System (INIS)

    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 ≅ 0.01%. Blot hybridization of electrophoretically fractionated poly(A) + RNA revealed a major mRNA ≅ 4 kilobases long and two minor forms of ≅ 3.1 and ≅ 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 γ-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 ≅ 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function

  9. Generation of longer 3' cDNA fragments from massively parallel signature sequencing tags.

    Science.gov (United States)

    Silva, Ana Paula M; Chen, Jianjun; Carraro, Dirce M; Wang, San Ming; Camargo, Anamaria A

    2004-07-06

    Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3'end of transcripts. A single MPSS experiment can generate over 10(7) tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (approximately 1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3' cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts.

  10. Generation of longer 3′ cDNA fragments from massively parallel signature sequencing tags

    Science.gov (United States)

    Silva, Ana Paula M.; Chen, Jianjun; Carraro, Dirce M.; Wang, San Ming; Camargo, Anamaria A.

    2004-01-01

    Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3′end of transcripts. A single MPSS experiment can generate over 107 tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (∼1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3′ cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts. PMID:15247327

  11. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

    Science.gov (United States)

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-09-03

    Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

  12. [Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa].

    Science.gov (United States)

    Zhang, Zhongyi; Fan, Huamin; Yang, Yanhui; Li, Mingjie; Li, Juan; Xu, Haixia; Chen, Junying; Chen, Xinjian

    2011-02-01

    To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

  13. CONSTRUCTION AND APPLICATIONS OF A MYCORRHIZAL ARBUSCULAR SPECIFIC cDNA LIBRARY.

    Science.gov (United States)

    Isayenkov, S; Maathuis, F J M

    2016-01-01

    To exploit the potential benefits of mycorrhizas, we need to investigate the processes that occur in these symbiotic interactions, particularly in the arbuscular compartment where nutrients are exchanged between the plant and the fungus. Progress in this area is restricted due to the intricacy and complexity of this plant-fungus interface and many techniques that have been employed successfully in other plants and animal systems cannot be used. An effective approach to study processes in arbuscules is to examine transcript composition and dynamics. We applied laser capture microdissection (LCM) to isolate approximately 3000 arbuscules from Glomus intraradices colonised Me- dicago truncatula roots. Total RNA was extracted from microdissected arbuscules and subjected to T7 RNA polymerase-based linear amplification. Amplified RNA was then usedfor construction of a cDNA library. The presence and level of enrichment of mycorrhiza-specific transcripts was determined by quantitative Real-time and conventional PCR. To improve enrichment a cDNA library subtraction was performed. Complementation of yeast mutants deficient in the uptake of.potassium, phosphate, sulphate, amino acids, ammonium and of a Mn²⁺sensitive strain, demonstrates the functionality of our cDNA library.

  14. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger.

    Science.gov (United States)

    Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

    2010-10-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.

  15. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    Science.gov (United States)

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.

  16. Effects of heavy metals on Drosophila larvae and a metallothionein cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Maroni, G.; Lastowski-Perry, D.; Otto, E.; Watson, D.

    1986-03-01

    Drosophila melanogaster larvae reared on food containing radioactive cadmium retained over 80% of it, mostly in the intestinal epithelium. The majority of this radioactivity was associated with a soluble protein of less than 10,000 molecular weight. Synthesis of this cadmium-binding protein was induced by the metal as demonstrated by incorporation of radioactive cysteine. Most copper ingested by larvae was also found to associate with a low molecular weight, inducible protein, but some of it was found in an insoluble fraction. A D. melanogaster cDNA clone was isolated based on its more intense hybridization to copies of RNA sequences from copper-fed larvae than from control larvae. This clone showed strong hybridization to mouse metallothionein-I cDNA at reduced stringency. Its nucleotide sequence includes an open-reading segment which codes for a 40 amino acid protein; this protein was identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The ten cysteine residues present occur in five pairs of near-vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels.

  17. Sequencing of first-strand cDNA library reveals full-length transcriptomes.

    Science.gov (United States)

    Agarwal, Saurabh; Macfarlan, Todd S; Sartor, Maureen A; Iwase, Shigeki

    2015-01-21

    Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 5' ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the 'full-length' transcriptome with a single library.

  18. An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.

    Science.gov (United States)

    Sterling, Catherine H; Veksler-Lublinsky, Isana; Ambros, Victor

    2015-01-01

    The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

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

  20. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

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

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (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 β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-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 β-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 β-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

  1. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  2. cDNA encoding a polypeptide including a hev ein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    2000-07-04

    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 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

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

    DEFF Research Database (Denmark)

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

    1985-01-01

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

  4. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

    Science.gov (United States)

    Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

    2010-01-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

  5. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng [National Yang Ming Univ., Taipei, Taiwan (China). Inst. of Radiological Sciences] (and others)

    2001-12-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

  6. cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

    Directory of Open Access Journals (Sweden)

    Valenzuela Jesus G

    2007-07-01

    Full Text Available Abstract Background The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions. Results We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST, including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6% with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions. Conclusion Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively

  7. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...

  8. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  9. Identification of a cryptic prokaryotic promoter within the cDNA encoding the 5' end of dengue virus RNA genome.

    Directory of Open Access Journals (Sweden)

    Dongsheng Li

    Full Text Available Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV 5' UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.

  10. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A) + RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  11. Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

    Science.gov (United States)

    Sakajo, S; Nakamura, K; Asahi, T

    1987-06-01

    A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

  12. Isolation and sequence analysis of a chalcone synthase cDNA of Matthiola incana R. Br. (Brassicaceae).

    Science.gov (United States)

    Epping, B; Kittel, M; Ruhnau, B; Hemleben, V

    1990-06-01

    A cDNA clone (pcM12) of the chalcone synthase (CHS) of Matthiola incana R. Br. (Brassicaceae) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5' and 3' untranslated regions. The deduced amino acid sequence shows that the Matthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.

  13. The alpha-kinase family: an exceptional branch on the protein kinase tree.

    NARCIS (Netherlands)

    Middelbeek, J.A.J.; Clark, K.; Venselaar, H.; Huynen, M.A.; Leeuwen, F.N. van

    2010-01-01

    The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in

  14. Treatment of Donor Cells and Reconstructed Embryos with a Combination of Trichostatin-A and 5-aza-2'-Deoxycytidine Improves the Developmental Competence and Quality of Buffalo Embryos Produced by Handmade Cloning and Alters Their Epigenetic Status and Gene Expression.

    Science.gov (United States)

    Saini, Monika; Selokar, Naresh L; Agrawal, Himanshu; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham S; Palta, Prabhat

    2017-06-01

    The application of cloning technology on a large scale is limited by very low offspring rate primarily due to aberrant or incomplete epigenetic reprogramming. Trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferases, are widely used for altering the epigenetic status of cloned embryos. We optimized the doses of these epigenetic modifiers for production of buffalo embryos by handmade cloning and examined whether combined treatment with these epigenetic modifiers offered any advantage over treatment with the individual epigenetic modifier. Irrespective of whether donor cells or reconstructed embryos or both were treated with 50 nM TSA +7.5 nM 5-aza-dC, (1) the blastocyst rate was significantly higher (71.6 ± 3.5, 68.3 ± 2.6, and 71.8 ± 2.4, respectively, vs. 43.1 ± 3.4 for controls, p < 0.05); (2) the apoptotic index was lower (5.4 ± 1.1, 9.5 ± 1.0, and 7.4 ± 1.3, respectively, vs. 19.5 ± 2.1 for controls, p < 0.05) and was similar to that of in vitro fertilization blastocysts (6.0 ± 0.8); (3) the global level of H3K18ac was higher (p < 0.01) and that of H3K27me3 lower (p < 0.05) than in controls and was similar among all treatment groups; and (4) the expression level of epigenetic-(HDAC1, DNMT1, and DNMT3a), pluripotency-(OCT4 and NANOG), and development-related (FGF4) genes, but not that of SOX2 and CDX2, was similar among all treatment groups. These results demonstrate that similar levels of beneficial effects can be obtained following treatment of either donor cells or reconstructed embryos or both with the combination of TSA +5-aza-dC. Therefore, there is no advantage in treating both donor cells and reconstructed embryos when the combination of TSA and 5-aza-dC is used.

  15. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Science.gov (United States)

    Soares, Marcelo Bento; Bonaldo, Maria de Fatima

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  16. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)

    Science.gov (United States)

    Morris, S W; Naeve, C; Mathew, P; James, P L; Kirstein, M N; Cui, X; Witte, D P

    1997-05-08

    Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.

  17. Construction and identification of subtracted cDNA library in bone marrow cells of radon-exposed mice

    International Nuclear Information System (INIS)

    Li Jianxiang; Nie Jihua; Tong Jian; Fu Chunling; Zhou Jianwei

    2008-01-01

    Objective: To construct and identify subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation. Methods: Adult male BALB/c mice, weighing 18-22 g, were placed in a multi- functional radon chamber. One group of mice was exposed to radon up to the accumulative dose of 105 work level month (WLM). The control group of mice was housed in a room with an accumulative dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and the suppression subtractive hybridization were performed. The obtained forward and reverse cDNA fragments were directly inserted into pMD18-T vector and transformed into E. coli JM109. The inserting cDNA fragments were screened by the blue-and-white blot screening and nested PCR of bacterium liquid. Results: The 244 of 285 white bacteria clones obtained randomly were positive clones contained 100-1100 bp inserted cDNA fragments. Conclusions: The forward and reverse subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation is successfully constructed. (authors)

  18. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a P-Glycoprotein cDNA.

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

    Full Text Available The efflux transporter P-glycoprotein (P-gp is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  19. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2004-06-01

    Full Text Available Abstract Background cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. Results We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. Conclusions The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb, when high-quality starting mRNA is used.

  20. [Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

    Science.gov (United States)

    Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

    2003-07-01

    Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.

  1. Tec protein tyrosine kinase inhibits CD25 expression in human T-lymphocyte.

    Science.gov (United States)

    Susaki, Kentaro; Kitanaka, Akira; Dobashi, Hiroaki; Kubota, Yoshitsugu; Kittaka, Katsuharu; Kameda, Tomohiro; Yamaoka, Genji; Mano, Hiroyuki; Mihara, Keichiro; Ishida, Toshihiko

    2010-01-04

    The Tec protein tyrosine kinase (PTK) belongs to a group of structurally related nonreceptor PTKs that also includes Btk, Itk, Rlk, and Bmx. Previous studies have suggested that these kinases play important roles in hematopoiesis and in the lymphocyte signaling pathway. Despite evidence suggesting the involvement of Tec in the T-lymphocyte activation pathway via T-cell receptor (TCR) and CD28, Tec's role in T-lymphocytes remains unclear because of the lack of apparent defects in T-lymphocyte function in Tec-deficient mice. In this study, we investigated the role of Tec in human T-lymphocyte using the Jurkat T-lymphoid cell line stably transfected with a cDNA encoding Tec. We found that the expression of wild-type Tec inhibited the expression of CD25 induced by TCR cross-linking. Second, we observed that LFM-A13, a selective inhibitor of Tec family PTK, rescued the suppression of TCR-induced CD25 expression observed in wild-type Tec-expressing Jurkat cells. In addition, expression of kinase-deleted Tec did not alter the expression level of CD25 after TCR ligation. We conclude that Tec PTK mediates signals that negatively regulate CD25 expression induced by TCR cross-linking. This, in turn, implies that this PTK plays a role in the attenuation of IL-2 activity in human T-lymphocytes.

  2. Growth factors and kinases in glioblastoma growth

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Peña-Ortiz

    2016-10-01

    Full Text Available Glioblastoma multiforme (GBM is the most aggressive type of brain cancer, having the highest invasion, migration, proliferation, and angiogenesis rates. Several signaling pathways are involved in the regulation of these processes including growth factors and their tyrosine kinase receptors, such as vascular endothelial growth factor (VEGF, transforming growth factor beta (TGFβ, fibroblast growth factor (FGF, platelet-derived growth factor (PDGF, and insulin-like growth factor–I (IGF–I. Different kinases and regulators also participate in signaling pathways initiated by growth factors, such as mitogen-activated kinases (MAPK, protein kinases C (PKC, phosphatidylinositol-3 kinases (PI3K, protein kinase B (PKB or Akt, glycogen synthase kinase 3β (GSK3β, the mTOR complex, and Bcl-2. In this review, we will focus on the role of these proteins as possible therapeutic targets in GBM.

  3. Characterization of a cDNA encoding metallothionein 3 from cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Jordan, Robin H; Turley, Rickie B; Defauw, Sherri L; Steele, Mark

    2005-04-01

    A cDNA encoding metallothionein (MT) was isolated from a library constructed with poly A(+) RNA purified from 48 h etiolated cotton (Gossypium hirsutum L.) cotyledons. This cDNA encodes a deduced protein with 63 residues and a molecular weight of 6.3 kDa. The protein has 10 cysteines of which 4 are within the CXXCXCXXXXXC amino-terminus motif and six are within the CXCXXXCXCXXCXC carboxyl-terminus motif characteristic of the type III MT (MT3). The cotton MT3 protein sequence is 76.2, 69.8, 66.7, 60.3 and 33.5% identical to MT3 from Carica papaya, Rubus idaeus, Ribes nigrum, Citrus unshiu, and Gossypium hirsutum type I MT, respectively. A fusion protein was constructed by producing PCR primers for the 5' and 3' ends of the cotton MT3 cDNA and ligating the PCR product inframe at the 3' end of a bacterial glutathione S-transferase (GST) gene in the pGEX3 vector. The 5' PCR primer incorporated a segment of the cotton MT3 noncoding region, resulting in an addition of 9 residues to the MT3 (after Factor Xa digestion site) which increased the size of the expressed protein to 72 residues and 7.6 kDa. Expression of the 7.6 kDa protein in bacteria was confirmed by SDS-PAGE. Induction and accumulation of the GST-MT3 protein began inhibiting bacterial growth after 1 h. Addition of Cu (1 muM to 1 mM), 1 mM cysteine, or 1 mM cystine to the media did not rescue growth. Additionally, this protein was evaluated for its ability to bind Cd, Cu, Ni and Zn in the bacterial expression system. We found that cotton MT3 preferentially binds Cu.

  4. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA.

    Science.gov (United States)

    Davis, Claytus; Barvish, Zeev; Gitelman, Inna

    2007-10-09

    The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

  5. Construction and characterization of a goat mammary gland cDNA library.

    Science.gov (United States)

    Han, Xue Feng; Luo, Jun; Wu, Ning; Matand, Kanyand; Yang, Bao Jin; Wu, Hui Juan; Zhang, Li Juan; Wang, Hai Bin

    2008-03-01

    A lactating goat mammary gland cDNA library was constructed by using a modified commercially available cDNA library construction kit protocol. The resulting clones were sequenced and functionally analyzed through cross-species genomic comparison to assess (1) the capacity and functional quality of the constructed library for subsequent research and (2) the efficiency of the procedural modifications. The study resulted in the construction of a high-quality mammary gland cDNA library, which was characterized by (1) the total recombinants number of 1.4 x 10(7) colony-forming units (cfus) that was at least 10 times greater than the number expected from the application of the standard kit protocol, (2) the recombinants rate of 96%, and (3) the average insert size of 1,082 bp. BLAST analysis of sequenced clones against GenBank databases determined 55.7% of clone redundancy, 22 known function gene clusters, and 29 novel gene clusters. The analysis of the primary gene expression profile showed that 59% of the tested clones were genes that coded for milk proteins while 16% of the clones coded for ribosomal, metabolism, immune response, and translation proteins. The remaining 25% of the tested clones were described as novel genes. Cross-species comparison showed that 77% of characterized gene clusters were successfully identified by using resources from other ruminants and unrelated species. This outcome is in consonance with the common belief that the genomic resources that have been generated across species are potentially powerful tools that could be used for enhancing the molecular understanding of less genomically studied species, such as goat.

  6. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA

    Directory of Open Access Journals (Sweden)

    Gitelman Inna

    2007-10-01

    Full Text Available Abstract Background The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. Results We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. Conclusion We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

  7. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  8. Synthesis of deoxycytidine oligomers containing phosphorodithioate linkages

    DEFF Research Database (Denmark)

    Grandas, Ana; Marshall, William S.; Nielsen, John

    1989-01-01

    Deoxydicytidine phosphoramidite, 4-chlorobenzylmercaptan and tetrazole reacted to form dinucleoside thiophosphite. Oxidation with sulfur yields phosphorodithioates which were used to synthesize pentadecadeoxyoligonucleotides containing nuclease resistant phosphorodithioate internucleotide linkages....

  9. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

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

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

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

  11. Cloning and Characterization of a cDNA Encoding a Novel Extracellular Peroxidase from Trametes versicolor

    Science.gov (United States)

    Collins, Patrick J.; O’Brien, Margaret M.; Dobson, Alan D. W.

    1999-01-01

    The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. PMID:10049906

  12. Construction of small RNA cDNA libraries for deep sequencing.

    Science.gov (United States)

    Lu, Cheng; Meyers, Blake C; Green, Pamela J

    2007-10-01

    Small RNAs (21-24 nucleotides) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are potent regulators of gene expression in both plants and animals. Several hundred genes encoding miRNAs and thousands of siRNAs have been experimentally identified by cloning approaches. New sequencing technologies facilitate the identification of these molecules and provide global quantitative expression data in a given biological sample. Here, we describe the methods used in our laboratory to construct small RNA cDNA libraries for high-throughput sequencing using technologies such as MPSS, 454 or SBS.

  13. Construction of small RNA cDNA libraries for high-throughput sequencing.

    Science.gov (United States)

    Lu, Cheng; Shedge, Vikas

    2011-01-01

    Small RNAs (smRNAs) play an essential role in virtually every aspect of growth and development, by regulating gene expression at the post-transcriptional and/or transcriptional level. New high-throughput sequencing technology allows for a comprehensive coverage of smRNAs in any given biological sample, and has been widely used for profiling smRNA populations in various developmental stages, tissue and cell types, or normal and disease states. In this article, we describe the method used in our laboratory to construct smRNA cDNA libraries for high-throughput sequencing.

  14. Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

    Science.gov (United States)

    Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

    2017-02-16

    Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

  15. Characterization of full-length sequenced cDNA inserts (FLIcs from Atlantic salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Lunner Sigbjørn

    2009-10-01

    Full Text Available Abstract Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP, the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91% of the transcripts were annotated using Gene Ontology (GO terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS. The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS. This

  16. Horse cDNA clones encoding two MHC class I genes

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  18. Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines.

    NARCIS (Netherlands)

    Bergman, A.M.; Kuiper, C.M.; Voorn, D.A.; Comijn, E.M.; Myhren, F.; Sandvold, ML; Hendriks, H.R.; Peters, G.J.

    2004-01-01

    1-beta-D-arabinofuranosylcytosine (ara-C) is a deoxycytidine analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate 1-beta-D-arabinofuranosylcytosine triphosphate, but can be deaminated by deoxycytidine deaminase.

  19. Spinach Pyruvate Kinase Isoforms 1

    Science.gov (United States)

    Baysdorfer, Chris; Bassham, James A.

    1984-01-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide Km values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The Ki for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a Ka of 0.05 millimolar, and glutamate is an inhibitor with a Ki of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation. PMID:16663425

  20. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

    Energy Technology Data Exchange (ETDEWEB)

    Hoskins, Roger A.; Stapleton, Mark; George, Reed A.; Yu, Charles; Wan, Kenneth H.; Carlson, Joseph W.; Celniker, Susan E.

    2005-04-22

    The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the gene models. The approach should be effective for improving cDNA collections for other model organisms and the human. It also provides a simple and rapid method for isolating cDNAs of interest in any system for which plasmid cDNA libraries and complete or partial gene sequences are available.

  1. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  2. Bioinformatic mining of kinase inhibitors that regulate autophagy through kinase signaling pathways.

    Science.gov (United States)

    Yang, Yang; Ma, Biao; Jin, Ye; Ben, Wei; Zhang, Dandan; Jiang, Keping; Feng, Shujun; Huang, Lu; Zheng, Jianhua

    2014-12-01

    The aim of this study was to predict the kinase inhibitors that may regulate autophagy. A total of 62 kinases were obtained through text mining by importing the keyword 'autophagy' and a 'protein kinase' Excel file to PubMed. Subsequently, 146 kinases were derivated through screening in the PubMed database by importing the 'autophagy‑associated gene' and 'protein kinase' files. Following intersection of the above two methods, 54 candidate autophagy‑associated kinases were obtained. Enrichment analysis indicated that these candidate autophagy‑associated kinases were mainly enriched in pathways such as the calcium, Wnt, HIF‑1 and mTOR signaling pathways. Among the 54 kinases, 24 were identified through text mining to have specific kinase inhibitors that regulate the corresponding functions; a total of 56 kinase inhibitors were found to be involved in the regulation of these 24 kinases. In total, nine of these 56 kinase inhibitors identified had been widely reported in autophagy regulation studies, 23 kinase inhibitors had been seldom reported and 24 had never been reported. Therefore, introducing these kinases into autophagy regulation analysis in subsequent studies may produce important results.

  3. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-01-20

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

  5. Evaluation of the gene-specific dye bias in cDNA microarray experiments.

    Science.gov (United States)

    Martin-Magniette, Marie-Laure; Aubert, Julie; Cabannes, Eric; Daudin, Jean-Jacques

    2005-05-01

    In cDNA microarray experiments all samples are labeled with either Cy3 or Cy5. Systematic and gene-specific dye bias effects have been observed in dual-color experiments. In contrast to systematic effects which can be corrected by a normalization method, the gene-specific dye bias is not completely suppressed and may alter the conclusions about the differentially expressed genes. The gene-specific dye bias is taken into account using an analysis of variance model. We propose an index, named label bias index, to measure the gene-specific dye bias. It requires at least two self-self hybridization cDNA microarrays. After lowess normalization we have found that the gene-specific dye bias is the major source of experimental variability between replicates. The ratio (R/G) may exceed 2. As a consequence false positive genes may be found in direct comparison without dye-swap. The stability of this artifact and its consequences on gene variance and on direct or indirect comparisons are addressed. http://www.inapg.inra.fr/ens_rech/mathinfo/recherche/mathematique

  6. Construction and characterization of a normalized cDNA library of Nannochloropsis oculata (Eustigmatophyceae)

    Science.gov (United States)

    Yu, Jianzhong; Ma, Xiaolei; Pan, Kehou; Yang, Guanpin; Yu, Wengong

    2010-07-01

    We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179, and obtained 905 nonredundant sequences (NRSs) ranging from 431-1 756 bp in length. Among them, 496 were very similar to nonredundant ones in the GenBank ( E ≤1.0e-05), and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups (KOG). Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs (>60%). We also identified the unigenes encoding phosphorus and nitrogen transporters, suggesting that N. oculata could efficiently transport and metabolize phosphorus and nitrogen, and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids (PUFAs), which will facilitate the demonstration of eicosapentaenoic acid (EPA) biosynthesis pathway of N. oculata. In comparison with the original cDNA library, the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering, and decreased the frequency of abundant gene sequences.

  7. [Cloning, sequencing and subcloning of cDNA coding for group I allergen of Dermatophagoides farinae].

    Science.gov (United States)

    Yang, Qing-gui; Li, Chao-pin

    2004-06-01

    To clone, sequence and subclone the cDNA coding for group 1 allergen of Dermatophagoides farinae (Der f 1). The cDNA of Der f 1 was amplified by RT-PCR and PCR. After purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Der f 1 was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digestion with restriction enzyme. The sequence of inserted Der f 1 gene fragment was also detected. Der f 1 was then subcloned into the vector of pET-32a(+). The Der f 1 gene fragment of Dermatophagoides farinae was specifically amplified from RNA by RT-PCR and PCR. The recombinant plasmid pMD-18T-Der f 1 and pET-32a(+)-Der f 1 was constructed and digested by Bam H I and Sac I, the size of gene fragment was 646 bp and in accordance with the expected one. The pET-32a(+)-Der f 1 subcloning has been constructed successfully.

  8. cDNA sequences of two inducible T-cell genes

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    Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (USA) Guthrie Research Institute, Sayre, PA (USA)); Weissman, S.M. (Yale Univ., New Haven, CT (USA))

    1989-03-01

    The authors have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

  9. Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

    Science.gov (United States)

    Chaves, Diego; Pinzón, Andrés; Grajales, Alejandro; Rojas, Alejandro; Mutis, Gabriel; Cárdenas, Martha; Burbano, Daniel; Jiménez, Pedro; Bernal, Adriana; Restrepo, Silvia

    2010-01-01

    Background Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. Methodology/Principal Findings We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. Conclusions/Significance We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta. PMID:20352100

  10. Discovery of Phytophthora infestans genes expressed in planta through mining of cDNA libraries.

    Directory of Open Access Journals (Sweden)

    Roberto Sierra

    Full Text Available BACKGROUND: Phytophthora infestans (Mont. de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora--Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. METHODOLOGY/PRINCIPAL FINDINGS: We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. CONCLUSIONS/SIGNIFICANCE: We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant--oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.

  11. Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

    Science.gov (United States)

    Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

    2017-02-15

    Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. High-quality RNA preparation from Rhodosporidium toruloides and cDNA library construction therewith.

    Science.gov (United States)

    Yang, Fan; Tan, Haidong; Zhou, Yongjin; Lin, Xinping; Zhang, Sufang

    2011-02-01

    Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A₂₆₀/A₂₈₀ of 2.09. A titer of 10⁵ cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

  14. Construction of cDNA library of cotton mutant Xiangmian-18 library during gland forming stage.

    Science.gov (United States)

    Xie, Yong-Fang; Wang, Bo-Chu; Li, Biao; Cai, Ying-Fan; Xie, Lei; Xia, Yu-Xian; Chang, Ping-An; Jiang, Huai-Zhong

    2007-11-15

    Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. This ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level. Here we described a simple and efficient method for constructing a normalized cDNA library from a cotton mutant, Xiangmian-18, during its pigments gland forming stage. It combined switching mechanism at 5'-end of RNA transcript (SMART) technique and duplex-specific nuclease (DSN) normalization methods. In a model experiment, double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E. coli JM109 by electroporation. Counting the number of colonies, the titer of the original library was 5.86x10(5)cfu/ml in this library. Electrophoresis gel results indicated the fragments ranged from 800bp to 2kb, with the average size of 1400bp. Random picking clones showed that the recombination rate was 94%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of pigments gland cottons.

  15. Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

    Science.gov (United States)

    Singh, Rupesh Kumar; Hou, Weina; Franklin, Gregory

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

  16. Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, M. S.; Han, B. J.; Cha, J. H.; Ryu, Y. M.; Shin, E. K.; Park, J. H.; Park, Y. H.; Kim, M. K. [Korea University Medical College, Seoul (Korea, Republic of)

    2002-07-01

    Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  17. Novel transcripts of human cytomegalovirus clinical strain found by cDNA library screening.

    Science.gov (United States)

    Ma, Y P; Ruan, Q; Ji, Y H; Wang, N; Li, M L; Qi, Y; He, R; Sun, Z R; Ren, G W

    2011-04-05

    Human cytomegalovirus (HCMV) is a double-stranded DNA virus with the largest genome (~235 kb) of the known human herpes viruses. The coding potential and transcript structures of most HCMV predicted genes have not been identified. New or unknown genes could exist in clinical strains. The SMART (switching mechanism at 5' end of RNA template of reverse transcriptase) technique was used to construct a full-length cDNA library of an HCMV clinical strain in the late expression phase. Randomly selected clones were sequenced. The sequenced expressed sequence tags were used to identify the expression and transcript structures of some predicted and unpredicted genes of HCMV. The transcripts of the UL99, TRL5/IRL5, UL73 to UL75, UL4, and UL115 genes, which were previously detected, were obtained with full-length structures from this library. Some novel transcripts, including several transcripts of UL/b' genes and three antisense transcripts of UL83, UL87 and UL31 were found. The novel transcripts that were found, particularly the antisense transcripts of UL83, UL87 and UL31, showed that the transcription of HCMV genes is more complex than previously predicted. Our study highlights the usefulness of the full-length cDNA library for discovering new genes and transcripts of HCMV.

  18. Purification, characterisation and cDNA cloning of an antimicrobial peptide from Macadamia integrifolia.

    Science.gov (United States)

    Marcus, J P; Goulter, K C; Green, J L; Harrison, S J; Manners, J M

    1997-03-15

    An antimicrobial peptide with no significant amino acid sequence similarity to previously described peptides has been isolated from the nut kernels of Macadcamia integrifolia. The peptide, termed MiAMP1, is highly basic with an estimated pI of 10.1, a mass of 8.1 kDa and contains 76 amino acids including 6 cysteine residues. A cDNA clone containing the entire coding region corresponding to the peptide was obtained. The deduced amino acid sequence of the cDNA indicated a 26-amino-acid signal peptide at the N-terminus of the preprotein. Purified MiAMP1 inhibited the growth of a variety of fungal, oomycete and gram-positive bacterial phytopathogens in vitro. Some pathogens exhibited close to 100% inhibition in less than 1 microM peptide (5 microg/ml). Antimicrobial activity was diminished against most, but not all, microbes in the presence of calcium and potassium chloride salts (1 mM and 50 mM, respectively). MiAMP1 was active against bakers yeast, was inactive against Escherichia coli and was non-toxic to plant and mammalian cells. Analysis of genomic DNA indicated that MiAMP1 was encoded on a single copy gene containing no introns. The MiAMP1 gene may prove useful in genetic manipulations to increase disease resistance in transgenic plants.

  19. De-novo transcriptome sequencing of a normalized cDNA pool from influenza infected ferrets.

    Directory of Open Access Journals (Sweden)

    Jeremy V Camp

    Full Text Available The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19,000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response.

  20. Primary analysis of the expressed sequence tags in a pentastomid nymph cDNA library.

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    Jing Zhang

    Full Text Available BACKGROUND: Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. METHODOLOGY/PRINCIPAL FINDINGS: Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19% were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. CONCLUSION/SIGNIFICANCE: We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis.

  1. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  2. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis.

    Science.gov (United States)

    van den Berg, Noëlani; Crampton, Bridget G; Hein, Ingo; Birch, Paul R J; Berger, Dave K

    2004-11-01

    Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

  3. The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1

    Directory of Open Access Journals (Sweden)

    Dong Sook Lee

    2017-10-01

    Full Text Available Receptor-like kinases are important signaling components that regulate a variety of cellular processes. In this study, an Arabidopsis cDNA microarray analysis led to the identification of the cysteine-rich receptor-like kinase CRK36 responsive to the necrotrophic fungal pathogen, Alternaria brassicicola. To determine the function of CRK36 in plant immunity, T-DNA-insertion knockdown (crk36 and overexpressing (CRK36OE plants were prepared. CRK36OE plants exhibited increased hypersensitive cell death and ROS burst in response to avirulent pathogens. Treatment with a typical pathogen-associated molecular pattern, flg22, markedly induced pattern-triggered immune responses, notably stomatal defense, in CRK36OE plants. The immune responses were weakened in crk36 plants. Protein-protein interaction assays revealed the in vivo association of CRK36, FLS2, and BIK1. CRK36 enhanced flg22-triggered BIK1 phosphorylation, which showed defects with Cys mutations in the DUF26 motifs of CRK36. Disruption of BIK1 and RbohD/RbohF genes further impaired CRK36-mediated stomatal defense. We propose that CRK36, together with BIK1 and NADPH oxidases, may form a positive activation loop that enhances ROS burst and leads to the promotion of stomatal immunity.

  4. In-frame cDNA library combined with protein complementation assay identifies ARL11-binding partners.

    Directory of Open Access Journals (Sweden)

    Sangkyou Lee

    Full Text Available The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5'-untranslated regions (UTRs that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5'-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an "in-frame cDNA library." We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11, cellular retinoic acid binding protein 2 (CRABP2, and phosphoglycerate mutase 1 (PGAM1. Thus, the in-frame cDNA library without 5'-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. (Univ. of Washington, Seattle (United States)); Chapline, C.; Crabb, J.W. (W.Alton Jones Cell Science Center, Lake Placid, NY (United States))

    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.

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

    Science.gov (United States)

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

    2006-08-01

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

  7. [Construction of a subtracted cDNA library of chronic intermittent hypoxia rabbit liver by suppression subtractive hybridization].

    Science.gov (United States)

    Wu, Yue-tao; Liu, Rui-hong; Yang, Yu; Luo, Ying-quan; Rong, Yao

    2007-12-01

    To construct a subtracted cDNA library of chronic intermittent hypoxia (CIH) rabbit liver by suppression subtractive hybridization (SSH). Twenty-four rabbits were divided into 4 groups: ordinary feeding group, full-fat food group, ordinary feeding in chronic intermittent hypoxia group, and full-fat food in chronic intermittent hypoxia group. The mRNAs were extracted from different rabbit livers and converted into double-strand cDNA. After digestion with restriction enzyme, the cDNA of hyperlipidemia-sensitive rabbit group was subdivided into 2 portions and each one was lighted with different adaptors. Two rounds of both hybridization and suppression PCR obtained the differentially expressed cDNA. The PCR products were inserted into T/A vector to set up the subtractive cDNA library. The clones were selected and amplified by PCR and identified. Based on the pathology of the abdominal aorta and liver, and the amplified library contained 500 positive bacteria clones, including 462 clones, which had inserts from 250 to 700 bp by PCR analysis. A novel rabbit gene, Cthrc1, involved in CHI had been cloned. The GenBank Accession Number is XM_418373. The molecular mechanism of CIH promoting atherogenesis formation is made clear.

  8. Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

    Science.gov (United States)

    Mahajan, Sonalika; Sharma, Gaurav Kumar; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati Keshari; Pattnaik, Bramhadev

    2015-05-01

    The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  9. [Construction and sequence analysis of a normalized full-length cDNA library of Dendrobium officinale].

    Science.gov (United States)

    Jiang, Min; Wang, Jiang; Wen, Guo-Song; Xu, Shao-Zhong; Zha, Ying-Hong; Rong, Tian-Ju; Qian, Xiong

    2013-02-01

    In order to obtain functional genes, a normalized stems cDNA library was constructed from medicinal plant Dendrobium officinale. SMART (switching mechanism at 5' end of RNA transcript) cDNA synthesis combined with DSN (duplex-specific nuclease) normalization was applied to construct the normalized full-length cDNA library of D. officinale. The titer of cDNA library was about 1.3 x 10(6) cfu x mL(-1) and the average insertion size was about 1.5 kb with high recombination rate (93.9%). Random selected 163 positive clones were sequenced at single side. Bio-information analysis indicated that 147 from 150 high-quality unique sequences matched corresponding homologous proteins, and they participated in various biological processes based on GO (gene ontology). There were 8 clones with complete coding sequence, which presumed to be full-length genes. These results showed preliminarily that we successfully constructed a normalized full-length cDNA library of D. officinale which could be used to screen the functional genes related to metabolic pathways of medicinal ingredients.

  10. A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.

    Science.gov (United States)

    Naimuddin, Mohammed; Kubo, Tai

    2016-02-08

    We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.

  11. Construction of cDNA libraries from Pseudocercospora fijiensis Morelet infected leaves of the cultivars Calcutta 4 and Niyarma Yik

    Directory of Open Access Journals (Sweden)

    Milady Mendoza-Rodríguez

    2004-01-01

    Full Text Available Molecular studies of plant-pathogen interaction are very important for the identification of gene (s related with the pathogenic process, as well as with the plant resistance. These gene (s could be use for the genetic improvement programs in order to obtain resistant cultivars. The aim of this work was to construct complementary DNA (cDNA libraries from infected leaves with Pseudocercospora fijiensis CCIBP-Pf1 isolated of two banana cultivars (a resistant one Calcutta4 and another one susceptible Niyarma Yik. First-strand cDNA synthesis, was made beginning with one microgram of total RNA by using oligo dT primer and cDNA quality was checked by Polimerase chain reaction (PCR with cytochrome b specific primers. Second-strand cDNA synthesis was performed by using the homopolymeric tailing with dC-BamH I + dT-Not I primer combination. Four cDNA libraries of infected plants at different times of infection with the pathogen were obtained. Forty one clones of one of the libraries of Niyarma Yik were sequenced and the obtained sequences correspond with genes related to fungi. Key words: Banana-Mycosphaerella fijiensis interaction,Black Sigatoka, Musa spp.

  12. Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes.

    Science.gov (United States)

    Hei, Y J; Farahbakhshian, S; Chen, X; Battell, M L; McNeill, J H

    1998-01-01

    To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.

  13. Lymphocyte responses of rats vaccinated with cDNA encoding a phosphoglycerate kinase of Fasciola hepatica (FhPGK) and F. hepatica infection.

    Science.gov (United States)

    Wesołowska, Agnieszka; Zawistowska-Deniziak, Anna; Norbury, Luke J; Wilkowski, Przemysław; Pyziel, Anna M; Zygner, Wojciech; Wędrychowicz, Halina

    2018-02-01

    Lymphocyte responses in the blood, peritoneal fluid and both mesenteric and hepatic lymph nodes of cDNA-FhPGK/pCMV vaccinated and/or Fasciola hepatica infected rats of both sexes were investigated to provide an insight into the immune responses that develop in different body compartments. The immune response that developed in cDNA-FhPGK/pCMV vaccinated females contributed to partial protection against F. hepatica infection (54% reduction in fluke recovery), while more liver flukes were found in the livers and bile ducts of cDNA-FhPGK/pCMV vaccinated male rats than in unvaccinated animals (increase of 13%). Rat sex not only affected the ultimate effectiveness of vaccination but also lymphocyte responses following vaccination and/or infection. Different CD4+ and CD8+ T cell profiles were noted in peritoneal fluid and lymph nodes, but not in blood, during acute and chronic fasciolosis. Moreover, independent lymphocyte responses developed in distinct body compartments. Immune responses of rats were polarized towards Th2/Treg with lymphocytes isolated from male rats showing higher IL-4 and IL-10 production than females. Lymphocyte proliferative capacities in response to mitogen (PHA) or vaccine antigen (FhPGK) were impaired in both sexes with a considerably higher reduction observed for males and restored lymphocyte proliferative capacities reported for females vaccinated with cDNA-FhPGK/pCMV during chronic fasciolosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. cDNA expression library screening and identification of two novel antigens, ubiquitin and receptor for activated C kinase (RACK) homologue, of the fish parasite Trypanosoma carassii

    NARCIS (Netherlands)

    Ruszczyk, A.; Joerink, M.; Guldenaar, C.; Hermsen, G.J.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2008-01-01

    Trypanosoma carassii is a kinetoplastid parasite infecting cyprinid fish with a high prevalence in nature. Antibodies have been shown to play a protective role in the immune response against this parasite in common carp, Cyprinus carpio. To identify immunogenic and putative protective T. carassii

  15. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  16. Conformational snapshots of Tec kinases during signaling.

    Science.gov (United States)

    Joseph, Raji E; Andreotti, Amy H

    2009-03-01

    The control of cellular signaling cascades is of utmost importance in regulating the immune response. Exquisitely precise protein-protein interactions and chemical modification of substrates by enzymatic catalysis are the fundamental components of the signals that alert immune cells to the presence of a foreign antigen. In particular, the phosphorylation events induced by protein kinase activity must be spatially and temporally regulated by specific interactions to maintain a normal and effective immune response. High resolution structures of many protein kinases along with supporting biochemical data are providing significant insight into the intricate regulatory mechanisms responsible for controlling cellular signaling. The Tec family kinases are immunologically important kinases for which regulatory details are beginning to emerge. This review focuses on bringing together structural insights gained over the years to develop an understanding of how domain interactions both within the Tec kinases and between the Tec kinases and other signaling molecules control immune cell function.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  18. Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. 1

    Science.gov (United States)

    Sánchez, Federico; Campos, Francisco; Padilla, Jaime; Bonneville, Jean-Marc; Enríquez, Consuelo; Caput, Daniel

    1987-01-01

    Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665575

  19. Cloning and characterization of a female genital complex cDNA from the liver fluke Fasciola hepatica.

    Science.gov (United States)

    Zurita, M; Bieber, D; Ringold, G; Mansour, T E

    1987-01-01

    A cDNA clone whose RNA is abundant in the female genital complex of the liver fluke Fasciola hepatica has been isolated from a cDNA library in lambda gt10 by differential screening. The pattern of expression in different fluke tissues and at different stages of miracidium formation suggests that this gene is expressed in the F. hepatica vitelleria. The nucleotide sequence of the cloned cDNA was determined and the primary structure of the putative protein was deduced. The proposed protein is rich in glycine, lysine, and tyrosine and its overall amino acid composition agrees with that reported for the F. hepatica egg shell. The clone has homology with DNA from other trematodes; this homology is higher in organisms in which egg development is similar to that of F. hepatica and suggests that the protein is conserved in organisms in which miracidium formation occurs in fresh water. Images PMID:3470798

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

    International Nuclear Information System (INIS)

    Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

    2001-01-01

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

  1. Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell.

    Science.gov (United States)

    Chuang, Pan; Shoichiro, Ishizaki; Yuji, Nagashima; Jialong, Gao; Shugo, Watabe

    2018-02-01

    In this article, we report original data on the designation of the primers for full-length cDNA amplification and the internal cDNA amplification of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C) on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental findings please refer to the article "Isolation and cDNA cloning of a novel red color-related pigment-binding protein derived from the shell of shrimp, Litopenaeus vannamei " (Chuang et al., 2017) [1].

  2. Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell

    Directory of Open Access Journals (Sweden)

    Pan Chuang

    2018-02-01

    Full Text Available In this article, we report original data on the designation of the primers for full-length cDNA amplification and the internal cDNA amplification of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental findings please refer to the article “Isolation and cDNA cloning of a novel red color-related pigment-binding protein derived from the shell of shrimp, Litopenaeus vannamei” (Chuang et al., 2017 [1].

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

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

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

  4. Measuring Kinase Activity-A Global Challenge.

    Science.gov (United States)

    Cann, Marissa L; McDonald, Ian M; East, Michael P; Johnson, Gary L; Graves, Lee M

    2017-11-01

    The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Tec family kinases in inflammation and disease.

    Science.gov (United States)

    Horwood, Nicole J; Urbaniak, Ania M; Danks, Lynett

    2012-04-01

    Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis.

  6. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei; Xing, Eric P; Myers, Connie; Mian, Saira; Bissell, Mina J

    2004-12-17

    Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Using LOOCV error of k-NNs as the evaluation criterion, three double

  7. ISOLASI cDNA SUCROSE TRANSPORTER (SUT DARI BATANG TANAMAN TEBU (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    - Slameto

    2010-09-01

    Full Text Available Sucrose Transporter (SUT is kind of protein transporter that control in sucrose translocation. Sucrose Transporter is intermediate in translocation of sucrose from apoplasmic to simplasmic. SUT facilitates sucrose transportation from vascular tissues to parenchyma cells toward in node sugarcane stem. This research was purposed to isolate cDNA SUT from sugarcane stem, and cloned in Escherichia coli strain DH5α. Total RNA of sugarcane stem was isolated by single step method, then add with oligo dT in order to obtain the first strand of SUT cDNA then used as template for PCR. The primer used for PCR is 5’ –ggg ctg att gtg gcc atg tc- ‘3 (SUT-F and 5’ –tgc cct ttg tct ccg gaa cc- ‘3 (SUT-R. PCR was programmed as follow denaturation at 94°C for 2 minutes and 30 second, annealing at 54°C for 30 s, extension at 72°C 2 min and 7 min, and storage at 4°C for unlimited, It was for 30 cycles. Complementary DNA SUT from PCR ligalized to pTOPO bunt-end, then it cloned in to E. coli strain DH5α. The cloning resulted then be sequenced in order to observe the homologues with other nucleotides sequences of some plant using BLASTn program in GENE BANK NCBI and the level of homology determined by Genetyx program. The concentrated of total RNA isolated was 5,024 μg/μl, with purity of 1,85. Complementary DNA SUT fragment from PCR with size 2037 bp appropriated to the both of primer was used. Complementary DNA SUT fragment showed by analyzed some of restriction enzyme e.g. EcoRI, PstI and BamHI. Homologues of this cDNA SUT fragment was 100% to SoSUT 2A of sugarcane stem and 84% to OsSUT of rice plant (Casu et al ., 2003.

  8. Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

    Science.gov (United States)

    Hou, Li; Tang, Jan-Wu; Cui, Xiao-Nan; Wang, Bo; Song, Bo; Sun, Lei

    2004-01-01

    AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential. PMID:15285011

  9. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    Science.gov (United States)

    2017-01-01

    prostate cancer patients have abnormalities in the AKT signaling pathway. These abnormalities are driven by mutations in the PTEN and AKT proteins as...AWARD NUMBER: W81XWH-12-1-0560 TITLE: Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway...2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase

  10. Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene.

    Science.gov (United States)

    Koiwai, O; Yasui, Y; Sakai, Y; Watanabe, T; Ishii, K; Yanagihara, S; Andoh, T

    1993-03-30

    The mouse cDNA encoding DNA topoisomerase I (TopoI) was cloned and the nucleotide sequence of 3512 bp was determined. The cDNA clone contained an open reading frame encoding a protein of 767 amino acids (aa), which is 2 aa longer than its human counterpart. Overall aa sequence homology between the mouse and human, and between the mouse and yeast (Saccharomyces cerevisiae) sequences was 96% and 42%, respectively. The mouse TopI gene was mapped at position 54.5 on chromosome 2 from linkage analyses of a three-point cross test with Geg, Ada, and a as marker genes.

  11. Nonreceptor Tyrosine Kinases in Prostate

    Directory of Open Access Journals (Sweden)

    Cancer Yu-Ming Chang

    2007-02-01

    Full Text Available BACKGROUND: Carcinoma of the prostate (CaP is the most commonly diagnosed cancer in men in the United States. Signal transduction molecules such as tyrosine kinases play important roles in CaP. Src, a nonreceptor tyrosine kinase (NRTK and the first proto-oncogene discovered is shown to participate in processes such as cell proliferation and migration in CaP. Underscoring NRTK's and, specifically, Src's importance in cancer is the recent approval by the US Food and Drug Administration of dasatinib, the first commercial Src inhibitor for clinical use in chronic myelogenous leukemia (CML. In this review we will focus on NRTKs and their roles in the biology of CaP. MATERIALS AND METHODS: Publicly available literature from PubMed regarding the topic of members of NRTKs in CaP was searched and reviewed. RESULTS: Src, FAK, JaK1/2, and ETK are involved in processes indispensable to the biology of CaP: cell growth, migration, invasion, angiogenesis, and apoptosis. CONCLUSIONS: Src emerges as a common signaling and regulatory molecule in multiple biological processes in CaP. Src's relative importance in particular stages of CaP, however, required further definition. Continued investigation of NRTKs will increase our understanding of their biological function and potential role as new therapeutic targets.

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

    International Nuclear Information System (INIS)

    Youssefian, S.; Kamada, I.; Sano, H.

    1990-01-01

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

  13. Genes expression by using cDNA Microarray in Whallak-tang

    Directory of Open Access Journals (Sweden)

    Cheol-kyung Sin

    2008-12-01

    Full Text Available Objective : This study was undertaken to determine the effect of Whallak-tang on expression of CD/cytokine Genes. Methods : The expression of CD/Cytokine Genes were examined by cDNA microarray using the human mast cell line(HMC-l. Results : The expression of ATP5F1, FLJ20671, unknown, KIAA0342, OAS2, unknown genes were increased in 200~300% range. The expression of unknown, MDS006, IFITM1, MRPL3, ZNF207, FTH1, FBP1, NRGN, NR1H2, KIAA0747 genes were decreased in 0~33% range. Conclusion : These results would provide important basic data on the possibility of the clinical treatment of Whallaktang in musculoskeletal disease.

  14. [Development of new SSR markers from EST of SSH cDNA libraries on rose fragrance].

    Science.gov (United States)

    Yan, Hui-Jun; Zhang, Hao; Xie, Ji-Rong; Li, Shu-Fa; Jian, Hong-Ying; Qiu, Xian-Qin; Wang, Qi-Gang; Wang, Ji-Hua; Tang, Kai-Xue

    2009-09-01

    The new SSR markers of rose related fragrance were developed based on the SSH cDNA libraries of rose floral scent mutant. In this study, 10 EST-SSRs (2.6%) from 391 ESTs in the libraries were identified. Six EST-SSRs primers were designed to sequence flanking SSRs. The primer pairs designed were screened on the wild-type Jinyindao, which has flowers full of pleasant scent, and the mutant-type Wangriqinghuai without perceivable floral scent. Five primer pairs were amplified effectively in Jinyindao and Wangriqinghuai, and 3 were polymorphic between Jinyindao and Wangriqinghuai. Eighteen rose cultivars including fragrant roses and nonfragrant roses were identified by the five prime pairs. These results proved that EST-SSR markers are effective markers to identify the polymorphism of the rose.

  15. Development of infectious cDNA clones of Salmonid alphavirus subtype 3

    Directory of Open Access Journals (Sweden)

    Karlsen Marius

    2010-09-01

    Full Text Available Abstract Background Salmonid alphavirus (SAV is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed. Results Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production. Conclusion We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.

  16. Analysis of cDNA libraries from developing seeds of guar (Cyamopsis tetragonoloba (L. Taub

    Directory of Open Access Journals (Sweden)

    Dixon Richard A

    2007-11-01

    Full Text Available Abstract Background Guar, Cyamopsis tetragonoloba (L. Taub, is a member of the Leguminosae (Fabaceae family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4-β-linked D-mannan backbone with single-unit, (1→6-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds. Results A database of 16,476 guar seed ESTs was constructed, with 8,163 and 8,313 ESTs derived from cDNA libraries I and II, respectively. Library I was constructed from seeds at an early developmental stage (15–25 days after flowering, DAF, and library II from seeds at 30–40 DAF. Quite different sets of genes were represented in these two libraries. Approximately 27% of the clones were not similar to known sequences, suggesting that these ESTs represent novel genes or may represent non-coding RNA. The high flux of energy into carbohydrate and storage protein synthesis in guar seeds was reflected by a high representation of genes annotated as involved in signal transduction, carbohydrate metabolism, chaperone and proteolytic processes, and translation and ribosome structure. Guar unigenes involved in galactomannan metabolism were identified. Among the seed storage proteins, the most abundant contig represented a conglutin accounting for 3.7% of the total ESTs from both libraries. Conclusion The present EST collection and its annotation provide a resource for understanding guar seed biology and galactomannan metabolism.

  17. Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

    International Nuclear Information System (INIS)

    Dewji, N.N.; Wenger, D.A.; O'Brien, J.S.

    1987-01-01

    Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

  18. Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR

    International Nuclear Information System (INIS)

    Kijima, Kazuyasu; Toyosawa, Kaoru; Yasuba, Masashi; Matsuoka, Nobuo; Adachi, Tetsuya; Komiyama, Masatoshi; Mori, Chisato

    2004-01-01

    To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment

  19. Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism.

    Science.gov (United States)

    Yano, Daichi; Suzuki, Takaya; Hirokawa, Saki; Fuke, Kyoko; Suzuki, Tomohiko

    2017-08-01

    The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Distinctive expression and functional regulation of the maize (Zea mays L.) TOR kinase ortholog.

    Science.gov (United States)

    Agredano-Moreno, Lourdes Teresa; Reyes de la Cruz, Homero; Martínez-Castilla, León Patricio; Sánchez de Jiménez, Estela

    2007-11-01

    TOR (Target of rapamycin) kinase is a central component of a signal transduction pathway that regulates cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. Knowledge of the TOR pathway in plants is scarce, and reports in agronomical relevant plants are lacking. Previous studies indicate that Arabidopsis thaliana TOR (AtTOR) activity is resistant to rapamycin whereas maize TOR (ZmTOR) is not, suggesting that plants might have different regulation mechanisms for this signal transduction pathway. In the present work maize ZmTOR cDNA was identified and its expression regulation was analyzed during germination on different tissues at various stages of differentiation and by the main ZmTOR regulators. Our results show that ZmTOR contains all functional domains characteristic of metazoan TOR kinase. ZmTOR expression is highly regulated during germination, a critical plant development period, but not on other tissues of contrasting physiological characteristics. Bioinformatic analyses indicated that maize FKBP12 and rapamycin form a functional structure capable of targeting the ZmTOR protein, similar to other non-plant eukaryotes, further supporting its regulation by rapamycin (in contrast with the rapamycin insensitivity of Arabidopsis thaliana) and the conservation of rapamycin regulation through plant evolution.

  1. cDNA cloning and mRNA expression of cat and dog Cdkal1

    OpenAIRE

    Sako T; Arai T; Kawasumi K; Mori A; Nakao N; Suzuki T; Hatano Y; Mori N; Fujiwara M; Takemitsu H; Gebin L; Ishikawa S; Yamamoto I

    2012-01-01

    Ichiro Yamamoto, Shingo Ishikawa, Li Gebin, Hiroshi Takemitsu, Megumi Fujiwara, Nobuko Mori, Yutaka Hatano, Tomoko Suzuki, Akihiro Mori, Nobuhiro Nakao, Koh Kawasumi, Toshinori Sako, Toshiro AraiLaboratory of Veterinary Biochemistry, Nippon Veterinary and Life Science University, Tokyo, JapanAbstract: The cyclin-dependent kinase 5 regulatory subunit–associated protein 1–like 1 (CDKAL1) gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabe...

  2. Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

    Science.gov (United States)

    Cavaney, D M; Rakoczy, P E; Constable, I J

    1995-05-01

    To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

  3. Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs

    Directory of Open Access Journals (Sweden)

    Lundkvist Åke

    2009-01-01

    Full Text Available Abstract Background Affecting both livestock and humans, Rift Valley Fever is considered as one of the most important viral zoonoses in Africa. However, no licensed vaccines or effective treatments are yet available for human use. Naked DNA vaccines are an interesting approach since the virus is highly infectious and existing attenuated Rift Valley Fever virus vaccine strains display adverse effects in animal trials. In this study, gene-gun immunisations with cDNA encoding structural proteins of the Rift Valley Fever virus were evaluated in mice. The induced immune responses were analysed for the ability to protect mice against virus challenge. Results Immunisation with cDNA encoding the nucleocapsid protein induced strong humoral and lymphocyte proliferative immune responses, and virus neutralising antibodies were acquired after vaccination with cDNA encoding the glycoproteins. Even though complete protection was not achieved by genetic immunisation, four out of eight, and five out of eight mice vaccinated with cDNA encoding the nucleocapsid protein or the glycoproteins, respectively, displayed no clinical signs of infection after challenge. In contrast, all fourteen control animals displayed clinical manifestations of Rift Valley Fever after challenge. Conclusion The appearance of Rift Valley Fever associated clinical signs were significantly decreased among the DNA vaccinated mice and further adjustment of this strategy may result in full protection against Rift Valley Fever.

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

    Directory of Open Access Journals (Sweden)

    Yujiao Yang

    2017-03-01

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

  5. Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

    Science.gov (United States)

    Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

    2003-01-01

    Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

  6. Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus)

    NARCIS (Netherlands)

    Vischer, H F; Bogerd, J.

    A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino

  7. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; Tamer, El M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the

  8. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus

  9. Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

    Science.gov (United States)

    Al-Taweel, Khaled; Dilantha Fernando, W G; Brûlé-Babel, Anita L

    2011-01-18

    Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

  10. Construction and Characterization of a cDNA Library from Wheat Infected with Fusarium graminearum Fg 2

    Directory of Open Access Journals (Sweden)

    Anita L. Brûlé-Babel

    2011-01-01

    Full Text Available Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

  11. Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient

    NARCIS (Netherlands)

    Huizing, M.; Iacobazzi, V.; IJlst, L.; Savelkoul, P.; Ruitenbeek, W.; van den Heuvel, L.; Indiveri, C.; Smeitink, J.; Trijbels, F.; Wanders, R.; Palmieri, F.

    1997-01-01

    The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC

  12. Bioinformatic methods for finding differentially expressed genes in cDNA libraries, applied to the identification of tumour vascular targets.

    Science.gov (United States)

    Herbert, John M J; Stekel, Dov J; Mura, Manuela; Sychev, Michail; Bicknell, Roy

    2011-01-01

    The aim of this method is to guide a bench scientist to maximise cDNA library analyses to predict biologically relevant genes to pursue in the laboratory. Many groups have successfully utilised cDNA libraries to discover novel and/or differentially expressed genes in pathologies of interest. This is despite the high cost of cDNA library production using the Sanger method of sequencing, which produces modest numbers of expressed sequences compared to the total transcriptome. Both public and propriety cDNA libraries can be utilised in this way, and combining biologically relevant data can reveal biologically interesting genes. Pivotal to the quality of target identification are the selection of biologically relevant libraries, the accuracy of Expressed Sequence Tag to gene assignment, and the statistics used. The key steps, methods, and tools used to this end will be described using vascular targeting as an example. With the advent of next-generation sequencing, these or similar methods can be applied to find novel genes with this new source of data.

  13. A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

    Science.gov (United States)

    Gadkar, Vijay J; Filion, Martin

    2013-06-01

    In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system. Using as little as 200 ng of total RNA, we developed a linear concatenation strategy to make the single-stranded cDNA template amenable for MDA. The concatenation, made possible by the template switching property of the reverse transcriptase enzyme, resulted in the amplified cDNA library with intact 5' ends. MDA generated micrograms of template, allowing large-scale polymerase chain reaction analyses or other large-scale downstream applications. As the amplified cDNA library contains intact 5' ends, it is also compatible with 5' RACE analyses of specific gene transcripts. Empirical validation of this protocol is demonstrated on a highly characterized (tomato) and an uncharacterized (corn gromwell) experimental system.

  14. Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors.

    Science.gov (United States)

    Kottke, Timothy; Errington, Fiona; Pulido, Jose; Galivo, Feorillo; Thompson, Jill; Wongthida, Phonphimon; Diaz, Rosa Maria; Chong, Heung; Ilett, Elizabeth; Chester, John; Pandha, Hardev; Harrington, Kevin; Selby, Peter; Melcher, Alan; Vile, Richard

    2011-06-19

    Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.

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

    DEFF Research Database (Denmark)

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

    1992-01-01

    The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3...

  16. Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labelled neurofilament cDNA probe.

    NARCIS (Netherlands)

    P. Liesi; J-P. Julien (Jean-Pierre); P. Vilja; F.G. Grosveld (Frank); L. Rechardt

    1986-01-01

    textabstractWe have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells is...

  18. Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization.

    Science.gov (United States)

    Lévesque, V; Fayad, T; Ndiaye, K; Nahé Diouf, M; Lussier, J G

    2003-07-01

    Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.

  19. Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data.

    Science.gov (United States)

    Zhou, Sun; Ji, Guoli; Liu, Xiaolin; Li, Pei; Moler, James; Karro, John E; Liang, Chun

    2012-05-03

    Expressed Sequence Tag (EST) sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be "unclean". Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3'-end terminal structures in designated 5'-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/) using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are "unclean" or abnormal, all of which could be cleaned or filtered by AFST. cDNA terminal pattern analysis, as

  20. Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

    Full Text Available Abstract Background Expressed Sequence Tag (EST sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be “unclean”. Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. Results After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3′-end terminal structures in designated 5′-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/ using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are “unclean” or abnormal, all of which could be cleaned

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  2. Kinase activity and specificity assay using synthetic peptides.

    Science.gov (United States)

    Wu, Xu Na; Schulze, Waltraud X

    2015-01-01

    Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase reaction. Phosphorylation of substrate peptides is then identified and quantified by mass spectrometry.

  3. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

    Science.gov (United States)

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

    1999-01-01

    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

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

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

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

  5. An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

    Directory of Open Access Journals (Sweden)

    Kaur Jatinder

    2010-05-01

    Full Text Available Abstract Background RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L. are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A+ RNA purification and cDNA synthesis. Findings A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. Conclusions This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.

  6. Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line.

    Science.gov (United States)

    Tan, W; Chen, Y; Zhang, L; Lu, Y; Li, S; Zeng, R; Zeng, Y; Li, Y; Cheng, J

    2006-09-01

    A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

  7. Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B.

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Yao, Hang-ping; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan

    2005-04-01

    To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

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

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    Utut Widyastuti Suharsono

    2008-11-01

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

  9. Full-length transcriptome analysis using a bias-free cDNA library prepared with the vector-capping method.

    Science.gov (United States)

    Kato, Seishi; Oshikawa, Mio; Ohtoko, Kuniyo

    2011-01-01

    Full-length complementary DNAs (cDNAs) are an essential resource for functional genomics. Recently, we have developed a simple and efficient method for preparing a full-length cDNA library from a small amount of total RNA, named the "vector-capping" method. The biggest advantage of this method is that the intactness of the cDNA can be assured by the presence of dG at the 5' end of the full-length cDNA. Furthermore, the cDNA library represents the mRNA population in the cell owing to a bias-free procedure. In this chapter, we describe not only the protocol for preparing the library but also the points for analyzing the 5'-end sequence of the obtained cDNA.

  10. Molecular Cloning and Characterization of a P38-Like Mitogen-Activated Protein Kinase fromEchinococcus granulosus.

    Science.gov (United States)

    Lü, Guodong; Li, Jing; Zhang, Chuanshan; Li, Liang; Bi, Xiaojuan; Li, Chaowang; Fan, Jinliang; Lu, Xiaomei; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

    2016-12-01

    Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus . We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus . Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-β1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus , as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-β1.

  11. Molecular cloning and in silico analysis of three somatic embryogenesis receptor kinase mRNA from date palm

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    Rekik Imen

    2013-01-01

    Full Text Available We report here the isolation and characterizations of three somatic embryogenesis receptor kinase (PhSERK genes from palm date by a rapid amplification of cDNA ends (RACE approach. PhSERKs belong to a small family of receptor kinase genes, share a conserved structure and extensive sequence homology with previously reported plant SERK genes. Sequence analysis of these genes revealed the sequence size of 11051 pb (PhSERK1, 7981 pb (PhSERK2 and 10510 pb (PhSERK3. The open reading frames of PhSERK1, PhSERK2 and PhSERK3 are 1914 pb, 1797 pb and 1719 pb respectively. PhSERKs belongs to the LRR-type cell surface RLKs, which possess a number of characteristic domains. These include an extracellular domain (EX containing a variable number of LRR units, signal pepetide (SP immediately followed by a single transmembrane domain (TM and an intracellular kinase domain. The phylogenetic tree shows that the protein PhSERK1, PhSERK2 and PhSERK3 clustered within monocots SERKs proteins groups. We also predicted the secondary and tertiary with ligand binding sites structure of the protein PhSERKs.

  12. Mitotic regulation by NIMA-related kinases

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    Blot Joelle

    2007-08-01

    Full Text Available Abstract The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets.

  13. Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

    Science.gov (United States)

    2010-01-01

    Background Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts

  14. [Identification of an auxin response factor-like protein cDNA from mango cotyledon section].

    Science.gov (United States)

    Xiao, Jie-Ning; Huang, Xue-Lin; Huang, Xia; Li, Xiao-Ju

    2004-01-01

    Auxin-responsive elements (AuxRE) interact with a new class of plant-specific transcription factors, auxin response factors (ARFs). Some of ARFs have been shown to repress or activate expression of genes with an AuxRE promotor element. In Arabidopsis, ARFs play important roles in early embryo development and vascular strand formation (ARF5), floral patterning (ARF3) and photo- and gravitropic responses (ARF7). Two cut surfaces (distal and proximal) of mango (Mangifera indica L. var. Zi-Hua) cotyledon showed different patterns of adventitious root formation, with only the proximal cut surface, but not the distal one, could be induced to form the roots. Thus, the mango cotyledon is a good system for studying adventitious root formation. A cDNA fragment homologous to the Arabidopsis auxin response factor-like protein and relates to adventitious root formation from the cut sections were isolated using suppressive subtractive hybridization (SSH). Two cDNA clones, designated as MiARF1 (mango auxin response factor 1 gene, GenBank accession number AY255705) and MiARF2 (mango auxin response factor 2 gene, GenBank accession number is AY300808), were identified by 3'RACE. MiARF1, 3 272bp long, contains an open reading frame (ORF) of 2 523bp, 5'UTR of 285bp and 3'UTR of 464bp, MiARF2, 1 474bp long, contains an ORF of 981bp, 5' UTR of 285bp and 3'UTR of 208bp. The deduced MiARF1 and MiARF2 are homologues of auxin response factor (ARF) family of transcriptional regulators, and show high similarity to ARF of Arabidopsis in conserved domains. The motifs of MiARF1 EL-WHACAGPL in DBD (DNA binding domain) and GDDPW in IV domain are identical to that of ARF-like protein of Arabidopsis. MiARF2 is identical to MiARF1 in a large part of DBD, but lacks a carboxyl-terminal domain containing conserved motifs III and IV. Virtual Northern blot showed that the expression of MiARF2 was high in rooting tissue of cultured cotyledon sections but low in non-rooting tissue, and the MiARF1 was

  15. [Construction of the female subtractive cDNA library and screening of the specific expressing genes].

    Science.gov (United States)

    Wang, Yan-hai; Peng, Hong-juan; Chen, Xiao-guang; Shen, Shu-man

    2006-02-28

    To screen the Schistosoma japonicum female specific expressing genes. S. japonicum adult worms were collected from the rabbits' vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms. Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40.5%) showed high identity with the S. japonicum egg-shell protein genes, 17 sequences (about 40.5%) were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protein. 8 fragments (about 19.0%) showed high identity with other S. japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S. japonicum actin gene as the internal standard. These fragments were highly homologous to S. japonicum egg shell protein gene AY222885, AY222895, AB

  16. Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target

    Science.gov (United States)

    Tokuhiro, Shinji; Nagataki, Mitsuru; Jarilla, Blanca R.; Nomura, Haruka; Kim, Tae Im; Hong, Sung-Jong; Agatsuma, Takeshi

    2013-01-01

    Background Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK) constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. Methology/Principal findings A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK) of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK) gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. Conclusion CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart, creatine. PMID:24278491

  17. Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

    Directory of Open Access Journals (Sweden)

    Jing-Ying Xiao

    2013-11-01

    Full Text Available BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart

  18. A comparison of parametric and nonparametric methods for normalising cDNA microarray data.

    Science.gov (United States)

    Khondoker, Mizanur R; Glasbey, Chris A; Worton, Bruce J

    2007-12-01

    Normalisation is an essential first step in the analysis of most cDNA microarray data, to correct for effects arising from imperfections in the technology. Loess smoothing is commonly used to correct for trends in log-ratio data. However, parametric models, such as the additive plus multiplicative variance model, have been preferred for scale normalisation, though the variance structure of microarray data may be of a more complex nature than can be accommodated by a parametric model. We propose a new nonparametric approach that incorporates location and scale normalisation simultaneously using a Generalised Additive Model for Location, Scale and Shape (GAMLSS, Rigby and Stasinopoulos, 2005, Applied Statistics, 54, 507-554). We compare its performance in inferring differential expression with Huber et al.'s (2002, Bioinformatics, 18, 96-104) arsinh variance stabilising transformation (AVST) using real and simulated data. We show GAMLSS to be as powerful as AVST when the parametric model is correct, and more powerful when the model is wrong. (c) 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  19. Cloning, Characterization, and Functional Expression of Phospholipase Dα cDNA from Banana (Musa acuminate L.

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    Li Li

    2017-01-01

    Full Text Available Phospholipase D (PLD plays a key role in adaptive responses of postharvest fruits. A cDNA clone of banana (Musa acuminate L. PLDα (MaPLDα was obtained by RT-PCR in this study. The MaPLDα gene contains a complete open reading frame (ORF encoding a 92-kDa protein composed of 832 amino acid residues and possesses a characteristic C2 domain and two catalytic H×K×××D (abbr. HKD motifs. The two HKD motifs are separated by 341 amino acid residues in the primary structure. Relatively higher PLD activity and expression of MaPLDα mRNA were detected in developing tissues compared to senescent or mature tissues in individual leaves, flower, stem, and fruit organs, respectively. The expression profile of PLDα mRNA in postharvest banana fruits at different temperatures was determined, and the MaPLDα mRNA reached the highest expression peak on day 5 at 25°C and on day 7 at 12°C. The results provide useful information for maintaining postharvest quality and extending the storage life of banana fruit.

  20. Frameshifting in the P6 cDNA Phage Display System

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    Veerle Somers

    2010-12-01

    Full Text Available Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zarlenga, D.; Gamble, H.R.

    1987-05-01

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

  2. Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA

    Science.gov (United States)

    1989-01-01

    A distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase that catalyzes the synthesis of the pigment melanin. A tyrosinase cDNA clone, designated BBTY-1, was isolated from a library constructed from the pigmented TA99+/CF21+ melanoma cell line SK-MEL-19. Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized and stored within membrane-bound vesicles in the cytoplasm of transfected fibroblasts. BBTY-1 detected a 2.4-kb mRNA transcript in nine of nine pigmented, tyrosinase-positive melanoma cell lines. Tyrosinase transcripts of the same size and abundance were detected in a subset (three of eight) of nonpigmented, tyrosinase-negative melanoma cell lines, suggesting that post-transcriptional events are important in regulating tyrosinase activity. Two melanocyte antigens, recognized by mAbs TA99 and CF21, that are specifically located within melanosomes and are coexpressed with tyrosinase activity, did not react with transfected mouse fibroblasts expressing human tyrosinase, supporting the conclusion that these antigenic determinants are distinct from the tyrosinase molecule coded for by BBTY-1. PMID:2499655

  3. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    International Nuclear Information System (INIS)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon

    2001-01-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with 33 P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells

  4. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

    Science.gov (United States)

    Aspán, A; Huang, T S; Cerenius, L; Söderhäll, K

    1995-02-14

    Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition system in crustaceans and insects, has been purified and cloned from a crayfish blood cell cDNA library. The deduced amino acid sequence codes for a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, the apparent mass of the purified enzyme. proPO contains two copper atoms, and two putative copper-binding sites were found in the deduced amino acid sequence. Sequence comparisons show that these putative copper-binding sites are similar to the corresponding sites in arthropod hemocyanins and also, although the sequence similarities are less extensive, similar to tyrosinases from vertebrates and microorganisms. The purified enzyme is a typical tyrosinase because it hydroxylates monophenols and oxidizes o-diphenols but does not oxidize p-diphenols. If a homogeneous preparation of crayfish proPO were incubated with a homogeneous sample of the proPO activating enzyme, a serine proteinase, the cleavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and Thr-177.

  5. Characterization of porcine ENO3: genomic and cDNA structure, polymorphism and expression

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    Xiong Yuanzhu

    2008-09-01

    Full Text Available Abstract In this study, a full-length cDNA of the porcine ENO3 gene encoding a 434 amino acid protein was isolated. It contains 12 exons over approximately 5.4 kb. Differential splicing in the 5'-untranslated sequence generates two forms of mRNA that differ from each other in the presence or absence of a 142-nucleotide fragment. Expression analysis showed that transcript 1 of ENO3 is highly expressed in liver and lung, while transcript 2 is highly expressed in skeletal muscle and heart. We provide the first evidence that in skeletal muscle expression of ENO3 is different between Yorkshire and Meishan pig breeds. Furthermore, real-time polymerase chain reaction revealed that, in Yorkshire pigs, skeletal muscle expression of transcript 1 is identical at postnatal day-1 and at other stages while that of transcript 2 is higher. Moreover, expression of transcript 1 is lower in skeletal muscle and all other tissue samples than that of transcript 2, with the exception of liver and kidney. Statistical analysis showed the existence of a polymorphism in the ENO3 gene between Chinese indigenous and introduced commercial western pig breeds and that it is associated with fat percentage, average backfat thickness, meat marbling and intramuscular fat in two different populations.

  6. Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

    Science.gov (United States)

    Rajput, S K; Kumar, P; Roy, B; Verma, A; Pandey, H P; Singh, D; De, S; Datta, T K

    2013-03-01

    A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

  7. Molecular cloning and characterization of the sheep malic enzyme cDNA.

    Science.gov (United States)

    Stefos, Georgios C; Argyrokastritis, Alexandros; Bizelis, Iosif; Rogdakis, Emmanuel

    2008-10-15

    Malic enzyme catalyzes decarboxylation of malate to pyruvate and CO(2), providing de novo biosynthesis of fatty acids with NADPH. Since lipogenesis in ruminants, contrarily to some monogastric species like human and rodents, occurs predominantly in adipose tissue, the activity of many lipogenic enzymes is higher in adipose tissue compared to liver. Expression of malic enzyme is regulated by nutrition; refeeding after a period of starvation results to an induction of the enzyme. Here we present the nucleotide sequence of two transcripts of the ovine cytosolic malic enzyme gene that differ at the length of the 3' UTR. These are the first published cDNA sequences for ruminant species and share high similarity with the corresponding sequences of other species. Malic enzyme mRNA was present in every ovine tissue that was examined. In agreement with the fact that adipose tissue is the major lipogenic site for ruminants, mRNA levels in adipose tissue were higher than in liver. Refeeding after two weeks of caloric restriction resulted in a two-fold increase of the mRNA level of malic enzyme in adipose tissue.

  8. Protein Kinase A in Cancer

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    Antonio Caretta

    2011-02-01

    Full Text Available In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA, that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors.

  9. Protein Kinase A in Cancer

    International Nuclear Information System (INIS)

    Caretta, Antonio; Mucignat-Caretta, Carla

    2011-01-01

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors

  10. Protein Kinase A in Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Caretta, Antonio; Mucignat-Caretta, Carla, E-mail: carla.mucignat@unipd.it [Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova (Italy)

    2011-02-28

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors.

  11. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Directory of Open Access Journals (Sweden)

    Heinz Ruth A

    2003-09-01

    Full Text Available Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4. The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60% did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences

  12. MAP kinase cascades in Arabidopsis innate immunity

    DEFF Research Database (Denmark)

    Rasmussen, Magnus Wohlfahrt; Roux, Milena Edna; Petersen, Morten

    2012-01-01

    Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs) by host transmembrane pattern recognition receptors which trigger MAPK-dependent innate ...

  13. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... including PKA, PKG, ribosomal S6 kinase, and epidermal growth factor receptor kinase (EGFRK) in an ATP- competitive manner, while having no effect on other kinases such as CK1, CK2, MAP kinase, and CSK (Meggio et al. 1995). The necessity for more specific inhibitors of PKC led to the discovery of ...

  14. A novel mutation (Cys145-->Stop) in Bruton's tyrosine kinase is associated with newly diagnosed X-linked agammaglobulinemia in a 51-year-old male.

    Science.gov (United States)

    Kornfeld, S. J.; Haire, R. N.; Strong, S. J.; Tang, H.; Sung, S. S.; Fu, S. M.; Litman, G. W.

    1996-01-01

    BACKGROUND: X-linked agammaglobulinemia (XLA) is a severe, life-threatening disease characterized by failure of B cell differentiation and antibody production and is associated with mutations in Bruton's tyrosine kinase (Btk). The proband in this study is a 51-year-old male presenting with chronic nasal congestion, recurrent sinusitis, sporadic pneumonia, and pronounced B cell deficiency. A family history suggestive of an X-linked immunodeficiency disease was noted. MATERIALS AND METHODS: cDNA was synthesized from mRNA prepared from peripheral blood mononuclear leukocytes. Btk cDNA amplified by polymerase chain reaction (PCR) was subjected to both manual and automated DNA sequencing. A DNA sequence corresponding to exons 6 and 7 of Btk was amplified from genomic DNA. Western blot analysis employed both polyclonal and monoclonal antibodies to Btk and reaction patterns were obtained both by chemiluminescence and an in vitro kinase assay. RESULTS: A mutation (Cys145-->Stop) was identified in Btk cDNA and was confirmed in amplified exon 6 of genomic DNA from both the proband and an affected nephew. Neither Btk nor a truncated peptide was detected in Western blot analyses of peripheral blood mononuclear cell lysates. CONCLUSIONS: The C145A mutation reported here is novel. This family study is extraordinary in that affected male members who did not undergo aggressive medical management either succumbed to complications in early life or survived into later life. The proband is the oldest de novo diagnosed patient with XLA reported to date. Images FIG. 2 FIG. 3 PMID:8898377

  15. The PIM kinases in hematological cancers.

    Science.gov (United States)

    Alvarado, Yesid; Giles, Francis J; Swords, Ronan T

    2012-02-01

    The PIM genes represent a family of proto-oncogenes that encode three different serine/threonine protein kinases (PIM1, PIM2 and PIM3) with essential roles in the regulation of signal transduction cascades, which promote cell survival, proliferation and drug resistance. PIM kinases are overexpressed in several hematopoietic tumors and support in vitro and in vivo malignant cell growth and survival, through cell cycle regulation and inhibition of apoptosis. PIM kinases do not have an identified regulatory domain, which means that these proteins are constitutively active once transcribed. They appear to be critical downstream effectors of important oncoproteins and, when overexpressed, can mediate drug resistance to available agents, such as rapamycin. Recent crystallography studies reveal that, unlike other kinases, they possess a hinge region, which creates a unique binding pocket for ATP, offering a target for an increasing number of potent small-molecule PIM kinase inhibitors. Preclinical studies in models of various hematologic cancers indicate that these novel agents show promising activity and some of them are currently being evaluated in a clinical setting. In this review, we profile the PIM kinases as targets for therapeutics in hematologic malignancies.

  16. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

    Science.gov (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

    1994-04-08

    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  17. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    International Nuclear Information System (INIS)

    Matsushima, H.; Bogenmann, E.

    1990-01-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment

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

    Science.gov (United States)

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2004-02-27

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

  19. (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

    Science.gov (United States)

    Prosser, Ian; Phillips, Andy L; Gittings, Simon; Lewis, Mervyn J; Hooper, Antony M; Pickett, John A; Beale, Michael H

    2002-08-01

    Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

  20. ALP gene expression in cDNA samples from bone tissue engineering using a HA/TCP/Chitosan scaffold

    Science.gov (United States)

    Stephanie, N.; Katarina, H.; Amir, L. R.; Gunawan, H. A.

    2017-08-01

    This study examined the potential use of hydroxyapatite (HA)/tricalcium phosphate (TCP)/Chitosan as a bone tissue engineering scaffold. The potential for using HA/TCP/chitosan as a scaffold was analyzed by measuring expression of the ALP osteogenic gene in cDNA from bone biopsies from four Macaque nemestrina. Experimental conditions included control (untreated), treatment with HA/TCP 70:30, HA/TCP 50:50, and HA/TCP/chitosan. cDNA samples were measured quantitively with Real-Time PCR (qPCR) and semi-quantitively by gel electrophoresis. There were no significant differences in ALP gene expression between treatment subjects after two weeks, but the HA/TCP/chitosan treatment gave the highest level of expression after four weeks. The scaffold using the HA/TCP/chitosan combination induced a higher level of expression of the osteogenic gene ALP than did scaffold without chitosan.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-12-01

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

  3. Acetylcholinesterase of the Sand Fly, Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression, and Biochemical Properties

    Science.gov (United States)

    2013-01-01

    and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus...distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Background: Millions of people and domestic animals around the world are affected by...nucleotides, [GenBank: DQ898276]) respectively, as well as to other arthropod AChEs. The P. papatasi AChE cDNA ORF encodes a 710-amino acid protein

  4. Comparative transcriptome maps: a new approach to the diagnosis of colorectal carcinoma patients using cDNA microarrays

    Czech Academy of Sciences Publication Activity Database

    Jansová, E.; Koutná, I.; Krontorád, P.; Svoboda, Z.; Křivánková, S.; Žaloudík, J.; Kozubek, M.; Kozubek, Stanislav

    2006-01-01

    Roč. 69, č. 3 (2006), s. 218-227 ISSN 0009-9163 R&D Projects: GA MZd(CZ) 1A8241; GA AV ČR(CZ) IAA5004306; GA AV ČR(CZ) IAA1065203; GA ČR(CZ) GA202/04/0907 Institutional research plan: CEZ:AV0Z50040507 Keywords : cDNA microarrays * colorectal carcinoma * expression profiles Subject RIV: BO - Biophysics Impact factor: 3.140, year: 2006

  5. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

    Full Text Available Abstract Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12 gene that is responsible for ricinoleate biosynthesis. The role(s of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2 gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at

  6. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    International Nuclear Information System (INIS)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

    1990-01-01

    A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

  7. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

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

    Science.gov (United States)

    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.

  9. A single cDNA encodes two isoforms of stathmin, a developmentally regulated neuron-enriched phosphoprotein.

    Science.gov (United States)

    Doye, V; Soubrier, F; Bauw, G; Boutterin, M C; Beretta, L; Koppel, J; Vandekerckhove, J; Sobel, A

    1989-07-25

    Stathmin, a 19-kDa neuron-enriched soluble phosphoprotein, has been recently proposed as an ubiquitous intracellular relay for the diverse extracellular signals regulating cell proliferation, differentiation, and functions through various second messenger pathways (Sobel, A., Boutterin, M.C., Beretta, L., Chneiweiss, H., Doye, V., and peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772). Internal sequences of the protein from rat brain were determined after purification by two-dimensional polyacrylamide gel electrophoresis, electrotransfer onto Immobilon, and in situ proteolysis. Oligonucleotide mixtures based on these sequences were used to clone a cDNA for stathmin from a rat PC12 cell lambda gt 10 library. The deduced amino acid sequence reveals partial homologies with the coiled coil structural regions of several intracellular matrix phosphoproteins. Using this cDNA as a probe, we show that the expression of stathmin mRNA parallels that of the protein during brain ontogenesis, reaching a maximum at the neonatal stage. In vitro translation of the derived cRNA yielded all the known molecular forms of stathmin, namely its alpha and beta isoforms in their unphosphorylated and phosphorylated states. Thus, a single cDNA codes for both biologically relevant isoforms of the protein, indicating that they differ by co- or post-translational modifications.

  10. Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification.

    Science.gov (United States)

    Carinelli, S; Kühnemund, M; Nilsson, M; Pividori, M I

    2017-07-15

    This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    International Nuclear Information System (INIS)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

    1988-01-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  12. Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach.

    Directory of Open Access Journals (Sweden)

    Mei Zhang

    Full Text Available Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants' acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway.

  13. Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach.

    Science.gov (United States)

    Zhang, Mei; Mo, Hui; Sun, Wen; Guo, Yan; Li, Jing

    2016-01-01

    Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd) stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants' acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway.

  14. Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

    Science.gov (United States)

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Ky, Huynh; Napis, Suhaimi

    2011-01-01

    Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

  15. Construction of a full-length cDNA library from castor endosperm for high-throughput functional screening.

    Science.gov (United States)

    Lu, Chaofu; Wallis, James G; Browse, John

    2011-01-01

    It is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet increasing demands by the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial fatty acids usually results in low levels of desired fatty acids in transgenic oilseeds. The abundance of unusual fatty acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor fatty acid hydroxylase (FAH12) to identify genes that can boost hydroxy fatty acid accumulation in transgenic seeds. We described previously a high-throughput approach that in principle can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into a FAH12-expressing Arabidopsis line. The resulting transgenic seeds can be screened by high-throughput gas chromatography. The most critical step of the approach is the construction of a full-length cDNA library. In this chapter, we describe in detail the construction of the cloning vectors and a full-length cDNA library from developing castor seed endosperms. The approach we describe has broad applicability in many areas of biology.

  16. Construction of a cDNA library from the ephemeral plant Olimarabidopsis pumila and preliminary analysis of expressed sequence tags.

    Science.gov (United States)

    Zhao, Yun-Xia; Wei, Yan-Ling; Zhao, Ping; Xiang, Cheng-Bin; Xu, Fang; Li, Chao; Huang, Xian-Zhong

    2013-01-01

    Olimarabidopsis pumila is a close relative of the model plant Arabidopsis thaliana but, unlike A. thaliana, it is a salt-tolerant ephemeral plant that is widely distributed in semi-arid and semi-salinized regions of the Xinjiang region of China, thus providing an ideal candidate plant system for salt tolerance gene mining. A good-quality cDNA library was constructed using cap antibody to enrich full-length cDNA with the gateway technology allowing library construction without traditional methods of cloning by use of restriction enzymes. A preliminary analysis of expressed sequence tags (ESTs) was carried out. The titers of the primary and the normalized cDNA library were 1.6 x 10(6) cfu/mL and 6.7 x 10(6) cfu/mL, respectively. A total of 1093 clones were randomly selected from the normalized library for EST sequencing. By sequence analysis, 894 high-quality ESTs were generated and assembled into 736 unique sequences consisting of 72 contigs and 664 singletons. The resulting unigenes were categorized according to the gene ontology (GO) hierarchy. The potential roles of gene products associated with stress-related ESTs are discussed. The 736 unigenes were similar to A. thaliana, A. lyrata, or Thellungiella salsuginea. This research provides an overview of the mRNA expression profile and first-hand information of gene sequence expressed in young leaves of O. pumila.

  17. [Construction and preliminary analysis of a full-length cDNA library for Paris polyphylla var. yunnanensis].

    Science.gov (United States)

    Zhao, Shuang; Dong, Xu; Ma, Teng

    2014-01-01

    A full-length cDNA library of Paris polyphylla var. yunnanensis was constructed in order to research the genes relating to growing development and the genes regulation of its secondary metabolite biosynthesis. The total RNA was extracted from Paris polyphylla var. yunnanensis using modified Trizol method. The SMART (switching mechanism at 5' end of RNA transcript )technology was appliedl to construct the full-length cDNA library. The library titer,recombinant rate and length of insert fragments were determined,the sequences of the library were analyzed by Blastx and were compared to GenBank database. The capacity of the library was 2. 5 x 107 cfu/mL, the recombinant rate was 98.5% and the average size of the inserted fragment was 1.5 kb. 9 ESTs (Expressed Sequence Tags) were relating to growing development and 5 ESTs were relating to regulation of secondary metabolite biosynthesis among 149 ESTs obtained from 192 clones sequenced. A full-length cDNA library of Paris polyphylla var. yunnanensis is constructed by SMART technology successfully, and the library has enough capacity, high recombinant rate and long insert fragment for the further research to screen and identify the functional genes of Paris polyphylla var. yunnanensis.

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

    Science.gov (United States)

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

    1990-10-01

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

  19. Characterization of MMP-9 gene from a normalized cDNA library of kidney tissue of yellow catfish (Pelteobagrus fulvidraco).

    Science.gov (United States)

    Ke, Fei; Wang, Yun; Hong, Jun; Xu, Chen; Chen, Huan; Zhou, Shuai-Bang

    2015-08-01

    Matrix metalloproteinase-9 (MMP-9), one of members of the MMP family, is important for the cleaving of structural extracellular matrix (ECM) molecules and involved in inflammatory processes. In this study, MMP-9 cDNA was isolated and characterized from a normalized cDNA library of kidney tissue of yellow catfish (designated as YcMMP-9). The complete sequence of YcMMP-9 cDNA consisted of 2561 nucleotides. The open reading frame potentially encoded a protein of 685 amino acids with a calculated molecular mass of approximately 77.182 kDa. Amino acid sequence of YcMMP-9 have typical characteristics of MMP-9 family and showed highest identity (85.3%) to channel catfish MMP-9. The YcMMP-9 genomic DNA contains 13 exons and 12 introns. Quantitative RT-PCR (qRT-PCR) analysis showed that YcMMP-9 mRNA was constitutively expressed in all examined tissues in normal fish with high expression in head kidney, trunk kidney, blood, and spleen. However, expression of YcMMP-9 mRNA was induced by Aeromonas hydrophila stimulation, especially in these four tissues mentioned above. It indicated that YcMMP-9 was involved in innate immune responses against bacterial infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Yunwi Heo

    2016-07-01

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

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

    Science.gov (United States)

    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

  2. Cloning and structural analysis of alpha-latroinsectotoxin cDNA. Abundance of ankyrin-like repeats.

    Science.gov (United States)

    Kiyatkin, N; Dulubova, I; Grishin, E

    1993-04-01

    alpha-Latroinsectotoxin (alpha-LIT), purified from venom glands of the black widow spider Latrodectus mactans tredecimguttatus, is a presynaptic neurotoxin selective only for insects. A cDNA encoding the putative alpha-LIT precursor was isolated from a spider venom gland cDNA library. The cDNA contains a 4236-base-pair open reading frame corresponding to a 157826-Da protein composed of 1411 amino acids. The mature alpha-LIT, with molecular mass approximately 130 kDa, is probably derived from double processing in the N-terminal and C-terminal regions of the primary translation product. The structure region, extending over residues 464-1176, is composed almost entirely of ankyrin-like repeats which represent a motif also found in the alpha-latrotoxin (alpha-LTX), which has selective action on vertebrates. Total alignment of the alpha-LIT and alpha-LTX amino acid sequences reveals an overall similarity of 34.1%. Strong sequence divergence is observed in analogous cysteine-rich regions situated within the ankyrin-repeat domains of both alpha-LIT and alpha-LTX.

  3. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

    International Nuclear Information System (INIS)

    Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

    1987-01-01

    To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

  4. Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination.

    Science.gov (United States)

    Weterings, K; Reijnen, W; van Aarssen, R; Kortstee, A; Spijkers, J; van Herpen, M; Schrauwen, J; Wullems, G

    1992-04-01

    This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-06-01

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

  6. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-07-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary lambdagt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/..beta..-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of /sup 125/I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

  7. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    International Nuclear Information System (INIS)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-01-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary λgt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/β-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of 125 I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene

  8. Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

    Science.gov (United States)

    Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A; Fagiolini, Michela; Hensch, Takao K; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide

    2003-06-01

    We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

  9. The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum.

    Science.gov (United States)

    De Smet, Lina; Dimitrov, Ivan; Debyser, Griet; Dolashka-Angelova, Pavlina; Dolashki, Aleksandar; Van Beeumen, Jozef; Devreese, Bart

    2011-11-10

    Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α(D)-HlH, α(N)-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α(D)-HlH and β-HlH was obtained, including the 5' and 3' UTR, 180bp of the 5' end and around 900bp at the 3' end are missing for the third subunit. The subunits α(D)-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α(N)-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α(D)-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Molecular cloning and expression of a hexamerin cDNA from the honey bee, Apis mellifera.

    Science.gov (United States)

    Cunha, Adriana D; Nascimento, Adriana M; Guidugli, Karina R; Simões, Zilá L P; Bitondi, Márcia M G

    2005-10-01

    A cDNA encoding a hexamerin subunit of the Africanized honey bee (Apis mellifera) was isolated and completely sequenced. In the deduced translation product we identified the N-terminal sequence typical of the honey bee HEX 70b hexamerin. The genomic sequence consists of seven exons flanked by GT/AT exon/intron splicing sites, which encode a 683 amino acid polypeptide with an estimated molecular mass of 79.5 kDa, and pI value of 6.72. Semi-quantitative RT-PCR revealed high levels of Hex 70b message in larval stages, followed by an abrupt decrease during prepupal-pupal transition. This coincides with decaying titers of juvenile hormone (JH) and ecdysteroids that is the signal for the metamorphic molt. To verify whether the high Hex 70b expression is dependent on high hormone levels, we treated 5th instar larvae with JH or 20-hydroxyecdysone (20E). In treated larvae, Hex 70b expression was maintained at high levels for a prolonged period of time than in the respective controls, thus indicating a positive hormone regulation at the transcriptional level. Experiments designed to verify the influence of the diet on Hex 70b expression showed similar transcript amounts in adult workers fed on a protein-enriched diet or fed exclusively on sugar. However, sugar-fed workers responded to the lack of dietary proteins by diminishing significantly the amount of HEX 70b subunits in hemolymph. Apparently, they use HEX 70b to compensate the lack of dietary proteins.

  11. Construction and analysis of cotton (Gossypium arboreum L. drought-related cDNA library

    Directory of Open Access Journals (Sweden)

    Zhang Chao-Jun

    2009-07-01

    Full Text Available Abstract Background Drought is one of the most important environmental factors causing water stress for cotton, and it greatly limits cotton growth and crop productivity. So far only a few drought-tolerance genes have been functionally characterized in details, and most efforts on this topic have been made in model organisms. Therefore, to identify more drought-related genes in cotton plays a crucial role in elucidating the underlying mechanisms of drought tolerance as well as utilizing bioengineering techniques to improve the tolerance in this organism. Findings Here we constructed a subtractive drought-tolerance cDNA library using suppressive subtractive hybridization (SSH. Through differential screening and bioinformatics analysis, we identified 392 positive clones with differential expression, corresponding 265 unique genes. By BLAST search against Genbank, we found that more than half of these EST sequences were homologous to those previously known drought-related genes and that there were 57 sequences with unknown functions, suggesting that many more genes are involved in this complex trait. Moreover, using RT-PCR, we examined the expression of nine representative candidate genes and confirmed that their expression levels were increased at different levels under drought stress. Conclusion Our results show that drought tolerance is a complex trait in cotton, which involves the coordination of many genes and multiple metabolism pathways. The candidate EST sequences we identified here would facilitate further functional studies of drought-related genes and provide important insights into the molecular mechanisms of drought-stress tolerance and genetic breeding in cotton.

  12. Normal uniform mixture differential gene expression detection for cDNA microarrays

    Directory of Open Access Journals (Sweden)

    Raftery Adrian E

    2005-07-01

    Full Text Available Abstract Background One of the primary tasks in analysing gene expression data is finding genes that are differentially expressed in different samples. Multiple testing issues due to the thousands of tests run make some of the more popular methods for doing this problematic. Results We propose a simple method, Normal Uniform Differential Gene Expression (NUDGE detection for finding differentially expressed genes in cDNA microarrays. The method uses a simple univariate normal-uniform mixture model, in combination with new normalization methods for spread as well as mean that extend the lowess normalization of Dudoit, Yang, Callow and Speed (2002 1. It takes account of multiple testing, and gives probabilities of differential expression as part of its output. It can be applied to either single-slide or replicated experiments, and it is very fast. Three datasets are analyzed using NUDGE, and the results are compared to those given by other popular methods: unadjusted and Bonferroni-adjusted t tests, Significance Analysis of Microarrays (SAM, and Empirical Bayes for microarrays (EBarrays with both Gamma-Gamma and Lognormal-Normal models. Conclusion The method gives a high probability of differential expression to genes known/suspected a priori to be differentially expressed and a low probability to the others. In terms of known false positives and false negatives, the method outperforms all multiple-replicate methods except for the Gamma-Gamma EBarrays method to which it offers comparable results with the added advantages of greater simplicity, speed, fewer assumptions and applicability to the single replicate case. An R package called nudge to implement the methods in this paper will be made available soon at http://www.bioconductor.org.

  13. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Directory of Open Access Journals (Sweden)

    Thomas Eden

    2018-01-01

    Full Text Available Nanobodies (Nbs are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully

  14. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Science.gov (United States)

    Eden, Thomas; Menzel, Stephan; Wesolowski, Janusz; Bergmann, Philine; Nissen, Marion; Dubberke, Gudrun; Seyfried, Fabienne; Albrecht, Birte; Haag, Friedrich; Koch-Nolte, Friedrich

    2018-01-01

    Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic

  15. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  16. Crystal structure of Cryptosporidium parvum pyruvate kinase.

    Directory of Open Access Journals (Sweden)

    William J Cook

    Full Text Available Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303-320 of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

  17. MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

    OpenAIRE

    Gardner, A M; Vaillancourt, R R; Lange-Carter, C A; Johnson, G L

    1994-01-01

    MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphory...

  18. Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Shimotohno, A.; Ohno, R.; Bišová, Kateřina; Sakaguchi, N.; Huang, J.; Koncz, C.; Uchimiya, H.; Umeda, M.

    2006-01-01

    Roč. 47, - (2006), s. 701-710 ISSN 0960-7412 Institutional research plan: CEZ:AV0Z50200510 Keywords : cyclin -dependent kinase * cdk-activating kinase * cyclin Subject RIV: EE - Microbiology, Virology Impact factor: 6.565, year: 2006

  19. A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  20. A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present