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Sample records for cell clones expressing

  1. cDNA expression cloning in mammalian cells.

    PubMed

    Hoffman, B J

    2001-05-01

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

  2. Gene expression analysis of the CD4+ T-cell clones derived from gingival tissues of periodontitis patients.

    PubMed

    Ito, H; Honda, T; Domon, H; Oda, T; Okui, T; Amanuma, R; Nakajima, T; Yamazaki, K

    2005-12-01

    The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating

  3. Expression of cloned human lactoferrin in baby-hamster kidney cells.

    PubMed Central

    Stowell, K M; Rado, T A; Funk, W D; Tweedie, J W

    1991-01-01

    Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin. Images Fig. 1. Fig. 4. PMID:2049066

  4. A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types.

    PubMed Central

    McMahan, C J; Slack, J L; Mosley, B; Cosman, D; Lupton, S D; Brunton, L L; Grubin, C E; Wignall, J M; Jenkins, N A; Brannan, C I

    1991-01-01

    cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin-1 (IL-1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL-1 receptor (type II receptor). The mature type II IL-1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin-like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL-1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL-1 receptor can bind all three forms of IL-1 (IL-1 alpha, IL-1 beta and IL-1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL-1 receptor. Images PMID:1833184

  5. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    PubMed

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors. PMID:24879460

  6. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    PubMed Central

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors. PMID:24879460

  7. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    PubMed

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. PMID:24462869

  8. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening. PMID:24395411

  9. Molecular cloning, expression and bioactivity of B cell activating factor (BAFF) in African ostrich.

    PubMed

    Yang, Keli; Xiao, Ke; Huang, Haibo; Lu, Shun; Zhong, Juming; Ansari, Abdur Rahman; Khaliq, Haseeb; Song, Hui; Liu, Huazhen; Peng, Kemei

    2015-09-01

    B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds. PMID:26256697

  10. CD38 expression labels an activated subset within chronic lymphocytic leukemia clones enriched in proliferating B cells

    PubMed Central

    Damle, Rajendra N.; Temburni, Sonal; Calissano, Carlo; Yancopoulos, Sophia; Banapour, Taraneh; Sison, Cristina; Allen, Steven L.; Rai, Kanti R.

    2007-01-01

    Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38+ and CD38− members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38+ fraction within a CLL clone, CD38+ subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38+ fraction. Despite these activation/proliferation differences, both CD38+ and CD38− fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38+ cells within clones are associated with poor clinical outcome. PMID:17684154

  11. Production of retroviral constructs for effective transfer and expression of T-cell receptor genes using Golden Gate cloning.

    PubMed

    Coren, Lori V; Jain, Sumiti; Trivett, Matthew T; Ohlen, Claes; Ott, David E

    2015-03-01

    Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins. PMID:25757546

  12. Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.

    PubMed

    Zhang, Ziguo; Yang, Jing; Barford, David

    2016-02-15

    The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells. PMID:26454197

  13. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.

    PubMed

    Neeper, M; Schmidt, A M; Brett, J; Yan, S D; Wang, F; Pan, Y C; Elliston, K; Stern, D; Shaw, A

    1992-07-25

    Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins. PMID:1378843

  14. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    PubMed Central

    Kondo, Hiroshi; Miyoshi, Keiko; Sakiyama, Shoji; Tangoku, Akira; Noma, Takafumi

    2015-01-01

    Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII) cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12) were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC), an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5), an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1) expression levels were enhanced. After treatment with dexamethasone (DEX), 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and keratinocyte growth factor (KGF), surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation. PMID:26167183

  15. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

    PubMed Central

    Funk, C D; Furci, L; FitzGerald, G A

    1990-01-01

    The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway. Images PMID:2377602

  16. Clonal analysis of the T-cell response to in vivo expressed Mycobacterium tuberculosis protein Rv2034, using a CD154 expression based T-cell cloning method.

    PubMed

    Commandeur, Susanna; Coppola, Mariateresa; Dijkman, Karin; Friggen, Annemieke H; van Meijgaarden, Krista E; van den Eeden, Susan J F; Wilson, Louis; van der Ploeg-van Schip, Jolien J; Franken, Kees L M C; Geluk, Annemieke; Ottenhoff, Tom H M

    2014-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death worldwide. A better understanding of the role of CD4+ and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81-100 and Mtb lysate. Remarkably, while the recognition of the dominant p81-100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88-107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations. PMID:24905579

  17. Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

    PubMed Central

    Commandeur, Susanna; Coppola, Mariateresa; Dijkman, Karin; Friggen, Annemieke H.; van Meijgaarden, Krista E.; van den Eeden, Susan J. F.; Wilson, Louis; van der Ploeg-van Schip, Jolien J.; Franken, Kees L. M. C.; Geluk, Annemieke; Ottenhoff, Tom H. M.

    2014-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death worldwide. A better understanding of the role of CD4+ and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81–100 and Mtb lysate. Remarkably, while the recognition of the dominant p81–100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88–107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations. PMID:24905579

  18. Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    PubMed Central

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

    2015-01-01

    Background CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Methods Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Results Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). Conclusion Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera. PMID:25926951

  19. A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

    PubMed

    Hornsby, P J; Yang, L; Lala, D S; Cheng, C Y; Salmons, B

    1992-02-01

    The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics. PMID:1616718

  20. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    PubMed

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. PMID:25792187

  1. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    SciTech Connect

    Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

    1989-01-01

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector /lambda/pSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB/sup +/ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA/sup +/ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB/sup +/ and lysA/sup +/. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes.

  2. [Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells].

    PubMed

    Iakorski, P; Kuntsova, M M; Loseva, E F; Khasanov, F K

    1991-06-01

    The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions. PMID:1944323

  3. Effects of dapoxetine on cloned Kv1.5 channels expressed in CHO cells.

    PubMed

    Jeong, Imju; Yoon, Shin Hee; Hahn, Sang June

    2012-07-01

    The effects of dapoxetine were examined on cloned Kv1.5 channels stably expressed in Chinese hamster ovary cells using the whole-cell patch clamp technique. Dapoxetine decreased the peak amplitude of Kv1.5 currents and accelerated the decay rate of current inactivation in a concentration-dependent manner with an IC ( 50 ) of 11.6 μM. Kinetic analysis of the time-dependent effects of dapoxetine on Kv1.5 current decay yielded the apparent association (k (+1 )) and dissociation (k (-1 )) rate constants of 2.8 μM(-1) s(-1) and 34.2 s(-1), respectively. The theoretical K ( D ) value, derived by k (-1 )/k (+1 ), yielded 12.3 μM, which was reasonably similar to the IC ( 50 ) value obtained from the concentration-response curve. Dapoxetine decreased the tail current amplitude and slowed the deactivation process of Kv1.5, which resulted in a tail crossover phenomenon. The block by dapoxetine is voltage-dependent and steeply increased at potentials between -10 and +10 mV, which correspond to the voltage range of channel activation. At more depolarized potentials, a weaker voltage dependence was observed (δ=0.31). Dapoxetine had no effect on the steady-state activation of Kv1.5 but shifted the steady-state inactivation curves in a hyperpolarizing direction. Dapoxetine produced a use-dependent block of Kv1.5 at frequencies of 1 and 2 Hz and slowed the time course for recovery of inactivation. These effects were reversible after washout of the drug. Our results indicate that dapoxetine blocks Kv1.5 currents by interacting with the channel in both the open and inactivated states of the channel. PMID:22538641

  4. In vitro Expression in Eukaryotic Cells of a Prion Protein Gene Cloned from Scrapie-Infected Mouse Brain

    NASA Astrophysics Data System (ADS)

    Caughey, Byron; Race, Richard E.; Vogel, Mari; Buchmeier, Michael J.; Chesebro, Bruce

    1988-07-01

    It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.

  5. [Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts Saccharomyces cerevisiae and Pichia pastoris].

    PubMed

    Kozlov, D G; Cheperigin, S E; Chestkov, A V; Krylov, V N; Tsygankov, Iu D

    2010-03-01

    Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form. PMID:20391778

  6. An expressed sequence tag database of T-cell-enriched activated chicken splenocytes: sequence analysis of 5251 clones.

    PubMed

    Tirunagaru, V G; Sofer, L; Cui, J; Burnside, J

    2000-06-01

    The cDNA and gene sequences of many mammalian cytokines and their receptors are known. However, corresponding information on avian cytokines is limited due to the lack of cross-species activity at the functional level or strong homology at the molecular level. To improve the efficiency of identifying cytokines and novel chicken genes, a directionally cloned cDNA library from T-cell-enriched activated chicken splenocytes was constructed, and the partial sequence of 5251 clones was obtained. Sequence clustering indicates that 2357 (42%) of the clones are present as a single copy, and 2961 are distinct clones, demonstrating the high level of complexity of this library. Comparisons of the sequence data with known DNA sequences in GenBank indicate that approximately 25% of the clones match known chicken genes, 39% have similarity to known genes in other species, and 11% had no match to any sequence in the database. Several previously uncharacterized chicken cytokines and their receptors were present in our library. This collection provides a useful database for cataloging genes expressed in T cells and a valuable resource for future investigations of gene expression in avian immunology. A chicken EST Web site (http://udgenome. ags.udel. edu/chickest/chick.htm) has been created to provide access to the data, and a set of unique sequences has been deposited with GenBank (Accession Nos. AI979741-AI982511). Our new Web site (http://www. chickest.udel.edu) will be active as of March 3, 2000, and will also provide keyword-searching capabilities for BLASTX and BLASTN hits of all our clones. PMID:10860659

  7. Production of interferon-gamma and tumour necrosis factor-alpha by human T-cell clones expressing different forms of the gamma delta receptor.

    PubMed Central

    Christmas, S E; Meager, A

    1990-01-01

    Panels of human T-cell clones bearing the gamma delta T-cell receptor (TcR) were obtained from peripheral blood and decidual tissue and maintained in the presence of interleukin-2 (IL-2). TcR V gamma and V delta gene expression was determined in 40 TcR delta 1+ clones using the gamma delta T-cell subset markers Ti gamma A and delta TCS1, in conjunction with Southern blot analysis using TcR J gamma and J delta probes. gamma delta T-cell clones, together with control alpha beta T-cell clones derived from the same lymphocyte populations, were stimulated with phytohaemagglutinin (PHA) and their ability to produce interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) tested using specific ELISA. Many clones representative of the major peripheral V gamma 9/V delta 2J1 subset produced high amounts of both cytokines and mean levels were not significantly different from those produced by alpha beta T-cell clones. Panels of clones expressing V gamma 9 and V delta 2J1 produced significantly higher levels of TNF-alpha than clones not expressing V delta 2J1 and those expressing V delta 1J1. There was no relationship between levels of IFN-gamma and TNF-alpha produced by individual gamma delta T-cell clones and also no relationship between their non-major histocompatibility complex (MHC)-restricted cytotoxic activity and levels of either cytokine. There was a significant tendency for gamma delta T-cell clones to produce more TNF-alpha than IFN-gamma in comparison to alpha beta T-cell clones. The significance of these findings is discussed in the light of the reported differences in distribution in vivo of V delta 1J1+ and V delta 2J1+ cells. Images Figure 1 PMID:2126252

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

    PubMed

    Udo, Hiroshi

    2015-01-01

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

  9. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone.

    PubMed Central

    Folks, T M; Clouse, K A; Justement, J; Rabson, A; Duh, E; Kehrl, J H; Fauci, A S

    1989-01-01

    Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, was demonstrated to induce the expression of human immunodeficiency virus (HIV) in a chronically infected T-cell clone (ACH-2). Concentrations of recombinant TNF-alpha as low as 50 pg/ml induced a significant increase over background of HIV expression in the ACH-2 cells as determined by supernatant reverse transcriptase activity. The HIV-inducing effects of TNF-alpha could not be explained by toxic effects on the cells. In addition, both the uninfected parental cell line (A3.01) and the infected ACH-2 cells were shown to have high-affinity receptors for TNF-alpha. Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat. These studies provide evidence that TNF-alpha may play a role in the mechanisms of pathogenesis of HIV infection. Images PMID:2784570

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

    PubMed Central

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

    2015-01-01

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

  11. Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells

    SciTech Connect

    Rogers, S.W.; Rogers, J.C. . Inst. of Biological Chemistry)

    1999-04-01

    The authors cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Ncotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of re-expression of mRNA for the aleurone RNase revealed that, like the pattern for [alpha]-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase [alpha]-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

  12. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis

    PubMed Central

    Guo, Xi-zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα–β–) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  13. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis.

    PubMed

    Guo, Xi-Zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)β(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  14. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    SciTech Connect

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  15. [Cloning of flavone synthase (FNSII) gene and expression in three cell lines of Saussurea medusa].

    PubMed

    Wang, Bingjie; Li, Houhua; Wang, Yajie; Gaol, Yan; Fu, Wany; Weil, Xincui

    2015-12-01

    Saussurea medusa is a rare traditional Chinese medicinal herb, of which luteolin is the niain active medicinal compound for cancer prevention and treatment. A full-length FNSII gene, namely SmFNSII (GenBank Accession No. KF170286), was obtained from green cell line of Saussurea medusa by RT-PCR and RACE-PCR. Sequence analysis indicated that SmFNSII is 1 710 bp in full length, containing a 34 bp 5'-untranslated region (5'-UTR), a 125 bp 3'-UTR, and a 1 551 bp open reading frame (ORF) encoding 516 amino acid residues. Amino acid sequence analysis indicated that SmFNSII belonged to subfamily CYP93B of plant cytochrome P450. Sequence alignment and phylogenetic analysis revealed that amino acid sequences of SmFNSII shared 87% homology with the protein in Hieracium pilosella. Quantitative real-time PCR analysis indicated that SmFNSII expression is the highest in red cell line and the lowest in white cell line, corresponding to quantitative analysis of luteolin concentration. pET-SmFNSII, a prokaryotic expression recombinant plasmid, was constructed and transferred into Escherichia coli, and the expressed protein band was the same size with predicted protein. Saussurea medusa cultivars with high anti-inflammatory, anti-cancer activities and health care function would be cultivated through filtering cell lines and plants with high expression level of FNSII gene and luteolin accumulation. PMID:27093835

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

    PubMed Central

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

    1983-01-01

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

  17. Differential expression of alpha-subunits of G-proteins in human neuroblastoma-derived cell clones.

    PubMed

    Klinz, F J; Yu, V C; Sadée, W; Costa, T

    1987-11-16

    The distribution of alpha- and beta-subunits of G-proteins was analyzed in membranes of three cell clones which are derived from the human neuroblastoma cell line SK-N-SH. The neuroblast-like clone SH-SY5Y shows a pattern of G-proteins very similar to that of human brain cortex with high levels of Gi alpha and Go alpha but low levels of G40 alpha. The intermediate clone SH-IN contains high levels of Go alpha and Gi alpha and moderate levels of G40 alpha. The non-neuronal clone SH-EP shows high levels of G40 alpha but lacks Go alpha. Differentiation of the neuroblast-like clone SH-SY5Y by retinoic acid or nerve growth factor does not change the amount of Gi alpha or Go alpha in the membrane. PMID:3119368

  18. Auto-transporter A protein of Neisseria meningitidis: a potent CD4+ T-cell and B-cell stimulating antigen detected by expression cloning.

    PubMed

    Ait-Tahar, K; Wooldridge, K G; Turner, D P; Atta, M; Todd, I; Ala'Aldeen, D A

    2000-09-01

    A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate. PMID:10972828

  19. Programmed cell death 4 in bacterially-challenged Apostichopus japonicus: Molecular cloning, expression analysis and functional characterization.

    PubMed

    Lv, Zhimeng; Li, Chenghua; Shao, Yina; Zhang, Weiwei; Wang, Zhenhui; Wang, Haihong

    2016-07-01

    Programmed cell death 4 (PDCD4) plays a crucial role in modulating cellular signals, mainly via TOLL cascades during the immune response. In the present study, a novel PDCD4 homologue gene (denoted as AjPDCD4) was cloned from the sea cucumber Apostichopus japonicus using RACE. The full-length AjPDCD4 cDNA comprised a 366bp 5'-UTR, a 418bp 3'-UTR, and a 1353bp open reading frame encoding a 450 amino acid residue protein with two typical MA3 domains. Phylogenetic analysis revealed that AjPDCD4 belonged to the invertebrate PDCD4 family. Spatial expression analysis indicated that AjPDCD4 mRNA transcripts are expressed at a high level in the tentacles and at a low level in muscle compared with coelomocytes. Vibrio splendidus challenge and LPS exposure could both significantly down-regulate AjPDCD4 mRNA expression. More importantly, we found that ultraviolet (UV)-induced ROS production and DNA damage were greatly repressed in AjPDCD4-knockdown coelomocytes. Meanwhile, the expression levels of the NF-kappa B homologue, p105, were synchronously up-regulated in the same conditions. All of these results indicated that AjPDCD4 is involved in modulating DNA damage and ROS production in sea cucumber, perhaps by affecting the TLR pathway. PMID:27262523

  20. Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface.

    PubMed

    Sarhan, Mohammed A A; Musa, Mustaffa; Zainuddin, Zainul F

    2011-09-01

    Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa). PMID:21941936

  1. CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

    PubMed Central

    Weaver, C T; Pingel, J T; Nelson, J O; Thomas, M L

    1991-01-01

    CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling. Images PMID:1652055

  2. Quality improvement of transgenic cloned bovine embryos using an aggregation method: Effects on cell number, cell ratio, embryo perimeter, mitochondrial distribution, and gene expression profile.

    PubMed

    Bang, J I; Jin, J I; Ghanem, N; Choi, B H; Fakruzzaman, M; Ha, A N; Lee, K L; Uhm, S J; Ko, D H; Koo, B C; Lee, J G; Kong, I K

    2015-09-01

    The production of cloned embryos using conventional methods has extremely low success rates owing to low embryo quality. To improve the quality of cloned bovine embryos expressing enhanced green fluorescent protein (EGFP), we applied an aggregation culture method. The EGFP gene was transfected into bovine fetal fibroblasts using a retroviral vector system. Somatic cell nuclear transfer was performed using these cells, and the resulting embryos were cultured in aggregates or individually. Gene expression was analyzed by a microarray, and differentially expressed genes were validated by quantitative real-time polymerase chain reaction. The total number of cells per blastocyst and the ratio of inner cell mass cells to trophectoderm cells were higher in aggregated transgenic cloned blastocysts (agBL; 368.7 ± 109.6 and 1:4.8, respectively) than in in vitro-fertilized blastocysts (ivfBL; 189.8 ± 65.8 and 1:2.6, respectively) and nonaggregated transgenic cloned blastocysts (sBL; 113.1 ± 36.3 and 1:1.5, respectively; P < 0.05 and P < 0.01, respectively). Moreover, the blastocyst perimeter was larger in the agBL group than in the ivfBL and sBL groups (1168.8 ± 200.23 vs. 887.33 ± 187.62 and 678 ± 226.1 μm; P < 0.05). In addition, mitochondrial fluorescence intensity was higher in the agBL group than in the ivfBL and sBL groups (P < 0.05). The number of apoptotic cells per blastocyst was lower in the ivfBL and agBL groups than in the sBL group (3.7 ± 2.2 and 3.4 ± 2.1 vs. 6.7 ± 6.8; P < 0.05). The genes identified in the microarray belonged to 18 categories. Expression of the Krüppel-like factor 4 gene, which is associated with cell proliferation, development, and transcription, was 7.2-fold higher in the agBL group than in the ivfBL group (P < 0.05) but did not differ between the sBL and ivfBL groups (P > 0.05). Expression of the heat shock 70-kDa protein 1A gene, which is associated with apoptosis, was 12-fold higher in the s

  3. Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

    PubMed Central

    Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

    2015-01-01

    Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites

  4. Single cell derived murine embryonic stem cell clones stably express Rex1-specific green fluorescent protein and their differentiation study

    SciTech Connect

    Chen Xiaopan; Wu Rongrong; Feng Shumei; Gu Bin; Dai Licheng; Zhang Ming; Zhao Xiaoli

    2007-10-19

    Embryonic stem cells (ESCs) often display high rates of apoptosis and spontaneous differentiation in routine culture, thus bring the proliferation of these cells highly inefficient. Moreover, little is known about the factors that are indispensable for sustaining self-renewal. To surmount these issues, we established transgenic mES cell lines expressing the enhanced green fluorescent protein (EGFP) under the control of the Rex1 promoter which is a key regulator of pluripotency in ES cells. In addition, we provided a simplified and improved protocol to derive transgenic mESCs from single cell. Finally, we showed that embryoid body (EB) development was faster than adherent differentiation in terms of differentiation ratio by real-time tracking of the EGFP expression. Therefore, these cell lines can be tracked and selected both in vitro and in vivo and should be invaluable for studying the factors that are indispensable for maintaining pluripotency.

  5. Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor

    NASA Astrophysics Data System (ADS)

    Ishikawa, Fuyuki; Miyazono, Kohei; Hellman, Ulf; Drexler, Hannes; Wernstedt, Christer; Hagiwara, Koichi; Usuki, Kensuke; Takaku, Fumimaro; Risau, Werner; Heldin, Carl-Henrik

    1989-04-01

    Cloning and sequencing of the complementary DNA for platelet-derived endothelial cell growth factor indicates that it is a novel factor distinct from previously characterized proteins. The factor, a protein with a relative molecular mass of about 45,000, stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo.

  6. Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

    PubMed

    Aceti, David J; Bingman, Craig A; Wrobel, Russell L; Frederick, Ronnie O; Makino, Shin-Ichi; Nichols, Karl W; Sahu, Sarata C; Bergeman, Lai F; Blommel, Paul G; Cornilescu, Claudia C; Gromek, Katarzyna A; Seder, Kory D; Hwang, Soyoon; Primm, John G; Sabat, Grzegorz; Vojtik, Frank C; Volkman, Brian F; Zolnai, Zsolt; Phillips, George N; Markley, John L; Fox, Brian G

    2015-06-01

    Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed. PMID:25854603

  7. Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model

    PubMed Central

    Garcia-Manteiga, Jose M.; Bonfiglio, Silvia; Malosio, Maria Luisa; Lazarevic, Dejan; Stupka, Elia; Cittaro, Davide; Meldolesi, Jacopo

    2015-01-01

    Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation. PMID:26413508

  8. Expression Profiles of Cloned Channel Catfish (Ictalurus punctatus) Lymphoid Cell Lines and Mixed Lymphocyte Cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLC) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date clonal catfish cell lines and MLC have been biologically and phenotypically characterized using a variety of techniq...

  9. Met-ase: Cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells

    SciTech Connect

    Smyth, M.J.; Trapani, J.A. ); Sayers, T.J.; Wiltrout, T. ); Powers, J.C. )

    1993-12-01

    A cDNA clone encoding a human NK serine protease was obtained by screening a [lambda]-gt10 library from the Lopez NK leukemia with the rat natural killer Met-ase (RNK-Met-1) cDNA clone. In Northern blot analysis human Met-ase (Hu-Met-1) cDNA hybridized with a 0.9-kb mRNA in two human NK leukemia cell lines, unstimulated human PBMC, and untreated purified CD3[sup [minus

  10. CLONING AND EXPRESSION OF RABBIT INTERLEUKIN-15

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA co...

  11. Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

    PubMed Central

    Mao, Mao; Fu, Gang; Wu, Ji-Sheng; Zhang, Qing-Hua; Zhou, Jun; Kan, Li-Xin; Huang, Qiu-Hua; He, Kai-Li; Gu, Bai-Wei; Han, Ze-Guang; Shen, Yu; Gu, Jian; Yu, Ya-Ping; Xu, Shu-Hua; Wang, Ya-Xin; Chen, Sai-Juan; Chen, Zhu

    1998-01-01

    Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34+ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34+ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation. PMID:9653160

  12. Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

    PubMed Central

    Agrawal, Neema; Malhotra, Pawan; Bhatnagar, Raj K.

    2002-01-01

    Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction. PMID:12200317

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

    PubMed

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

    2000-01-01

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

  14. PAF-receptor is preferentially expressed in a distinct synthetic phenotype of smooth muscle cells cloned from human internal thoracic artery: Functional implications in cell migration

    SciTech Connect

    Stengel, Dominique; O'Neil, Caroline; Brocheriou, Isabelle; Karabina, Sonia-Athina; Durand, Herve; Caplice, Noel M.; Pickering, J. Geoffrey; Ninio, Ewa . E-mail: ninio@chups.jussieu.fr

    2006-08-04

    Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation.

  15. Cloning

    MedlinePlus

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

  16. Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2).

    PubMed

    Hou, J; Wang, F; McKeehan, W L

    1994-02-01

    The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp 130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line. PMID:8012652

  17. A Novel Ubiquitin-Specific Protease, UBP43, Cloned from Leukemia Fusion Protein AML1-ETO-Expressing Mice, Functions in Hematopoietic Cell Differentiation

    PubMed Central

    Liu, Li-Qin; Ilaria, Robert; Kingsley, Paul D.; Iwama, Atsushi; van Etten, Richard A.; Palis, James; Zhang, Dong-Er

    1999-01-01

    Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation. PMID:10082570

  18. A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation.

    PubMed

    Liu, L Q; Ilaria, R; Kingsley, P D; Iwama, A; van Etten, R A; Palis, J; Zhang, D E

    1999-04-01

    Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation. PMID:10082570

  19. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    PubMed Central

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

  20. Lack of correlation between membrane CD30 expression and cytokine secretion pattern in allergen-primed naive cord blood T-cell lines and clones.

    PubMed

    Spinozzi, F; Agea, E; Piattoni, S; Falini, B; Grignani, F; Bertotto, A

    1997-04-01

    Various surface molecules are expressed by activated T cells. Among them, the CD30 antigen has been proposed as a reproducible marker that identifies a subset of differentiated and/or activated T lymphocytes that produce T helper (Th)-2-type cytokines, i.e. interleukin (IL)-4 and IL-5. However, because CD30 has mainly been detected on established T-cell clones, it is still unclear whether a priming allergen and/or cytokine can induce its membrane expression on naive T cells, perhaps in parallel with the up-regulation of other relevant activation markers, such as CD25, HLA-DR and L-selectin. It is also unknown whether proper allergen stimulation affects the cytokine secretion pattern by CD30+ T-cell clones derived from antigen-unprimed (naive) T lymphocytes. More information on these questions was sought by adopting a model that used cord blood as a source of virgin T cells and exposing them to native cypress allergen or cytokine (IL-2 or IL-4) stimulation, as well as to conventional polyclonal activators such as PHA or anti-CD3. Peripheral blood MC from four adult cypress-sensitive patients was also assayed and used as controls for all culture experiments. Freshly isolated cord and adult T cells did not express the CD30 antigen on their membrane. Many of the stimulating agents tested were able to up-regulate the expression of CD30. However, despite high expression of this molecule, cloned allergen-specific cord CD4+ T lymphocytes were unable to produce IFN-gamma and/or IL-4. In contrast, they retained the capability to produce IL-2. Thus, expression of the CD30 antigen on virgin T cells does not correlate with a polarized model of T helper (Th)-1 or Th-2 cytokine-producing cells, suggesting that these types of lymphokine-secreting lymphocytes are not a paradigmatic example of T-cell subpopulations that display stable phenotypical features. PMID:9105430

  1. Molecular cloning of a novel NF2/ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells.

    PubMed

    Shimizu, K; Nagamachi, Y; Tani, M; Kimura, K; Shiroishi, T; Wakana, S; Yokota, J

    2000-04-15

    We have cloned a novel gene, Ehm2, that is expressed in high-metastatic but not in low-metastatic K-1735 murine melanoma cells. The Ehm2 gene encodes a protein of 527 amino acid residues, showing up to 41% amino acid identity with the FERM domain of NF2/ERM/4.1 superfamily proteins, which have the function of connecting cell surface transmembrane proteins to cytoskeletal molecules. The Ehm2 gene was mapped to chromosome 4 and was expressed in the liver, lung, kidney, and testis and in 7- to 17-day embryos. The highest level of homology was observed with NBL4, which is a new subfamily protein of the NF2/ERM/4.1 superfamily. A human homologue of the mouse Ehm2 gene, showing significant homology (83% identity), was identified in the genomic DNA and EST databases. Furthermore, seven rat EST clones and one pig EST clone in the GenBank EST database were identified as having 83-92% sequence homology with the cDNA sequence of the mouse Ehm2 gene. Thus, Ehm2 is a highly conserved gene that encodes a novel member of the NF2/ERM/4.1 superfamily proteins. PMID:10783258

  2. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    PubMed Central

    Sasso, Emanuele; Paciello, Rolando; D'Auria, Francesco; Riccio, Gennaro; Froechlich, Guendalina; Cortese, Riccardo; Nicosia, Alfredo; De Lorenzo, Claudia; Zambrano, Nicola

    2015-01-01

    Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications. PMID:26649313

  3. Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA

    SciTech Connect

    Tsurimoto, T.; Fujiyama, A.; Matsubara, K.

    1987-01-01

    A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

  4. Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells.

    PubMed Central

    Trobridge, G D; Chiou, P P; Leong, J A

    1997-01-01

    Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells. PMID:9188599

  5. Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line

    PubMed Central

    Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

    2012-01-01

    Background: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Methods: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting. Results: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. Conclusion: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models. PMID:22977370

  6. A simple improvement in expression cloning.

    PubMed

    Takemoto, Y; Furuta, M; Sato, M; Hashimoto, Y

    1997-06-01

    Expression cloning is an effective approach for isolating genes encoding proteins that associate with a target species. Several molecules have been isolated by expression cloning, including CRE-BP1 associating with Jun (Macgregor et al., 1990); Grb1, identical to p85 PI3-kinase, with the EGF receptor (Skolnik et al., 1991); and Max with Myc (Blackwood and Eisenman, 1991). Expression cloning involves induction of proteins from a lambda gt11 cDNA expression library and screening the proteins on nitrocellulose membranes using a peptide probe (Macgregor et al., 1990). With this method, we previously isolated an Lck tyrosine kinase-associated protein, LckBP1, which is identical to HS1 (Kitamura et al., 1989, 1995; Takemoto et al., 1995). In those experiments, we used a glutathione S-transferase (GST)-Lck SH3 domain fusion protein as a probe, followed by detection of the complex with anti-GST polyclonal antibody. Whereas the ease of obtaining the fusion construct and high-titer anti-GST polyclonal antibody represented clear advantages, the system suffered from high background and low sensitivity. Here we show that pretreatment of nitrocellulose filters with NaDodSO4 reduces background and, in turn, increases sensitivity. PMID:9212173

  7. Molecular cloning, expression pattern analysis of porcine Rb1 gene and its regulatory roles during primary dedifferentiated fat cells adipogenic differentiation.

    PubMed

    Hu, Xiaoming; Luo, Pei; Peng, Xuewu; Song, Tongxing; Zhou, Yuanfei; Wei, Hongkui; Peng, Jian; Jiang, Siwen

    2015-04-01

    Adipocytes are the main constituent of adipose tissue and are considered to be a corner stone in the homeostatic control of whole body metabolism. Recent reports evidenced that retinoblastoma 1 (Rb1) gene plays an important role in fat development and adipogenesis in mice. Here, we cloned the partial cDNA sequences of the porcine Rb1 gene which contains the complete coding sequences (CDS) of 2820bp encoding a protein of 939 amino acids. Bioinformatic analysis revealed that the CDS of porcine Rb1 was highly identical with those of cattle, human and mice. The porcine Rb1 has three typical conserved structural domains, including Rb-A pocket domain, CYCLIN domain and C-terminus domain, and the phylogenetic tree indicates a closer genetic relationship with cattle and human. Tissue distribution analysis showed that Rb1 expression appeared to be ubiquitously in various tissues, being higher in heart, liver, muscle, and stomach. Furthermore, significant downregulation of Rb1 was found at the initial stage of dedifferentiated fat (DFAT) cells adipogenic differentiation. With the knockdown of the Rb1 expression by siRNA, the number of DFAT cells recruited to white rather than brown adipogenesis was promoted, and mRNA levels of adipogenic markers, such as PPARγ, aP2, LPL and adiponectin and protein expression of PPARγ and adiponectin were increased after hormone stimulation. The underlying mechanisms may be that knockdown of Rb1 promotes the mitotic clonal expansion and PPARγ expression by derepressing the transcriptional activity of E2F so as to facilitate the first steps of adipogenesis. In summary, we cloned and characterized an important negative regulator in adipogenic commitment of porcine DFAT cells. PMID:25626122

  8. Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

    PubMed Central

    Erickson, J D; Eiden, L E; Hoffman, B J

    1992-01-01

    A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules. Images PMID:1438304

  9. DQw3 variants defined by cloned alloreactive T cells.

    PubMed

    Mickelson, E M; Nepom, G T; Nisperos, B; Hansen, J A

    1988-01-01

    The polymorphism of HLA class II molecules expressing the serologically defined alloantigen DQw3 was studied using cloned proliferative T lymphocytes. Two clones, IG9 and IC3, were selectively primed against DQw3-associated determinants and tested against a panel of 92 HLA-D homozygous cells. Both clones were specific for DQw3, but each showed a distinct response pattern. Clone IG9 recognized a DQw3-associated determinant expressed on a subset of DR4 and DR5 haplotypes and on all DRw6, 7, w8, and w9 haplotypes tested. In contrast, clone IC3 recognized a distinct DQw3-associated determinant expressed only on a subset of DR4 haplotypes. In monoclonal antibody inhibition experiments, anti-DQ, but not anti-DR or anti-DP antibodies, blocked reactivity of both clones IG9 and IC3, further demonstrating that the determinants defined by these clones are associated with DQ molecules. In DNA hybridization studies using a DQ beta probe, a correlation was observed between restriction site polymorphisms in the DQ beta gene, designated DQw"3.1" and "3.2," and the expression of the T-cell-defined IG9 and IC3 determinants. It is, thus, possible to demonstrate by cloned T-cell reactivity functionally relevant recognition sites on DQw3+ molecules that are associated with structural polymorphisms defined by molecular and genomic analysis. PMID:2452816

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

    PubMed

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

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide-derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human--hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and its gene regulation. PMID:1907252

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

    SciTech Connect

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

    1991-06-01

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

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

    PubMed

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

    2009-05-01

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

  13. Cloning and expression in Escherichia coli cells of a plasmid pBS195 gene that determines the activity of oxygenase

    SciTech Connect

    Kozlova, E.V.; Suvorova, E.S.; Romanov, V.P.; Boronin, A.M.

    1995-02-01

    Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not been detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a {sup 32}P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I-BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli minicells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa. 9 refs., 3 figs., 1 tab.

  14. [Cloning and expression of the transmembranic glycoprotein from human T cell lymphotropic virus in a prokaryotic system].

    PubMed

    Russo-Carbolante, Elisa Maria de Sousa; Penteado, Flora Cristina Lobo; Medeiros, Luciene; Kashima, Simone; Takayanagui, Oswaldo M; Covas, Dimas Tadeu

    2007-01-01

    HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology. PMID:17653460

  15. Automated Cell-Cutting for Cell Cloning

    NASA Astrophysics Data System (ADS)

    Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

    We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

  16. [Cloning and expression of variants of the glutaryl-7-aminocephalosporic acid acylase of the bacterium Brevundimonas diminuta in Escherichia coli cells].

    PubMed

    Khatuntseva, S A; El'darov, M A; Lopatin, S A; Zeĭnalov, O A; Skriabin, K G

    2007-01-01

    The gene coding for glutaryl-7-aminocephalosporic acid acylase (Gl7ACA acylase) of the bacterium Brevundimonas diminuta (BrdGl7ACA), a commercial enzyme widely used in modem biocatalytic technologies for manufacture of b-lactam antibiotics, was cloned. Efficient expression systems for producing a "native" recombinant BrdGl7ACA and its analogs modified by attaching affinity groups--the chitin-binding domain of chitinases A1 and hexahistidine sequence--were designed. It was demonstrated that both the recombinant hybrid proteins and the native Gl7ACA acylase produced in E. coli cells underwent a correct autoproteolytic processing with generation of functionally active enzymes and could be isolated with a high yield using one-step affinity chromatography. PMID:17929575

  17. Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells.

    PubMed

    Wu, S; Wright, R A; Rockey, P K; Burgett, S G; Arnold, J S; Rosteck, P R; Johnson, B G; Schoepp, D D; Belagaje, R M

    1998-01-01

    Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67-70% amino acid similarity with each other and 42-45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist L-AP4 varied greatly between the group III clones, being mGluR8>mGluR4 > mGluR7. The reported group II mGluR agonist L-CCG-I was a highly potent mGluR8 agonist (EC50=0.35 microM), with significant agonist activities at both mGluR4 (EC50=3.7 microM) and mGluR7 (EC50=47 microM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8 > mGluR4 = mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands. PMID:9473604

  18. Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line.

    PubMed

    Kojima, T; Shworak, N W; Rosenberg, R D

    1992-03-01

    The cloned rat fat pad endothelial cell (RFP-EC) line synthesizes anticoagulantly active heparan sulfate proteoglycans (HSPGact) and anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact), both of which exhibit 25-, 30-, and 50-kDa core proteins of extremely similar structure. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kDa component from the HSPGinact core proteins demonstrate that the 30-kDa component is a previously unidentified species, designated as ryudocan, with the 25-kDa component representing a proteolytic degradation product, while the 50-kDa component is the rat homolog of syndecan (Saunders, S. Jalkanen, M., O'Farrell, S., and Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556). Specific oligonucleotide probes were obtained for ryudocan and syndecan by polymerase chain reaction, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only three potential glycosaminoglycan attachment sites within the extracellular region while syndecan has five glycosaminoglycan attachment sites within the same domain. Both species are expressed in RFP-EC lines, primary rat aortic smooth muscle cells and primary rat skin fibroblast cells. The levels of ryudocan and syndecan mRNA were measured by quantitative polymerase chain reaction in primary microvascular endothelial cells and closely associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA. PMID:1537865

  19. Cloning, expression and sequencing of Helicobacter felis urease genes.

    PubMed

    Ferrero, R L; Labigne, A

    1993-07-01

    Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease. PMID:8412683

  20. Cloning, heterologous expression and characterization of ascorbate peroxidase (APX) gene in laticifer cells of rubber tree (Hevea brasiliensis Muell. Arg.).

    PubMed

    Chao, Jinquan; Zhang, Shixin; Chen, Yueyi; Tian, Wei-Min

    2015-12-01

    Ascorbate peroxidases (APXs) are a kind of crucial enzymes for removing reactive oxygen species (ROS) in plant cell. In the present study, a full-length cDNA encoding an APX, designated HbAPX, was isolated from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbAPX was 1174-bp in length and contained a 912-bp open reading frame (ORF) encoding a putative protein of 304 amino acids. The predicted molecular mass of HbAPX was 27.6 kDa (kDa) with an isoelectric point (pI) of 6.73. The phylogenetic analysis showed that HbAPX belonged to the cytosolic subgroup and was more relative to PtAPX and MdAPX2. By using PlantCare online analysis, such cis-acting elements as W-box and MRE were detected in the promoter region of HbAPX. Overproduction of recombinant HbAPX protein either in Escherichia coli or yeast enhanced their tolerance to such abiotic stresses as Cu(2+), Zn(2+), Na(2+) and hydrogen peroxide (H2O2). Ethrel application significantly down-regulated the expression of HbAPX and inhibited the activity of HbAPX in vivo. The ethrel-caused down-regulation of HbAPX may disturb the redox homeostasis in laticifer cells of rubber tree. PMID:26519821

  1. Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression.

    PubMed Central

    Schrewe, H; Thompson, J; Bona, M; Hefta, L J; Maruya, A; Hassauer, M; Shively, J E; von Kleist, S; Zimmermann, W

    1990-01-01

    Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region. Images PMID:2342461

  2. Cloning

    MedlinePlus

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

  3. Single cell-derived clones from human adipose stem cells present different immunomodulatory properties

    PubMed Central

    Sempere, J M; Martinez-Peinado, P; Arribas, M I; Reig, J A; De La Sen, M L; Zubcoff, J J; Fraga, M F; Fernández, A F; Santana, A; Roche, E

    2014-01-01

    Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single-cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, five single-cell clones were isolated (generally called 1.X and 3.X) from two volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1·10 and 1·22 expressed the lowest amounts, while clones 3·10 and 3·5 expressed more CD105 than the rest and clone 1·7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of interleukin (IL)-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore, and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of proinflammatory cytokines such as IL-1β, while clones 1·10 and 1·22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are, together, more potent inhibitors than clones 3.X for proliferation of total, CD3+T, CD4+T and CD8+T lymphocytes and natural killer (NK) cells. The results of this work indicate that the adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies. PMID:24666184

  4. Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

    PubMed Central

    Sharma, Ashu; Sojar, Hakimuddin T.; Glurich, Ingrid; Honma, Kiyonobu; Kuramitsu, Howard K.; Genco, Robert J.

    1998-01-01

    Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response. PMID:9826345

  5. Seed coat-associated invertases of fava bean control both unloading and storage functions: cloning of cDNAs and cell type-specific expression.

    PubMed

    Weber, H; Borisjuk, L; Heim, U; Buchner, P; Wobus, U

    1995-11-01

    We have studied the molecular physiology of photosynthate unloading and partitioning during seed development of fava bean (Vicia faba). During the prestorage phase, high levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat. Three cDNAs were cloned. Sequence comparison revealed genes putatively encoding one soluble and two cell wall-bound isoforms of invertase. Expression was studied in different organs and tissues of developing seeds by RNA gel analysis, in situ hybridization, enzyme assay, and enzyme activity staining. One extracellular invertase gene is expressed during the prestorage phase in the thin-walled parenchyma of the seed coat, a region known to be the site of photoassimilate unloading. We propose a model for an invertase-mediated unloading process during early seed development and the regulation of cotyledonary sucrose metabolism. After unloading from the seed coat, sucrose is hydrolyzed by cell wall-bound invertases. Thus, invertase contributes to establish sink strength in young seeds. The resultant hexoses are loaded into the cotyledons and control carbohydrate partitioning via an influence on the sucrose synthase/sucrose-phosphate synthase pathway. The developmentally regulated degradation of the thin-walled parenchyma expressing the invertase apparently initiates the storage phase. This is characterized by a switch to a low sucrose/hexoses ratio. Feeding hexoses to storage-phase cotyledons in vitro increases the sucrose-phosphate synthase/sucrose synthase ratio and changes carbohydrate partitioning in favor of sucrose. Concomitantly, the transcript level of the major storage product legumin B is downregulated. PMID:8535137

  6. Expression cloning of a candidate gene for Mucolipidosis type IV

    SciTech Connect

    Gama Sosa, M.A.; De Gasperi, R.; Battistini, S.

    1994-09-01

    Mucolipidosis IV is an autosomal recessive lysosomal storage disease characterized by progressive psychomotor retardation and opthalmological abnormalities, namely corneal opacity and retinal degeneration. Biochemically, it is characterized by the lysosomal accumulation of diverse compounds such as gangliosides, phospholipids and acidic mucopolysaccharides. To date, the basic biochemical defect causing this storage disease is still unknown and the relevant gene has also not been identified. An expression cloning strategy was used to identify human kidney cDNA clones capable of reverting in transient gene expression assays the PAS+ phenotype typical of Mucolipidosis IV cells to the normal PAS- phenotype. By this method, a candidate cDNA clone (Mu cDNA) capable of clearing Mucolipidosis IV fibroblasts of their PAS+ positive storage material was isolated. Nucleotide sequence analysis indicated the presence of 2 open reading frames. In vitro translation of T7 transcribed Mu RNA showed protein products of 7,000 and 6,000 mw. Altered expression of the Mu gene may result in the onset of Mucolipidosis type IV.

  7. Cloning, Expression and Biological Analysis of Recombinant Chicken IFN-gamma Expressed in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The interferon-gamma (CHIFN-') derived from the spleen cells of White Leghorns chicken, a local Chinese breeding species was amplified by RT-PCR. The gene encoding CHIFN-' with the deletion of the N-terminal signal peptide was cloned into prokaryotic expression vector pET30a, resulting in a recombin...

  8. Cloning, sequence, and expression of bovine interleukin 2.

    PubMed Central

    Cerretti, D P; McKereghan, K; Larsen, A; Cantrell, M A; Anderson, D; Gillis, S; Cosman, D; Baker, P E

    1986-01-01

    Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system. Images PMID:3517854

  9. Cloning and expression analysis of murine phospholipase D1.

    PubMed Central

    Colley, W C; Altshuller, Y M; Sue-Ling, C K; Copeland, N G; Gilbert, D J; Jenkins, N A; Branch, K D; Tsirka, S E; Bollag, R J; Bollag, W B; Frohman, M A

    1997-01-01

    Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells. PMID:9307024

  10. Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones.

    PubMed

    Oomen, Ronald J F J; Tzitzikas, Emmanouil N; Bakx, Edwin J; Straatman-Engelen, Irma; Bush, Maxwell S; McCann, Maureen C; Schols, Henk A; Visser, Richard G F; Vincken, Jean-Paul

    2004-03-01

    Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis. PMID:15003416

  11. [Production of porcine blastocysts expressed EGFP by handmade cloning].

    PubMed

    Zhang, Peng; Yang, Zhen-Zhen; Dou, Hong-Wei; Li, Wei-Hang; Lv, Bo; Bolund, Lars; DU, Yu-Tao; Tan, Ping-Ping; Ma, Run-Lin

    2011-05-01

    Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production. PMID:21586400

  12. Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System

    PubMed Central

    Pirdel, Leila; Hosseini, Ahmad Zavaran; Kazemi, Bahram; Rasouli, Manoochehr; Bandehpour, Mojgan; Soudi, Sara

    2012-01-01

    Background Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). Methods The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Results Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. Conclusion These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis. PMID:23407850

  13. Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2.

    PubMed

    Cangelosi, Chris; Hannagan, Susan; Santiago, Clayton P; Wilson, James W

    2015-11-01

    Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria. PMID:26505312

  14. Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.

    PubMed Central

    Schwartz, R S; Rybicki, A C; Nagel, R L

    1997-01-01

    We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and beta-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton. PMID:9359436

  15. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    PubMed

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed. PMID:25239739

  16. Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.

    PubMed

    Price, H P; Doenhoff, M J; Sayers, J R

    1997-05-01

    A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine. PMID:9149415

  17. Keith's MAGIC: Cloning and the Cell Cycle.

    PubMed

    Wells, D N

    2013-10-01

    Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material. PMID:24020700

  18. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing

    PubMed Central

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L.

    2016-01-01

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from −938 to −337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells. PMID:27570841

  19. Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster

    PubMed Central

    Bilyk, Oksana; Sekurova, Olga N.; Zotchev, Sergey B.; Luzhetskyy, Andriy

    2016-01-01

    Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the “capture” vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties. PMID:27410036

  20. Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.

    PubMed

    Bilyk, Oksana; Sekurova, Olga N; Zotchev, Sergey B; Luzhetskyy, Andriy

    2016-01-01

    Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the "capture" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties. PMID:27410036

  1. Noncytotoxic T cell clones obtained from a human mixed leukocyte culture.

    PubMed

    Chu, M H; Wee, S L; Bach, F H

    1990-02-01

    Peripheral blood mononuclear cells from a DQW-1 homozygous individual were cocultured with irradiated lymphoblastoid cell line from a DQW-1 homozygous unrelated donor bearing BW35-DW1 haplotype. From T cell cloning of primary and twice-stimulated mixed leukocyte cultures (MLC), 7 and 11 T cell clones were obtained respectively. None of the 18 clones showed specific cytotoxic activity against the alloantigen of the stimulator cell as well as natural killer (NK)-like activity against K562 cells. However, most T cell clones from both primary and re-stimulated MLC demonstrated moderate cytotoxic activity in lectin-dependent cell-mediated cytolysis (LDCC) assay. Screening assay for cell-mediated lympholysis (CML) performed on growing microcultures obtained from restimulated MLC cloning confirmed the non-cytotoxic status of these T cell clones by showing that 41 out of 44 growing microcultures were not cytotoxic against the stimulator cell; the other 3 clones lyzed the target cell mildly. The cells from all 5 T cell clones detected for indirect fluorescence expressed CD3 and CD4 surface markers. Taken together, the results suggested that proliferation-regulating T cell subsets or factor(s) may be generated during the course of MLCs under the present responder-stimulator combination, and may suppress the development of alloreactive cytotoxic T cells and NK-like cells. PMID:2144231

  2. Expression of X-linked Genes in Deceased Neonates and Surviving Cloned Female Piglets

    PubMed Central

    Jiang, Le; Jobst, Pete; Lai, Liangxue; Samuel, Melissa; Prather, Randall S.; Ayares, David; Yang, Xiangzhong; Tian, X. Cindy

    2008-01-01

    Animal cloning through somatic cell nuclear transfer is very inefficient, probably due to insufficient reprogramming of the donor nuclei, which in turn would cause the dysregulation of gene expression. X-Chromosome inactivation (XCI) is a multi-step epigenetic process utilized by mammals to achieve dosage compensation in females. Our aim was to determine if any dysregulation of X-linked genes, which would be indicative of unfaithful reprogramming of donor nuclei, was present in cloned pigs. Real time reverse transcription polymerase chain reaction (RT-PCR) was performed to quantify the transcript levels of five X-linked genes, XIST, TSIX, HPRT1, G6PD, ARAF1 and one autosomal gene, COL4A1 in major organs of neonatal deceased and surviving female cloned pigs and age-matched control pigs from conventional breeding. Aberrant expression level of these genes was prevalent in the neonatal deceased clones, while it was only moderate in cloned pigs that survived after birth. These results suggest a correlation between the viability of the clones and the normality of their gene expression and provide a possible explanation for the death of a large portion of cloned animals around birth. PMID:17474099

  3. Indigofera suffruticosa Mill extracts up-regulate the expression of the π class of glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 in rat Clone 9 liver cells.

    PubMed

    Chen, Chun-Chieh; Liu, Chin-San; Li, Chien-Chun; Tsai, Chia-Wen; Yao, Hsien-Tsung; Liu, Te-Chung; Chen, Haw-Wen; Chen, Pei-Yin; Wu, Yu-Ling; Lii, Chong-Kuei; Liu, Kai-Li

    2013-09-01

    Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent. PMID:23831193

  4. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    PubMed Central

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  5. Cloning and expression of special F protein from human liver

    PubMed Central

    Liu, Shu-Ye; Yu, Xin-Da; Song, Chun-Juan; Lu, Wei; Zhang, Jian-Dong; Shi, Xin-Rong; Duan, Ying; Zhang, Ju

    2007-01-01

    AIM: To clone human liver special F protein and to express it in a prokaryotic system. METHODS: Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein’s cDNA was subcloned into the expression vector pET-15b and transformed into E. coli BL21 (DE3) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein. RESULTS: The cDNA clone of human liver special F protein (1134bp) was successfully produced, with the cDNA sequence being published in Gene-bank: DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION: F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein. PMID:17465469

  6. Cloning and expression of the functional human anti-vascular endothelial growth factor (VEGF) using the pcDNA3.1 vector and the human chronic myelogenous leukemia cell line K562.

    PubMed

    Hajirezaei, Masoumeh; Darbouy, Mojtaba; Kazemi, Bahram

    2014-02-01

    In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies. PMID:24399318

  7. Pharmacology of a cloned potassium channel from mouse brain (MK-1) expressed in CHO cells: effects of blockers and an 'inactivation peptide'.

    PubMed Central

    Robertson, B.; Owen, D. G.

    1993-01-01

    1. Chinese hamster ovary cells (CHO), maintained in cell culture, were stably transfected with DNA for the MK-1 voltage-activated potassium channel, previously cloned from a mouse brain library. 2. Voltage-activated currents were recorded by the whole cell patch clamp method. In CHO cells transfected with the vector only, there were no significant outward voltage activated currents. However, large outward voltage-activated potassium currents were always observed in those cells which had been transfected with the vector containing the DNA encoding for MK-1. 3. These potassium currents activated from -40 mV, and reversed at the potassium equilibrium potential. The half-maximal conductance of MK-1 was at -10 mV and had a slope factor of 11 mV when fitted with a Boltzmann function. There was only very slight (< 10%) inactivation of MK-1 even at very large positive voltages. 4. MK-1 was reversibly blocked by: 4-aminopyridine (4-AP, 0.1-4 mM), Toxin I 10-100 nM), mast cell degranulating peptide (1 microM), tetraethylammonium (TEA, 4-10 mM), tedisamil (100 microM), quinine (100 microM) and ciclazindol (100 microM); all applied to the outside of the cell from a 'U tube' rapid perfusion system. 4-AP may block closed as well as open MK-1 potassium channels. 5. A synthetic 20 amino acid peptide derived from the N-terminus sequence of the Shaker B potassium channel (the 'inactivation peptide') produced dramatic inactivation of MK-1 when applied to the inside, but not the outside of the cell. Reducing peptide concentration or 'degrading' the peptide produced less inactivation. 6. The block of MK-1 by the synthetic inactivation peptide was quite different in time dependence from block by internal TEA (0.4-4 mM), which probably blocks much more quickly but less potently than the peptide. PMID:8358568

  8. Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

    PubMed Central

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in

  9. Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells.

    PubMed

    Si, Shao-Yan; Song, Shu-Jun; Zhang, Jian-Zhong; Liu, Jun-Li; Liang, Shuang; Feng, Kai; Zhao, Gang; Tan, Xiao-Qing

    2011-08-01

    Telomerase is a ribonucleoprotein complex, whose function is to add motif-specific nucleotides to the end of chromosomes. Telomerase consists of three major subunits, the telomerase RNA template (hTR), the telomerase-associated protein (TEP1) and telomerase reverse transcriptase (TERT). TERT is the most important component responsible for the catalytic activity of telomerase and a rate-limiting determinant of the activity. Telomerase activities were at high levels in approximately 90% of mouse cancers or tumor-derived cell lines through TERT transcriptional up-regulation. Unlike human telomerase, telomerase activity exists in colon, liver, ovary and testis but not in brain, heart, stomach and muscle in normal mouse tissues. In this study, we prepared 5' truncations of 1086 bp fragments upstream of the initiating ATG codon of the mTERT gene to construct luciferase reporter gene plasmids, and transfected these plasmids into a normal mouse cell line and several cancer lines to identify the core promoter region essential for transcriptional activation in cancer cells by a luciferase assay. We constructed a eukaryotic expression vector of membrane-expressing staphylococcal endotoxin A (SEA) gene driven by the core promoter region of the mTERT gene and observed if the core promoter region could express the SEA gene in these cancer cells, but not in normal cells following transfection with the construct. The results showed that the transcriptional activities of each fragment of the mTERT gene promoter in the cancer cell lines Hepa1-6, B16 and CT26 were higher than those in NIH3T3 cells, and the proximal 333-bp fragment was the core promoter of the mTERT gene in the cancer cells. The proximal 333-bp fragment was able to make the SEA express on the surface of the cancer cells, but not in NIH3T3 cells. It provides a foundation for cancer targeting gene therapy by using the mTERT gene promoter. PMID:21567104

  10. A comparative study on expression profile of developmentally important genes during pre-implantation stages in buffalo hand-made cloned embryos derived from adult fibroblasts and amniotic fluid derived stem cells.

    PubMed

    Em, Sadeesh; Shah, Fozia; Kataria, Meena; Yadav, P S

    2016-08-01

    Abnormal gene expression in somatic cell nuclear transfer embryos due to aberrant epigenetic modifications of the donor nucleus may account for much of the observed diminished viability and developmental abnormalities. The present study compared the developmentally important gene expression pattern at 4-cell, 8- to 16-cell, morula, and blastocyst stages of buffalo nuclear transfer (NT) embryos from adult fibroblasts (AFs) and amniotic fluid stem cells (AFSCs). In vitro fertilized embryos were used as control embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling, and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), oxidative stress (MnSOD), metabolism (GLUT1) regulation were observed in cloned embryos. The expression of transcripts in AFSC-NT embryos more closely followed that of the in vitro fertilized embryos compared with AF-NT embryos. It is concluded that AFSCs with a relatively undifferentiated genome may serve as suitable donors which could be reprogrammed more efficiently to reactivate expression of early embryonic genes in buffalo NT. PMID:26224482

  11. Molecular cloning and expression analysis of pig CD138.

    PubMed

    Bae, Joonbeom; Jeong, Seonah; Lee, Ju Yeon; Lee, Hyun-Jeong; Choi, Bong-Hwan; Kim, Ji-Eun; Choi, Inho; Chun, Taehoon

    2013-12-01

    CD138 (syndecan-1) interacts with various components of the extracellular matrix and associates with the actin cytoskeleton. In this study, we cloned pig CD138 cDNA and determined its complete cDNA sequence. Pig CD138 cDNA contained an open reading frame (930 bp) encoding 309 amino acids with five well conserved putative glycosaminoglycan attachment sites, a putative cleavage site for matrix metalloproteinases, and conserved motifs involved in signal transduction among mammalian species. Pig CD138 mRNA was detected in various tissues, including lymphoid and non-lymphoid organs, indicating the multicellular functions of CD138 in pigs. Western blot and flow cytometry analyses detected an approximate 35 kDa pig CD138 protein expressed on the cell surface. Further immunohistochemistry analysis revealed that CD138 expression was mainly observed in submucosa and lamina propria of the pig small intestine. Further study will be necessary to define the functional importance of CD138 during specific infectious diseases in pigs. PMID:24128845

  12. A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

    PubMed Central

    2013-01-01

    Background Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. Results Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15–25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3’ end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services. Conclusion This new

  13. Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

    PubMed Central

    Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

    2015-01-01

    Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554

  14. Cloning, expression, and purification of galectins for in vitro studies.

    PubMed

    Poland, Paul A; Kinlough, Carol L; Hughey, Rebecca P

    2015-01-01

    Galectins are best known for their ability to bind glycoconjugates containing β-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds, and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions, and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1. PMID:25253131

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

    PubMed Central

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

    1985-01-01

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

  16. Cloning and expression of chicken erythrocyte transglutaminase.

    PubMed Central

    Weraarchakul-Boonmark, N; Jeong, J M; Murthy, S N; Engel, J D; Lorand, L

    1992-01-01

    We report the sequences of cDNAs encoding chicken erythrocyte transglutaminase (EC 2.3.2.13). The complete mRNA consists of 3345/3349 nucleotides and predicts a single open reading frame. Nine peptide sequences derived from partial digests of the isolated protein agreed with the corresponding translation of the open reading frame. Approximately 60% identities between the avian protein and three related mammalian enzymes were found. Chicken erythrocyte transglutaminase mRNA is most abundant in red blood cells and kidney, and it accumulates during erythroid cell differentiation. Images PMID:1357669

  17. Cloning of human lung cancer cells.

    PubMed Central

    Walls, G. A.; Twentyman, P. R.

    1985-01-01

    We have carried out a comparison of two different methods for cloning human lung cancer cells. The method of Courtenay & Mills (1978) generally gave higher plating efficiencies (PE) than the method of Carney et al. (1980). The number of colonies increased with incubation time in both methods and the weekly medium replenishment in the Courtenay method was advantageous for longer incubation times of several weeks. In the Courtenay method, the use of August rat red blood cells (RBC) and low oxygen tension were both found to be necessary factors for maximum plating efficiency. The usefulness of heavily irradiated feeder cells in improving PE is less certain; each cell type may have its own requirement. PMID:3904799

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

    PubMed Central

    Kluxen, F W; Bruns, C; Lübbert, H

    1992-01-01

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

  19. Cloning and Optimization of Soluble Vascular Endothelial Growth Factor165 Expression in Escherichia coli

    PubMed Central

    Salimi, Ali; Babashamsi, Mohammad

    2016-01-01

    Background: Vascular Endothelial Growth Factor (VEGF) is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF165. VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF165 in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor165 expression in Escherichia coli (E. coli). Methods: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF165 gene was amplified by reverse transcription polymerase chain reaction (RTPCR), and then VEGF165 was subcloned into prokaryotic expression vectors pET-32a(+) and transformed into BL21 (DE3) E. coli strain. VEGF165 expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF165 was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using His•tag specific polyclonal antibody. Results: Our results demonstrated that VEGF165 was successfully cloned and expressed in pET-32a(+) vector. Optimization of the expression procedure showed that, induction by 1 mM IPTG at OD600=0.7 and overnight incubation at 22°C resulted in the highest expression levels of soluble VEGF165. Conclusion: In this study, the expression of VEGF165 in a high soluble level was successfully cloned and optimized. PMID:26855732

  20. High-Throughput Cloning and Expression Library Creation for Functional Proteomics

    PubMed Central

    Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

    2013-01-01

    The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

  1. Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line.

    PubMed

    Kovacs, I; Yamamura, H I; Waite, S L; Varga, E V; Roeske, W R

    1998-02-01

    The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms. PMID:9454790

  2. Human T-Cell Clones from Autoimmune Thyroid Glands: Specific Recognition of Autologous Thyroid Cells

    NASA Astrophysics Data System (ADS)

    Londei, Marco; Bottazzo, G. Franco; Feldmann, Marc

    1985-04-01

    The thyroid glands of patients with autoimmune diseases such as Graves' disease and certain forms of goiter contain infiltrating activated T lymphocytes and, unlike cells of normal glands, the epithelial follicular cells strongly express histocompatability antigens of the HLA-DR type. In a study of such autoimmune disorders, the infiltrating T cells from the thyroid glands of two patients with Graves' disease were cloned in mitogen-free interleukin-2 (T-cell growth factor). The clones were expanded and their specificity was tested. Three types of clones were found. One group, of T4 phenotype, specifically recognized autologous thyroid cells. Another, also of T4 phenotype, recognized autologous thyroid or blood cells and thus responded positively in the autologous mixed lymphocyte reaction. Other clones derived from cells that were activated in vivo were of no known specificity. These clones provide a model of a human autoimmune disease and their analysis should clarify mechanisms of pathogenesis and provide clues to abrogating these undesirable immune responses.

  3. Tightly regulated vectors for the cloning and expression of toxic genes.

    PubMed

    Anthony, Larry C; Suzuki, Hideki; Filutowicz, Marcin

    2004-08-01

    A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems. PMID:15234522

  4. Molecular cloning of Reteplase and its expression in E. coli using tac promoter

    PubMed Central

    Aghaabdollahian, Safieh; Rabbani, Mohammad; Ghaedi, Kamran; Sadeghi, Hamid Mir Mohammad

    2014-01-01

    Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression. Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli. PMID:25298959

  5. Molecular cloning and expression analysis on LPL of Coilia nasus.

    PubMed

    Wang, Meiyao; Xu, Dongpo; Liu, Kai; Yang, Jian; Xu, Pao

    2016-06-01

    Coilia nasus is one important commercial anadromous species which mainly distributed in the Yangtze River in China. At present, it has been on the "National Key Protective Species List" because of its severe resource damage. Lipid metabolism is very important during its long-distance migration. To make further research on lipid metabolism of C. nasus, we cloned lipoprotein lipase gene with homologous cloning method. A full-length cDNA of LPL of C. nasus was cloned from liver which covered 3537 bp with a 1519 bp open reading frame encoding 505 deduced amino acids whose molecular mass was 57.5 kDa and theoretical isoelectric point was 7.58. The deduced amino acids had high similarity with the reported LPL sequence of other species. It had typical conserved domain of LPL protein containing catalytic triad, N-linked glycosylation sites and conserved heparin-binding site, etc. We adopted quantitative real-time RT-PCR method to detect the mRNA expression of LPL of C. nasus in ten tissues including mesenteric adipose, liver, muscle, stomach, spleen, heart, head kidney, trunk kidney, gill and brain with β-actin as internal reference. LPL expressed in all the detected tissues. The highest expression was in mesenteric adipose, and followed by liver, muscle, stomach. Lipid expressed lowly in spleen, heart, head kidney, trunk kidney, gill and brain. The research on the cloning and differential expression of LPL of C. nasus will lay foundation for further research on lipid metabolism of C. nasus. PMID:26877109

  6. Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage.

    PubMed

    Siriboon, Chawalit; Tu, Ching-Fu; Kere, Michel; Liu, Ming-Sing; Chang, Hui-Jung; Ho, Lin-Lin; Tai, Miao-En; Fang, Wen-Der; Lo, Neng-Wen; Tseng, Jung-Kai; Ju, Jyh-Cherng

    2014-03-01

    The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P<0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P<0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass:total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P<0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P<0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term. PMID:23544704

  7. Immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes

    PubMed Central

    Gomez, German G.; Hickey, Michelle J.; Tritz, Richard; Kruse, Carol A.

    2008-01-01

    Summary We previously reported the cellular, functional and cytogenetic characterization of immunoresistant (IR) 13-06-IR29 and 13-06-IR30 human glioma cell clones isolated after immunoselection with alloreactive cytotoxic T lymphocytes (aCTL). Relative to the 13-06-MG parental cells, both clones resisted aCTL lysis at multiple effector to target ratios; the resistant phenotype was maintained for 13-41 cell doublings after cloning and when selective pressure was removed; cross-resistance to other inducers of apoptosis/cell death was also observed (Gomez et al, 2006; Gomez and Kruse, 2007). In this study we further characterize the IR clones for factors that may contribute to the resistance. Data obtained by in-vitro quantitative morphologic and 7-amino actinomycin D flow cytometric assays revealed reduced apoptotic cell death when IR clones were coincubated with aCTL, relative to the parental cells. Since changes in apoptosis were observed, we examined the expression patterns of apoptosis-related genes in several extracts of parental cells and IR clones using pathway-specific cDNA microarray analysis. In general, the apoptotic factors were downregulated in the IR clones. From three separate extracts analyzed separately on microarrays, three factors, ATM, caspases 3 and 8, were statistically downregulated in both IR clones. Immunoblotting of the proteins confirmed the findings. Therefore, a possible mechanism for immunoresistance in gliomas may be achieved by the downregulation of one or more genes in the apoptotic pathway. PMID:19066635

  8. Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

    PubMed Central

    Laplante, J M; O'Rourke, F; Lu, X; Fein, A; Olsen, A; Feinstein, M B

    2000-01-01

    A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation. PMID:10794731

  9. Cloning and expression pattern of akirin2 gene in broiler.

    PubMed

    Man, Chaolai; Chang, Yang; Mu, Weitao; Zhao, Dongxue

    2014-12-01

    Akirin2 is an important nuclear factor which plays functions in innate immune response, myogenesis, muscle development, and carcinogenesis. In this study, akirin2 genes were cloned from 4-day-old Sanhuang and AA(+) broiler, and its expression patterns were analyzed by RT-PCR. The results showed that there were four SNPs in the 5'-terminal region of akirin2 coding sequences. Expression profile analysis showed that the akirin2 transcripts were constitutively expressed in 15 tissues tested, and similar expression patterns were found between the two breeds of broilers. In addition, one of the interesting findings was that the akirin2 gene is highly expressed in blood and lowly expressed in heart, respectively. These data can serve as a foundation for further studying functions of akirin2 gene. PMID:25098451

  10. Molecular cloning and nucleotide sequence of the 1,2-alpha-D-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells.

    PubMed

    Inoue, T; Yoshida, T; Ichishima, E

    1995-12-01

    A full-length cDNA encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) from Aspergillus saitoi was cloned. Analysis of the 1718 bp nucleotide sequence of the cDNA revealed a single open reading frame with 1539 nucleotides of 1,2-alpha-D-mannosidase gene, msdS. The predicted amino-acid sequence of 1,2-alpha-D-mannosidase consists of 513 residues with a molecular mass of 55,767 and is 70%, 26% and 35% identity with those of Penicillium citrinum 1,2-alpha-D-mannosidase, yeast alpha-mannosidase, and mouse alpha-mannosidase. The cDNA of the msdS gene has been cloned and expressed in yeast cells. To identify the activity of expression product methyl-2-O-alpha-mannopyranosyl-alpha-mannopyranoside (Man alpha 1-->2Man-OMe) was used as a substrate at pH 5.0. PMID:8519794

  11. Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

    PubMed

    Knight, C; Simmons, D; Gu, T T; Gluhak-Heinrich, J; Pavlin, D; Zeichner-David, M; MacDougall, M

    2001-10-01

    Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis. PMID:11706948

  12. Cloning and Expression of a Hexose Transporter Gene Expressed during the Ripening of Grape Berry1

    PubMed Central

    Fillion, Laurent; Ageorges, Agnès; Picaud, Sarah; Coutos-Thévenot, Pierre; Lemoine, Rémi; Romieu, Charles; Delrot, Serge

    1999-01-01

    The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after véraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells. PMID:10444092

  13. Molecular cloning of Salmo salar Toll-like receptors (TLR1, TLR22, TLR5M and TLR5S) and expression analysis in SHK-1 cells during Piscirickettsia salmonis infection.

    PubMed

    Salazar, C; Haussmann, D; Kausel, G; Figueroa, J

    2016-02-01

    In fish, the innate immune system is the primary response against infection. Toll-like receptors (TLRs) recognize pathogens through pathogen-associated molecular patterns (PAMPs), and some target molecules of TLRs are homologous between fish and mammals. Piscirickettsia salmonis is one of the main pathogens affecting the salmon industry in Chile. Better knowledge of mechanisms underlying its invasive capacity and recognition of target cells is crucial for vaccine development. Therefore, Salmo salar L. TLR1, TLR22, membrane TLR5M and soluble TLR5S sequences were cloned, and expression kinetics were analysed by RT-qPCR in salmon head kidney cells (SHK-1) infected with three different P. salmonis preparations: alive, formaldehyde treated, extract. Clearly, all analysed TLRs were expressed and transcription level changes were revealed at 2 hpi, 12 or 16 hpi and 24 hpi depending on P. salmonis infection scheme. Increased IL1-beta expression confirmed TLR pathway response. Furthermore, significant expression modulations of several members of the TLR pathway in this in vitro model suggest that P. salmonis extract rather than formaldehyde-inactivated bacteria might strengthen the salmon immune system. PMID:25903926

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

    SciTech Connect

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

    1987-02-01

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

  15. Transplantation and differentiation of donor cells in the cloned pigs

    SciTech Connect

    Shimada, Arata; Tomii, Ryo; Kano, Koichiro; Nagashima, Hiroshi . E-mail: hnagas@isc.meiji.ac.jp

    2006-06-02

    The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal.

  16. Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System

    PubMed Central

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Pourhassan-Moghaddam, Mohammad; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Purpose: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. Methods: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. Results: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. Conclusion: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space. PMID:27478780

  17. Transcriptional profiling of phenotypically different Epo-Fc expressing CHO clones by cross-species microarray analysis.

    PubMed

    Trummer, Evelyn; Ernst, Wolfgang; Hesse, Friedemann; Schriebl, Kornelia; Lattenmayer, Christine; Kunert, Renate; Vorauer-Uhl, Karola; Katinger, Hermann; Müller, Dethardt

    2008-07-01

    Chinese hamster ovary (CHO) cells exhibit large variabilities regarding growth, recombinant protein production and post-translational processing during cell line development and clone selection. To accelerate the development of stable high quality cell factories, new efficient strategies for cell screening and clone selection are required. In our work, we combined phenotypic characterisation of recombinant CHO clones during early cell line development with transcription profile analysis using cross-species microarrays. The objective was to identify genes or gene patterns that correlate with clone specific alterations in terms of productivity, sialylation capacity and stress resistance. In all high producer clones transcriptional profiling revealed a common enrichment of gene ontology categories related to protein metabolism, transcription, nucleus and nucleolus, whereas no common genes were differentially regulated in clones showing higher sialylation capacities. Furthermore, we identified predictive stress-related marker genes that were up-regulated in one clone without showing the corresponding phenotype at an early stage of development. Thus, we successfully applied gene expression profiling to allocate transcriptomal differences to specific phenotypes that changed during cell line development. These promising results will further increase our efforts to develop CHO specific microarrays that deliver information about the suitability of a clone candidate for industrial production. PMID:18481264

  18. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    SciTech Connect

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  19. Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

    SciTech Connect

    Lee, Eugine; Lee, So Hyun; Kim, Sue

    2006-10-06

    Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.

  20. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  1. Cloning and expression of the potato alternative oxidase gene

    SciTech Connect

    Hiser, C.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Mitochondria from 24-hour-aged potato slices possess an alternative path capacity and a 36kD protein not present in fresh potato mitochondria. This 36kD protein was identified by a monoclonal antibody against the Sauromatum guttatum alternative oxidase. These results suggest de novo synthesis of the 36kD protein during the aging process. To investigate this phenomenon, a clone containing a potato alternative oxidase gene was isolated from a cDNA library using the S. guttatum gene as a probe. This clone shows areas of high homology to the S. guttatum gene. Norther blots of RNA from fresh and 24-hour-aged potato slices are being probed with the potato gene to examine its expression in relation to the appearance of the 36kD protein.

  2. Cloning and expression of calmodulin gene in Scoparia dulcis.

    PubMed

    Saitoh, Daisuke; Asakura, Yuki; Nkembo, Marguerite Kasidimoko; Shite, Masato; Sugiyama, Ryuji; Lee, Jung-Bum; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2007-06-01

    A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis. PMID:17541174

  3. Gene Expression Profile Normalization in Cloned Mice by Trichostatin A Treatment

    PubMed Central

    Kohda, Takashi; Kishigami, Satoshi; Kaneko-Ishino, Tomoko; Wakayama, Teruhiko

    2012-01-01

    Abstract Cloning mammals by somatic cell nuclear transfer (SCNT) has become an established procedure, but the success rate remains low and gene expression abnormalities are also observed. In addition, SCNT pups exhibited an abnormal gene expression profile with a high degree of heterogeneity among individuals. Recently, we reported that somatic clones treated with trichostatin A (TSA) exhibited a significantly improved success rate, probably due to its effects on chromatin remodeling and histone modification in early embryos. Here we show that the TSA treatment also improves the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up- or downregulation in the TSA clone pups decreased to half of the conventional SCNT pups, and the variation among individuals observed in the SCNT pups was also reduced to the level of the pups produced by the intracytoplasmic sperm injection (ICSI) method. Interestingly, the total gene expression profile of the TSA clones came to resemble that of the ICSI pups. PMID:22257163

  4. Transfer of experimental autoimmune thyroiditis with T cell clones

    SciTech Connect

    Romball, C.G.; Weigle, W.O.

    1987-02-15

    We have investigated three T lymphocyte clones isolated from CBA/CaJ mice primed with mouse thyroid extract (MTE) in adjuvant. All three clones are L3T4+, Ig-, and Lyt2- and proliferate to MTE, mouse thyroglobulin (MTG) and rat thyroid extract. Clones A7 and B7 transfer thyroiditis to irradiated (475 rad) syngeneic mice, but not to normal recipients. The thyroid lesion induced by the B7 clone is characterized by the infiltration of both mononuclear and polymorphonuclear cells. The thyroiditis is transient in that lesions are apparent 7 and 14 days after transfer, but thyroids return to normal by day 21. Clone B7 showed helper activity for trinitrophenyl-keyhole limpet hemocyanin-primed B cells in vitro when stimulated with trinitrophenyl-MTG and also stimulated the production of anti-MTG antibody in recipient mice. Clone A7 induced thyroid lesions characterized by infiltration of the thyroid with mononuclear cells, with virtually no polymorphonuclear cell infiltration. This clone has shown no helper activity following stimulation with trinitrophenyl-MTG. The third clone (D2) proliferates to and shows helper activity to MTG, but fails to transfer thyroiditis to syngeneic, irradiated mice. On continuous culture, clone B7 lost its surface Thy. The loss of Thy appears unrelated to the ability to transfer thyroiditis since subclones of B7 with markedly different percentages of Thy+ cells transferred disease equally well.

  5. Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus).

    PubMed

    Bhat, Narayani; Hong, Yunhan

    2014-05-01

    The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia. PMID:24607036

  6. Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

    NASA Astrophysics Data System (ADS)

    Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

    1985-12-01

    A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

  7. Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts.

    PubMed

    Wan, Yongjie; Deng, Mingtian; Zhang, Guomin; Ren, Caifang; Zhang, Hao; Zhang, Yanli; Wang, Lizhong; Wang, Feng

    2016-01-01

    Somatic cell nuclear transfer (SCNT) is a useful way to produce cloned animals. However, SCNT animals exhibit DNA methylation and genomic imprinting abnormalities. These abnormalities may be due to the faulty epigenetic reprogramming of donor cells. To investigate the consequence of SCNT on the genomic imprinting and global methylation in the donor cells, growth patterns and apoptosis of cloned goat fibroblast cells (CGFCs) at passage 7 were determined. Growth patterns in CGFCs were similar to the controls; however, the growth rate in log phase was lower and apoptosis in CGFCs were significantly higher (P < 0.01). In addition, quantitative expression analysis of three DNA methyltransferases (Dnmt) and two imprinted genes (H19, IGF2R) was conducted in CGFCs: Dnmt1 and Dnmt3b expression was significantly reduced (P < 0.01), and H19 expression was decreased sixfold (P < 0.01); however, the expression of Dnmt3a was unaltered and IGF2R expression was significantly increased (P < 0.05). Finally, we used bisulfite sequencing PCR to compare the DNA methylation patterns in differentially methylated regions (DMRs) of H19 and IGF2R. The DMRs of H19 (P < 0.01) and IGF2R (P < 0.01) were both highly methylated in CGFCs. These results indicate that the global genome might be hypomethylated. Moreover, there is an aberrant expression of imprinted genes and DMR methylation in CGFCs. PMID:26314395

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

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

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

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

    DOEpatents

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

    1998-10-20

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

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

    DOEpatents

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

    1998-11-03

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

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

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

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

  12. Molecular cloning and expression vector construction of bovine TRIM28.

    PubMed

    Ma, X; Zhai, Z C; Zhang, M L; Song, B H; Zhu, Y R; Yang, S B; Dong, X Q; Su, L Y; Wang, C F; Ma, H X; Luan, W M

    2016-01-01

    The bovine TRIM28 gene was amplified from ovary tissue by using RT-PCR. The TRIM28 gene was inserted into the eukaryotic expression vector pIRES2-EGFP and transfected into bovine fetal fibroblasts by using Lipofectamine 3000. TRIM28 mRNA and protein were detected by fluorescence microscope and western blotting. The results showed that the full length of TRIM28 was cloned and pIRES2-EGFP-TRIM28 was constructed successfully. EGFP expression was observed, and the pIRES2-EGFP-TRIM28 transfected group expressed more TRIM28 protein than that by the pIRES2-EGFP group. The TIMR28 gene has been successfully transferred into bovine fetal fibroblasts. PMID:27420979

  13. [Cloning and expression of Tachypleus tridentatus factor C].

    PubMed

    Wang, Dong-Ning; Liu, Jie-Wu; Chen, Lin; Wang, Lie; Yang, Guan-Zhen; Wu, Xiang-Fu; Zhang, Wei-Jie

    2002-01-01

    Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the horseshoe crab hemolymph. The special lipopolysaccharide (LPS) binding activation of FC makes it a potential drug for anti-LPS treatment and has a high commercial value. Based on the sequence of reported FC from Japan horseshoe crab, two pairs of primers were designed. The total RNA was extracted from amebocytes of Chinese Tachypleus tridentatus and the cDNA was separated into two parts and were cloned using RT-PCR, respectively. FCs from different geographical areas showed high homology in sequence. The whole FC cDNA was cloned into pET-28a (+) containing T7 promoter and recombinant expression plasmid pET-FC was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3). Recombinant FC was expressed as inclusion body when the expression strain was induced with 1 mmol/L IPTG. Refolded recombinant FC was confirmed to be active by bacteriostatic assay in vitro. The results of Western blot also suggested the recombinant FC may be able to cleave itself partly and produced an extra immunoblot band. PMID:11958140

  14. Functional cloning of a gp100-reactive T-cell receptor from vitiligo patient skin.

    PubMed

    Klarquist, Jared; Eby, Jonathan M; Henning, Steven W; Li, Mingli; Wainwright, Derek A; Westerhof, Wiete; Luiten, Rosalie M; Nishimura, Michael I; Le Poole, I Caroline

    2016-05-01

    We isolated gp100-reactive T cells from perilesional skin of a patient with progressive vitiligo with superior reactivity toward melanoma cells compared with tumor-infiltrating lymphocytes 1520, a melanoma-derived T-cell line reactive with the same cognate peptide. After dimer enrichment and limited dilution cloning, amplified cells were subjected to reverse transcription and 5' RACE to identify the variable TCRα and TCRβ subunit sequences. The full-length sequence was cloned into a retroviral vector separating both subunits by a P2A slippage sequence and introduced into Jurkat cells and primary T cells. Cytokine secreted by transduced cells in response to cognate peptide and gp100-expressing targets signifies that we have successfully cloned a gp100-reactive T-cell receptor from actively depigmenting skin. PMID:26824221

  15. Cloning and Expression of Yak Active Chymosin in Pichia pastoris.

    PubMed

    Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

    2016-09-01

    Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

  16. Cloning and Expression of Yak Active Chymosin in Pichia pastoris

    PubMed Central

    Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

    2016-01-01

    Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

  17. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity. PMID:18154441

  18. Molecular Cloning and Gene Expression of Canine Apoptosis Inhibitor of Macrophage

    PubMed Central

    TOMURA, Shintaro; UCHIDA, Mona; YONEZAWA, Tomohiro; KOBAYASHI, Masato; BONKOBARA, Makoto; ARAI, Satoko; MIYAZAKI, Toru; TAMAHARA, Satoshi; MATSUKI, Naoaki

    2014-01-01

    Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM. PMID:25649949

  19. Molecular cloning and expression of a human heat shock factor, HSF1

    SciTech Connect

    Rabindran, S.K.; Giorgi, G.; Clos, J.; Wu, C. )

    1991-08-15

    Human cells respond to heat stress by inducing the binding of a preexisting transcriptional activator (heat shock factor, HSF) to DNA. The authors isolated recombinant DNA clones for a human cDNA fragment. The human HSF1 probe was produced by the PCR with primers deduced from conserved amino acids in the Drosophila and yeast HSF sequences. The human HSF1 mRNA is constitutively expressed in HeLa cells under nonshock conditions and encodes a protein with four conserved leucine zipper motifs. Like its counterpart in Drosophila, human HSF1 produced in Escherichia coli in the absence of heat shock is active as a DNA binding transcription factor, suggesting that the intrinsic activity of HSF is under negative control in human cells. Surprisingly, an independently isolated human HSF clone, HSF2, is related to but significantly different from HSF.

  20. Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis.

    PubMed

    Mohanty, Ashok K; Fisher, Andrew J; Yu, Zhihao; Pradeep, Mangottil A; Janjanam, Jagdeesh; Kaushik, Jai K

    2009-04-01

    Breast regression protein (BRP39) is a glycoprotein, which is expressed during mammary gland involution in mouse. The physiological function of BRP39 is not known. High levels of expression of BRP39 have also been associated with breast cancer development. In the present investigation a cDNA encoding rBRP39 (recombinant BRP39) was cloned by PCR techniques. It consists of 1,143 nucleotides and encodes an open reading frame of 381 amino acid residues including a signal sequence of 21 amino acids. Recombinant BRP39 was produced in E. coli in a soluble form at low temperature (15 degrees C). Expression and purification of rBRP39 was confirmed by western blot analysis. Purified rBRP39 showed high chitin-binding activity but no chitinase activity. The lack of chitinase activity may be attributed to the mutation of critical active site residue Glu120 to Leu120 and Asp118 to Ala118 in BRP39. However, a mutant in which the residue was reverted back to Glu, by site directed mutagenesis, displayed no chitinase activity. Purified recombinant BRP39 was crystallized and the crystals diffracted X-rays to 2.8A resolution. The crystals belonged to the space group C2 with unit cell parameters a=130.4A, b=81.3A, c=229.2A, beta=105.9 degrees. The structure refinement is in progress. PMID:19041398

  1. Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

    PubMed Central

    Norgard, M V; Chamberlain, N R; Swancutt, M A; Goldberg, M S

    1986-01-01

    Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials. Images PMID:3021631

  2. Stromal-cell and cytokine-dependent lymphocyte clones which span the pre-B- to B-cell transition.

    PubMed

    Ishihara, K; Medina, K; Hayashi, S; Pietrangeli, C; Namen, A E; Miyake, K; Kincade, P W

    1991-01-01

    Five stromal-cell-dependent lymphocyte clones are described that correspond to late pre-B or early B-cell stages of differentiation. They are useful for determining the molecular requirements for pre-B replication, for studying the stromal cells that supply those factors, and for delineating the final sequence of differentiation events as newly formed lymphocytes prepare to exit the bone marrow. The efficiency of lymphocyte growth at limiting dilution varied substantially on different stromal-cell clones and may reflect functional heterogeneity of stromal cells. Most lymphocyte clones were similar to uncloned lymphocytes from Whitlock-Witte cultures in that they responded only transiently to interleukin-7 (IL-7) and then died, unless maintained on a stromal-cell clone. One unusual lymphocyte clone (2E8) was propagated for more than 1 year in IL-7 alone and was selectively responsive to that cytokine. Most of the lymphocyte clones were not tumorigenic in immunodeficient mice. However, one pre-B clone (1A9) grew autonomously in culture when held at high density, responded to conditioned medium from a number of cell lines, and was tumorigenic. Tumors derived from this clone were infiltrated by stromal cells and lymphocytes taken from the tumors' retained characteristics of the original clone. Ly-6 antigens were inducible on 2E8 and 1A9 cells, but the lymphocytes were otherwise arrested in differentiation. The 2E8 cells had rearranged and expressed kappa light-chain genes but displayed them on the surface along with surrogate light chains and mu heavy chains. Thus, expression of authentic light chain need not coincide with termination of surrogate light-chain utilization in newly formed B cells. Several glycoproteins have recently been demonstrated to be associated with surface immunoglobulin (Ig) on mature B-lineage cells and plasma-cell tumors. We now show that one member of this family (approximately 33 kD) was associated with the mu+surrogate light-chain complex on

  3. Cloning and expression of buffalo active chymosin in Pichia pastoris.

    PubMed

    Vallejo, Juan Andres; Ageitos, Jose Manuel; Poza, Margarita; Villa, Tomas G

    2008-11-26

    To date, only recombinant chymosin has been obtained in its active form from supernatants of filamentous fungi, which are not as good candidates as yeasts for large-scale fermentations. Since Bos taurus chymosin was cloned and expressed, the world demand for this protease has increased to such an extent that the cheesemaking industry has been looking for novel sources of chymosin. In this sense because buffalo chymosin has properties that are more stable than those of B. taurus chymosin, it may occupy a space of its own in the chymosin market. The main objective of the present work was the production of active recombinant buffalo chymosin in the culture supernatant of Pichia pastoris . This yeast has demonstrated its usefulness as an excellent large-scale fermentation tool for the secretion of recombinant foreign proteins. RNA was extracted from the abomasum of a suckling calf water buffalo ( Bubalus arnee bubalis ). Preprochymosin, prochymosin, and chymosin DNA sequences were isolated and expressed into P. pastoris. Only the recombinant clones of P. pastoris containing the prochymosin sequence gene were able to secrete the active form of the chymosin to the culture supernatant. This paper describes for the first time the production of active recombinant chymosin in P. pastoris without the need of a previous in vitro activation. The new recombinant yeast strain could represent a novel and excellent source of rennet for the cheesemaking industry. PMID:18975968

  4. [Cloning and expression analysis of GGPPS gene from Panax notoginseng].

    PubMed

    Min, Dan-dan; Tang, Mei-qiong; Li, Gang; Qu, Xiao-sheng; Miao, Jian-hua

    2015-06-01

    According to the transcriptome dataset of Panax notoginseng, the key geranylgeranyl pyrophosphate synthase gene (GGPPS) in terpenoid backbone biosynthesis was selected to be cloned. Using specific primer pairs combining with RACE (rapid amplification of cDNA ends) technique, the full-length cDNA sequence with 1 203 bp, which containing a 1 035 bp open reading frame, was cloned and named as PnGGPPS. The corresponding full-length DNA sequence contained 2 370 bp, consisted of 1 intron and 2 exons. The deduced protein PnGGPPS contained 344 amino acids and shared more than 73% identity with GGPPS from Ricinus communis and Salvia miltiorrhiza. PnGGPPS also had specific Aspartic acid enrichment regions and other conserved domains, which belonged to the Isoprenoid-Biosyn-C1 superfamily. The quantitative real-time PCR showed that PnGGPPS expressed in different tissues of 1, 2, 3 years old root, stem, leaf and 3 years old flower, and the expression level in 3 years old leaf was significant higher than that in other organs, which suggested that it might not only be involved in the regulation of the growth and development, but also be associated with the biosynthesis of chlorophyll and carotenoids, the development of chloroplast, the shade habit and the quality formation of P. notoginseng. PMID:26552162

  5. Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of water-permeable epithelial cells in osmoregulatory organs of the tree frog.

    PubMed

    Akabane, Gen; Ogushi, Yuji; Hasegawa, Takahiro; Suzuki, Masakazu; Tanaka, Shigeyasu

    2007-06-01

    An aquaporin (Hyla AQP-h3BL), consisting of 292 amino acid residues, has been cloned from the urinary bladder of Hyla japonica. In a swelling assay using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a sevenfold and 2.8-fold higher permeability to water and glycerol, respectively, than the water-injected oocytes. This permeability was inhibited by HgCl2. Immunofluorescence revealed that AQP-h3BL is localized in the basolateral plasma membrane of both granular cells in the ventral pelvic and dorsal skins and the secretory cells in the mucous glands. Immunopositive cells were also observed in the basolateral membrane of principal cells in the collecting ducts and in a portion of the late distal tubules in the kidneys, as well as in the principal cells of the urinary bladder. Sequence homology suggests that AQP-h3BL is a homolog to mammalian AQP3. This conclusion is supported by the observed localization of AQP-h3BL to the basolateral membrane in water- and glycerol-permeable epithelial cells. In ventral pelvic skins and urinary bladders, water enters into the cytoplasm through the apical plasma membrane at sites where AQP-h2, sometimes in association with AQP-h3, responds to stimulation by vasotocin; the water exits throughout AQP-h3BL to extracellular spaces. In the mucous glands, on the other hand, water enters throughout this AQP-h3BL and exits through AQP-x5, which is in the apical membrane of secretory cells. Thus, water homeostasis in the frog body is regulated by AQP-h3BL expressed in the basolateral membrane in concert with arginine vasotocin (AVT)-dependent or AVT-independent AQP. PMID:17332153

  6. Cloning, expression and characterisation of a promising mosquitocidal gene.

    PubMed

    Zhang, Lingling; Huang, Enjiong; Gelbic, Ivan; Guan, Chunyu; Guan, Yi; Guan, Xiong

    2009-10-01

    A new mosquitocidal gene, cyt1Aa6, was isolated and cloned from the novel Bacillus thuringiensis strain LLP29, previously isolated from the phylloplane of Magnolia denudata. Nucleotide sequence analysis of cyt1Aa6 indicated that the open reading frame consisted of 750 base pairs, encoding 249 amino acid sequences with a calculated molecular weight of 27.3681 kDa and a PI value of 4.77. An homological comparison revealed that the cyt1Aa6 amino acid sequence was 99% identical with those of known Cyt1Aa proteins. In addition, the cyt1Aa6 gene was successfully expressed in Escherichia coli BL21. Bioassays on Aedes albopictus showed that Bt LLP29 and the expressed BL21 were both toxic to 3rd-instar mosquito larvae. The isolation of cyt1Aa6 provides new opportunities for selecting new strains and to obtain novel B. thuringiensis products based on its toxins. PMID:20112806

  7. GLUT3 is present in Clone 9 liver cells and translocates to the plasma membrane in response to insulin.

    PubMed

    Defries, Danielle M; Taylor, Carla G; Zahradka, Peter

    2016-08-26

    Clone 9 cells have been reported to express only the GLUT1 facilitative glucose transporter; however, previous studies have not examined Clone 9 cells for GLUT3 content. The current study sought to profile the presence of glucose transporters in Clone 9 cells, H4IIE hepatoma cells, and L6 myoblasts and myotubes. While the other cell types contained the expected complement of transporters, Clone 9 cells had GLUT3 which was previously not reported. Interestingly, both GLUT3 mRNA and protein were detected in Clone 9 cells, but only mRNA for GLUT1 was detected. Glucose transport in Clone 9 cells was insulin-sensitive in a concentration-dependent manner, concomitant with the presence of GLUT3 in the plasma membrane after insulin treatment. Although basal glucose uptake was unaffected, insulin-stimulated glucose uptake was abolished with siRNA-mediated GLUT3 knockdown. These results contradict previous reports that Clone 9 cells exclusively express GLUT1 and suggest GLUT3 is a key insulin-sensitive glucose transporter required for insulin-stimulated glucose uptake by Clone 9 cells. PMID:27320866

  8. Express primer tool for high-throughput gene cloning and expression.

    SciTech Connect

    Yoon, J. R.; Laible, P. D.; Gu, M.; Scott, H. N.; Collart, F. R.; Biosciences Division

    2002-12-01

    High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for the simultaneous amplification of multiple target genes.

  9. Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3.

    PubMed Central

    Huston, E; Lumb, S; Russell, A; Catterall, C; Ross, A H; Steele, M R; Bolger, G B; Perry, M J; Owens, R J; Houslay, M D

    1997-01-01

    5'-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5' sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue 'long-form' PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 microM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram ¿4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone¿, the selective inhibitor of PDE4 (IC50 0.05-0.1 microM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 microM) to rolipram inhibition than its cytosolic form (IC50 0.2 microM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed

  10. Positive-selection and ligation-independent cloning vectors for large scale in planta expression for plant functional genomics.

    PubMed

    Oh, Sang-Keun; Kim, Saet-Byul; Yeom, Seon-In; Lee, Hyun-Ah; Choi, Doil

    2010-12-01

    Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligation-independent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3' to 5' exonuclease activity in the presence of only one dNTP (dGTP for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants. PMID:21340673

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

    PubMed

    Kato, N; Esaka, M

    1996-02-01

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

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

    PubMed Central

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

    1982-01-01

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

  13. Enhanced activation of human T cell clones specific for virus-like particles expressing the HIV V3 loop in the presence of HIV V3 loop-specific polyclonal antibodies

    PubMed Central

    Peifang, S.; Pira, G. L.; Fenoglio, D.; Harris, S.; Costa, M. G.; Venturino, V.; Dessì, V.; Layton, G.; Laman, J.; Huisman, J. G.; Manca, F.

    1994-01-01

    Recombinant virus-like particles (VLP), formed by the yeast Ty p1 protein, carrying the HIV gp120 V3 loop on their surface (V3-VLP) have been tested in vitro for immunogenicity and antigenicity by using VLP p1-specific human CD4+ T cell lines and clones. VLP-specific human T cell lines and clones were generated from normal individuals, indicating that VLP-specific precursor cells present in the peripheral lymphocyte pool can be induced to expand clonally upon antigen challenge in vitro, in the absence of previous immunization. It was also shown that V3-specific polyclonal antibodies enhance V3-VLP-induced activation of VLP-specific T cell clones. Antibody-dependent potentiation has been shown previously in other antigen systems, and it depends on enhanced uptake of complexed antigen by Fc receptor-positive antigen-presenting cells. Since in this case antigen is internalized by presenting cells as a complex, it can be inferred that a similar event of antibody-mediated antigen uptake can take place with V3-specific B cells, resulting in presentation by the B cells of T helper epitopes derived from processing of the VLP p1 moiety. This suggests that T helper cells specific for the carrier VLP p1 protein can be activated to provide help to V3-specific B cells in the presence of the appropriate antigen construct. PMID:7915974

  14. Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

    PubMed Central

    Itoh, Keiji; Sokol, Sergei Y.

    2014-01-01

    Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology. PMID:25285446

  15. Human GluR6 kainate receptor (GRIK2): Molecular cloning, expression, polymorphism, and chromosomal assignment

    SciTech Connect

    Paschen, W.; Blackstone, C.D.; Huganir, R.L. ); Ross, C.A. Max-Planck-Institute for Neurological Research, Koeln )

    1994-04-01

    Glutamate receptors mediate the majority of excitatory neurotransmission in the brain, and molecular cloning studies have revealed several distinct families. Because neuropathological states and possibly human disorders may involve kainate-preferring glutamate receptors, the authors have isolated a cDNA clone for the human GluR6 kainate-preferring receptor. This clone shows a very high sequence similarity with that of the rat, except for a part of the 3[prime] untranslated region in which there is a TAA triplet repeat. When the protein was overexpressed in human embryonic kidney 293 cells, it had a molecular weight, an antibody recognition, and a glutamate ligand-binding profile similar to those of the rate GluR6 receptor. Northern analysis showed expression in both human cerebral and cerebellar cortices. By PCR analysis of rodent-human monochromosomal cell lines, the human GluR6 could be assigned to chromosome 6. The length of the TAA triplet repeat was polymorphic in the normal population, with at least four alleles and an observed heterozygosity of about 45%. These studies should provide the basis for expression or linkage studies of the GluR6 kainate receptor in human disease or neuropathologic states. 53 refs., 7 figs.

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

    PubMed

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

    1985-08-01

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

  17. Cloning and expression of the sponge longevity gene SDLAGL.

    PubMed

    Schröder, H C; Kruse, M; Batel, R; Müller, I M; Müller, W E

    2000-07-01

    Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation. One gene, SDLAGL, was identified in the marine sponge Suberites domuncula whose deduced polypeptide showed high sequence similarity to the longevity assurance genes from other Metazoa. While in single cells no transcripts of SDLAGL could be identified, high expression was seen after re-aggregation of single cells and in proliferating cells of primmorphs. PMID:10906466

  18. Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.

    PubMed

    Lo, R Y; Shewen, P E; Strathdee, C A; Greer, C N

    1985-12-01

    A clone bank of Pasteurella haemolytica A1 was constructed by partial digestion of the genomic DNA with Sau3A and ligation of 5- to 10-kilobase-pair fragments into the BamHI site of the plasmid vector pBR322. After transformation into Escherichia coli K-12, a total of 4 X 10(3) recombinant clones was obtained. These were screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens were then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones were identified, and subsequent restriction analysis of the recombinant plasmids showed that the same 6.3 kilobase pairs of insert DNA was cloned in either of the two orientations into the plasmid vector pBR322. One of the clones was selected for further characterization of the leukotoxin as produced in E. coli. Tests for heat lability and target cell species specificity with canine, porcine, and human peripheral blood lymphocytes indicated that the activity of the cloned leukotoxin was identical to that of the P. haemolytica leukotoxin. Furthermore, the E. coli-produced leukotoxin was also neutralized by bovine or rabbit antiserum known to have antitoxic activity. When cellular proteins from the E. coli clones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, a 100,000-dalton protein was identified which corresponded to one of the soluble antigens found in the leukotoxic culture supernatant of P. haemolytica. These results demonstrated that the gene(s) for the P. haemolytica leukotoxin have been cloned and that the leukotoxin was expressed in E. coli. PMID:3905610

  19. Stem cell research: cloning, therapy and scientific fraud.

    PubMed

    Rusnak, A J; Chudley, A E

    2006-10-01

    Stem cell research has generated intense excitement, awareness, and debate. Events in the 2005-2006 saw the rise and fall of a South Korean scientist who had claimed to be the first to clone a human embryonic stem cell line. From celebration of the potential use of stem cells in the treatment of human disease to disciplinary action taken against the disgraced scientists, the drama has unfolded throughout the world media. Prompted by an image of therapeutic cloning presented on a South Korean stamp, a brief review of stem cell research and the events of the Woo-suk Hwang scandal are discussed. PMID:16965321

  20. Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase.

    PubMed

    Rey, Michael W; Brown, Kimberly M; Golightly, Elizabeth J; Fuglsang, Claus C; Nielsen, Bjarne R; Hendriksen, Hanne V; Butterworth, Amy; Xu, Feng

    2003-12-01

    Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/ cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae alpha-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 micromol/(min x mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60 degrees C. The enzyme was stable at 60 degrees C for 30 min. The Km and kcat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20 degrees C and pH 4.0, the enzyme had a Km of 0.33 +/- 0.07 mM and a kcat of (5.5 +/- 0.5) x 103 min(-1) for maltotriose. The temperature dependence of kcat/Km indicated an activation free energy of 2.8 kJ/mol across the range of 20-70 degrees C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi. PMID:14665735

  1. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. PMID:20347821

  2. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    USGS Publications Warehouse

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  3. In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs

    PubMed Central

    Brenner, Sydney; Williams, Steven R.; Vermaas, Eric H.; Storck, Thorsten; Moon, Keith; McCollum, Christie; Mao, Jen-I; Luo, Shujun; Kirchner, James J.; Eletr, Sam; DuBridge, Robert B.; Burcham, Timothy; Albrecht, Glenn

    2000-01-01

    We describe a method for cloning nucleic acid molecules onto the surfaces of 5-μm microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry ≈106 strands complementary to one of the tags. About 105 copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays. PMID:10677516

  4. Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.

    PubMed

    Singh, Manika Indrajit; Jain, Vikas

    2013-01-01

    We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag. PMID:23691118

  5. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  6. AEG-1/MTDH/LYRIC, the Beginning: Initial Cloning, Structure, Expression Profile, and Regulation of Expression

    PubMed Central

    DeSalle, Rob; Sarkar, Devanand; Fisher, Paul B.

    2014-01-01

    Since its initial identification as a HIV-1-inducible gene in 2002, astrocyte elevated gene-1 (AEG-1), subsequently cloned as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), has emerged over the past 10 years as an important oncogene providing a valuable prognostic marker in patients with various cancers. Recent studies demonstrate that AEG-1/MTDH/LYRIC is a pleiotropic protein that can localize in the cell membrane, cytoplasm, endoplasmic reticulum (ER), nucleus, and nucleolus, and contributes to diverse signaling pathways such as PI3K–AKT, NF-κB, MAPK, and Wnt. In addition to tumorigenesis, this multifunctional protein is implicated in various physiological and pathological processes including development, neurodegeneration, and inflammation. The present review focuses on the discovery of AEG-1/MTDH/LYRIC and conceptualizes areas of future direction for this intriguing gene. We begin by describing how AEG-1, MTDH, and LYRIC were initially identified by different research groups and then discuss AEG-1 structure, functions, localization, and evolution. We conclude with a discussion of the expression profile of AEG-1/MTDH/LYRIC in the context of cancer, neurological disorders, inflammation, and embryogenesis, and discuss how AEG-1/MTDH/LYRIC is regulated. This introductory discussion of AEG-1/MTDH/LYRIC will serve as the basis for the detailed discussions in other chapters of the unique properties of this intriguing molecule. PMID:23889986

  7. Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

    PubMed Central

    Su, J Y; Sclafani, R A

    1991-01-01

    (Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity. Images PMID:2017365

  8. Stimulus-dependent modulation of human B cell function by T cell clones.

    PubMed

    Patel, S S; Thiele, D L; Lipsky, P E

    1993-02-01

    T cells exert both positive and negative regulatory effects on B cell function. To determine whether the nature of the stimulus could alter the immunoregulatory effects of T cells, the capacity of a battery of human T cell clones to modulate B cell function after stimulation with either pokeweed mitogen or a mAb to the CD3 molecular complex was examined. It was observed that most clones induced B cell differentiation when stimulated with immobilized mAb to CD3 (64.1) regardless of their phenotype. Moreover, the majority of clones (42 of 51) augmented the generation of immunoglobulin-secreting cells (ISC) supported by anti-CD3-stimulated blood CD4+ T cells. By contrast, none of the clones induced B cell differentiation when stimulated with PWM and 48 of 51 clones suppressed the generation of ISC induced by blood CD4+ T cells. This suppression could not be accounted for by the depletion of essential molecules or factors or by secretion of suppressive factors. Suppressive activity of clones did not correlate with the CD4 or CD8 phenotype and was not overcome by the addition of supernatants generated from mitogen-stimulated T cells or recombinant IL-2. Suppression by most clones, however, was abrogated when the clones were treated with mitomycin C or irradiated. A number of suppressive mechanisms by individual PWM-stimulated clones was identified, including direct inhibition of B cell function by cytotoxic and non-lytic means and suppression of helper T cell function. The failure of anti-CD3-stimulated clones to suppress the differentiation of B cells appeared to reflect the capacity of this stimulus to induce apoptosis by the clones after initial activation. These results indicate that T cell clones can provide help for B cell differentiation or can suppress B cell function by a variety of mechanisms depending upon the mode of stimulation. PMID:8174175

  9. The atypical antipsychotic, olanzapine, potentiates ghrelin-induced receptor signaling: An in vitro study with cells expressing cloned human growth hormone secretagogue receptor.

    PubMed

    Tagami, Keita; Kashiwase, Yohei; Yokoyama, Akinobu; Nishimura, Hitomi; Miyano, Kanako; Suzuki, Masami; Shiraishi, Seiji; Matoba, Motohiro; Ohe, Yuichiro; Uezono, Yasuhito

    2016-08-01

    The growth hormone secretagogue receptor (GHS-R) belongs to Gαq-coupled G protein-coupled receptor (GPCR) that mediates growth hormone release, food intake, appetite, glucose metabolism and body composition. Ghrelin has been identified as an endogenous ligand for GHS-R, and it is the only orexigenic peptide found in the peripheral organs. Olanzapine, an atypical antipsychotic agent that binds to and inhibits the activation of GPCR for several neurotransmitters, has metabolic side effects such as excessive appetite and weight gain. Recently, studies have revealed that the orexigenic mechanism of olanzapine is mediated via GHS-R signaling, although the precise mechanisms have not been clarified. In this study, we investigated the effect of olanzapine on ghrelin-mediated GHS-R signaling by using an electrical impedance-based receptor biosensor assay system (CellKey™). Olanzapine at concentrations of 10(-7) and 10(-6)mol/L enhanced ghrelin-induced (10(-10)-10(-8)mol/L) GHS-R activation. A Ca(2+) imaging assay revealed that olanzapine (10(-7) and 10(-6)mol/L) enhanced ghrelin (10(-7) M)-induced GHS-R activity. In contrast, haloperidol (an antipsychotic agent) failed to enhance this ghrelin-mediated GHS-R activation, as demonstrated by both the CellKey™ and Ca(2+) imaging assays. Together, these results suggest that olanzapine, but not haloperidol, promotes appetite by enhancing ghrelin-mediated GHS-R signaling. PMID:26775231

  10. CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

    PubMed

    Borovsky, Dov; Sterner, Andeas; Powell, Charles A

    2016-01-01

    The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh. PMID:26440910

  11. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

    PubMed Central

    Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

  12. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    PubMed

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

  13. mu opiate receptor: cDNA cloning and expression.

    PubMed Central

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

    1993-01-01

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

  14. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.

    PubMed

    Unger, Tamar; Jacobovitch, Yossi; Dantes, Ada; Bernheim, Reut; Peleg, Yoav

    2010-10-01

    Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system. PMID:20600952

  15. Cloning and expression of canine interferon-alpha genes in Escherichia coli.

    PubMed

    Taira, Osamu; Watanugi, Itsuki; Hagiwara, Yuko; Takahashi, Masaki; Arai, Setsuo; Sato, Hisaaki; Maehara, Nobutoshi

    2005-10-01

    We cloned five new subtypes of cDNA encoding canine interferon-alpha (CaIFN-alpha) from a canine epithelial cell line. CaIFN-alphas were divided into two groups by amino acid sequences and a molecular phylogenic tree. Two subtypes of them were expressed in Escherichia coli, and IFN proteins were purified. Recombinant CaIFN-alphas were highly species-specific and showed antiviral activity against Vesicular stomatitis New Jersey virus and canine adenovirus-1 , but not against canine herpesvirus-1. PMID:16276065

  16. Cloning and expression of Che a 1, the major allergen of Chenopodium album in Escherichia coli.

    PubMed

    Vahedi, Fatemeh; Sankian, Mojtaba; Moghadam, Malihe; Mohaddesfar, Maryam; Ghobadi, Sirous; Varasteh, Abdol Reza

    2011-04-01

    Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b+ vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni-NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1. PMID:20872185

  17. Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli

    PubMed Central

    Shoja, Zahra; Rajabi Memari, Hamid; Roayaei Ardakani, Mohammd

    2015-01-01

    Background: C-Phycocyanin (C-PC) from blue-green algae such as Spirulina has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-tumor activities. Recombinant β-subunit of C-PC (C-PC/β) is an inhibitor of cell proliferation and an inducer of cancer cell apoptosis. Objectives: Since C-PC/β has a big potential to be used as a promising cancer prevention or therapy agent, the purpose of this study was to clone and express Spirulina platensis cpcB gene in a bacterial expression system. This is a significant step for the production of this compound. Materials and Methods: The cpcB gene was amplified using specific primers and cloned in a bacterial expression vector, namely pET43.1a+. Gene expression of cpcB was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the dot blotting technique. Results: The SDS-PAGE analysis and dot blotting confirmed the production of recombinant C-PC/β in the bacterial expression system. Over-expression of cpcB gene was optimized in induction by 1 mM Isopropyl-β-D-Thiogalactoside (IPTG), after four hours of inoculation at 30°C. Conclusions: Over-expression of the synthetic CPC/β protein in the bacterial system (Escherichia coli BL-21) showed that E. coli can be used as a basis for further research to produce this desired protein in large quantities. PMID:26464761

  18. Cloning and expression of an A1 adenosine receptor from rat brain

    SciTech Connect

    Mahan, L.C.; McVittie, L.D.; Smyk-Randall, E.M.; Nakata, H.; Monsma, F.J. Jr.; Gerfen, C.R.; Sibley, D.R. )

    1991-07-01

    The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.

  19. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

    PubMed

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease. PMID:26147378

  20. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma

    PubMed Central

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)–transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease. PMID:26147378

  1. College Students' Conceptions of Stem Cells, Stem Cell Research, and Cloning

    ERIC Educational Resources Information Center

    Concannon, James P.; Siegel, Marcelle A.; Halverson, Kristy; Freyermuth, Sharyn

    2010-01-01

    In this study, we examined 96 undergraduate non-science majors' conceptions of stem cells, stem cell research, and cloning. This study was performed at a large, Midwest, research extensive university. Participants in the study were asked to answer 23 questions relating to stem cells, stem cell research, and cloning in an on-line assessment before…

  2. Treating Cloned Embryos, But Not Donor Cells, with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

    PubMed Central

    HUAN, Yan Jun; ZHU, Jiang; XIE, Bing Teng; WANG, Jian Yu; LIU, Shi Chao; ZHOU, Yang; KONG, Qing Ran; HE, Hong Bin; LIU, Zhong Hua

    2013-01-01

    The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression. PMID

  3. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    PubMed

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. PMID:19444989

  4. Molecular cloning, characterization, and expression of sheep FGF5 gene.

    PubMed

    Zhang, Lihua; He, Sangang; Liu, Mingjun; Liu, Guosong; Yuan, Zheng; Liu, Chenxi; Zhang, Xumei; Zhang, Ning; Li, Wenrong

    2015-01-25

    The fibroblast growth factor 5 gene (FGF5) is a member of the FGF gene family, and represents a candidate gene for hair length because of its role in the regulation of the hair follicle growth cycle. In our current study, we cloned, sequenced, and characterized the full-length FGF5 cDNA of Chinese Merino sheep. We obtained the complete genomic sequence of the FGF5 gene from sheep blood samples, and compared it to other FGF5 sequences in GenBank. We found that the FGF5 gene spanned 21,743bp of genomic DNA, and consisted of 3 exons and 2 introns, both of which differed from those of a previously annotated FGF5 genomic sequence from sheep. We also identified a previously undescribed FGF5 mRNA splicing variant, FGF5S, and the western blot analysis showed that the molecular weights of the FGF5 (34kDa) and FGF5s (17kDa) proteins were consistent with the estimates based on the genomic and cDNA sequence data. We examined the expression of both FGF5 mRNAs in various tissues of sheep, and found that the expression of the FGF5S mRNA was restricted to the brain, spleen, and skin tissue. The single-nucleotide polymorphism analysis of the genomic sequence revealed 72 genetic variants of the FGF5 gene. Our findings provide insight into the functions of the FGF5 gene in Chinese Merino. PMID:25445274

  5. Cloning, sequencing, and expression of bacteriophage BF23 late genes 24 and 25 encoding tail proteins.

    PubMed Central

    Nakayama, S; Kaneko, T; Ishimaru, H; Moriwaki, H; Mizobuchi, K

    1994-01-01

    Two bacteriophage BF23 late genes, genes 24 and 25, were isolated on a 7.4-kb PstI fragment from the phage DNA, and their nucleotide sequences were determined. Gene 24 encodes a minor tail protein with the expected M(r) of 34,309, and gene 25 located 4 bp upstream of gene 24 encodes a major tail protein with the expected M(r) of 50,329. When total cellular RNA isolated from either phage-infected cells or cells bearing the cloned genes was analyzed by the primer extension method using the primers specific to either gene 25 or gene 24, we identified a possible late gene promoter, designated P25, in the 5'-flanking region of gene 25. This promoter was similar in structure to Escherichia coli promoters for sigma 70. Studies of the translational gene 25- and gene 24-lacZ fusions in the cloned gene system revealed that the promoter P25 was responsible for the expression of both genes 25 and 24 even in the absence of the regulatory genes which were absolutely required for late gene expression in the normal phage-infected cells. These results indicate that the two genes constitute an operon under the control of P25 and that the regulatory gene products of BF23 do not participate directly in specifying the late gene promoter. Images PMID:7961500

  6. Molecular cloning and expression analysis of CmMlo1 in melon.

    PubMed

    Cheng, Hong; Kun, Weiping; Liu, Dongshun; Su, Yongquan; He, Qiwei

    2012-02-01

    Mlo gene encodes an important transmembrane protein that is involved in biotic/abiotic stresses. Using the method of homologous, we cloned a Mlo gene from melon, named CmMlo1. The gene is 1551 bp in length, encoding 516 amino acids; it has seven-transmembrane domain topology and is a typical transmembrane protein. Localization analysis in onion epidermal cells showed that CmMlo1-GFP is localized to the plasma membrane. RT-PCR results indicated that CmMlo1 is mainly expressed in melon cotyledon and flower, with a tissue-specific distribution manner. CmMlo1 expression is not obvious under powdery mildew stress, but under cadmium stress, its expression was significantly up-regulated, indicating that CmMlo1 is possibly involved in abiotic stress. PMID:21660472

  7. Cloning, expression, and regulation of tissue-specific genes in Drosophila

    SciTech Connect

    Korochkin, L.I.

    1995-08-01

    The family of esterase genes was studied in various Drosophilia species. These genes are classified as tissue-specific and housekeeping ones. The expression of tissue-specific esterases in the male reproductive system of Drosophilia species from the virilis and melanogaster groups was thoroughly examined. Modifier genes controlling activity level, time of synthesis, and distribution in cells of the tissue-specific esterase isozyme from the ejaculatory bulb were revealed. The structural gene coding of this enzyme was isolated, cloned, and sequenced. This gene was shown to be similar in different Drosophilia species; the transcriptional level of tissue specificity of this gene was determined. The possibility of transformating the tissue-specific gene into a housekeeping one was demonstrated. In different Drosophilia species, this gene can be expressed in different parts of the reproductive system. In transgenic males carrying the gene of another species, the foreign gene is expressed as in the donor. 68 refs., 11 figs.

  8. Molecular cloning, characterization, and expression of pannexin genes in chicken.

    PubMed

    Kwon, Tae-Jun; Kim, Dong-Bin; Bae, Jae Woong; Sagong, Borum; Choi, Soo-Young; Cho, Hyun-Ju; Kim, Un-Kyung; Lee, Kyu-Yup

    2014-09-01

    Pannexins (Panx) are a family of proteins that share sequences with the invertebrate gap junction proteins, innexins, and have a similar structure to that of the vertebrate gap junction proteins, connexins. To date, the Panx family consists of 3 members, but their genetic sequences have only been completely determined in a few vertebrate species. Moreover, expression of the Panx family has been reported in several rodent tissues: Panx1 is ubiquitously expressed in mammals, whereas Panx2 and Panx3 expressions are more restricted. Although members of the Panx family have been detected in mammals, their genetic sequences in avian species have not yet been fully elucidated. Here, we obtained the full-length mRNA sequences of chicken PANX genes and evaluated the homology of the amino acids from these sequences with those of other species. Furthermore, PANX gene expression in several chicken tissues was investigated based on mRNA levels. PANX1 was detected in the brain, cochlea, chondrocytes, eye, lung, skin, and intestine, and PANX2 was expressed in the brain, eye, and intestine. PANX3 was observed in the cochlea, chondrocytes, and bone. In addition, expression of PANX3 was higher than PANX1 in the cochlea. Immunofluorescent staining revealed PANX1 in hair cells, as well as the supporting cells, ganglion neurons, and the tegmentum vasculosum in chickens, whereas PANX3 was only detected in the bone surrounding the cochlea. Overall, the results of this study provide the first identification and characterization of the sequence and expression of the PANX family in an avian species, and fundamental data for confirmation of Panx function. PMID:25002553

  9. Cloning and expression in Escherichia coli of Mycoplasma gallisepticum antigens recognized by sera from infected chickens.

    PubMed

    Krause, D C; Kleven, S H; Lee, K K

    1990-01-01

    A clone bank of Mycoplasma gallisepticum (MG) strain A5969 DNA was prepared in the expression vector phage lambda gt11. Approximately 75% of the resulting phages were recombinants, based upon the insertional inactivation of the lacZ gene of the vector. Clones were screened immunologically with serum prepared from specific-pathogen-free white leghorn chickens that had been infected with aerosolized MG. Approximately 250 clones, or less than 1% of the recombinant phage, reacted positively to various degrees with the test serum and failed to react with serum from uninfected specific-pathogen-free control chickens. A single clone was chosen at random for comparison with a vector control by western immunoblot, revealing a polypeptide of 140,000 molecular weight in the clone profile but not the control profile that reacted with immune serum. Clones expressing MG antigens recognized during infection may provide an improved means for antigen preparation for serologic diagnosis of mycoplasmosis. PMID:2142422

  10. Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein.

    PubMed

    Ivessa, N Erwin; Rehberg, Edward; Kienzle, Bernadette; Seif, Fridolin; Hermann, Robert; Hermann, Marcela; Schneider, Wolfgang J; Gordon, David A

    2013-07-01

    During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle. PMID:23542778

  11. Cloning and variation of ground state intestinal stem cells.

    PubMed

    Wang, Xia; Yamamoto, Yusuke; Wilson, Lane H; Zhang, Ting; Howitt, Brooke E; Farrow, Melissa A; Kern, Florian; Ning, Gang; Hong, Yue; Khor, Chiea Chuen; Chevalier, Benoit; Bertrand, Denis; Wu, Lingyan; Nagarajan, Niranjan; Sylvester, Francisco A; Hyams, Jeffrey S; Devers, Thomas; Bronson, Roderick; Lacy, D Borden; Ho, Khek Yu; Crum, Christopher P; McKeon, Frank; Xian, Wa

    2015-06-11

    Stem cells of the gastrointestinal tract, pancreas, liver and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, 'ground state' stem cells of the human intestine and colon. We show that derived stem-cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells is likely to enforce the functional specificity of the adult intestinal tract. Using clonally derived colonic epithelia, we show that toxins A or B of the enteric pathogen Clostridium difficile recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modelling and regenerative medicine. PMID:26040716

  12. Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

    PubMed

    Kianmehr, Anvarsadat; Golavar, Raziyeh; Rouintan, Mandana; Mahrooz, Abdolkarim; Fard-Esfahani, Pezhman; Oladnabi, Morteza; Khajeniazi, Safoura; Mostafavi, Seyede Samaneh; Omidinia, Eskandar

    2016-02-01

    Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. PMID:26546410

  13. Functional and phenotypic analysis of human T-cell clones which stimulate IgE production in vitro.

    PubMed Central

    Quint, D J; Bolton, E J; McNamee, L A; Solari, R; Hissey, P H; Champion, B R; MacKenzie, A R; Zanders, E D

    1989-01-01

    Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-gamma) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-gamma, although the amounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the mouse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29. PMID:2525520

  14. Molecular cloning and developmental expression of plakophilin 2 in zebrafish

    SciTech Connect

    Moriarty, Miriam A.; Martin, Eva D.; Byrnes, Lucy; Grealy, Maura

    2008-02-29

    Armadillo proteins are involved in providing strength and support to cells and tissues, nuclear transport, and transcriptional activation. In this report, we describe the identification and characterisation of the cDNA of the desmosomal armadillo protein plakophilin 2 in zebrafish. The 2448 bp coding sequence encodes a predicted 815 amino acid protein, with nine armadillo repeats characteristic of the p120-catenin subfamily. It shares conserved N-glycosylation, myristoylation, and glycogen synthase kinase 3, casein kinase 2, and protein kinase C phosphorylation sites with mammalian armadillo proteins including plakoglobin and {beta}-catenin. Semi-quantitative reverse transcription polymerase chain reaction and whole mount in situ hybridisation show that it is expressed both maternally and zygotically. It is ubiquitously expressed during blastula stages but becomes restricted to epidermal and cardiac tissue during gastrulation. These results provide evidence that zebrafish plakophilin 2 is developmentally regulated with potential roles in cell adhesion, signalling, and cardiac and skin development.

  15. Cloning mammary cell cDNAs from 17q12-q23 using interspecific somatic cell hybrids and subtractive hybridization

    SciTech Connect

    Cerosaletti, K.M.; Shapero, M.H.; Fournier, R.E.K.

    1995-01-01

    We have cloned human genes that are encoded in the region 17q12-q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12-q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12-q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12-q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12-q23 region. 47 refs., 6 figs.

  16. Cloned Hemoglobin Genes Enhance Growth Of Cells

    NASA Technical Reports Server (NTRS)

    Khosla, Chaitan; Bailey, James E.

    1991-01-01

    Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

  17. A novel approach to sequence validating protein expression clones with automated decision making

    PubMed Central

    Taycher, Elena; Rolfs, Andreas; Hu, Yanhui; Zuo, Dongmei; Mohr, Stephanie E; Williamson, Janice; LaBaer, Joshua

    2007-01-01

    Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE) system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence verification projects. The

  18. Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs.

    PubMed

    Parks, Christopher L; Wang, Hai-Ping; Kovacs, Gerald R; Vasilakis, Nikos; Kowalski, Jacek; Nowak, Rebecca M; Lerch, Robert A; Walpita, Pramila; Sidhu, Mohinderjit S; Udem, Stephen A

    2002-02-26

    A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 degrees C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells. PMID:11864746

  19. Cloning, sequencing and expression of the Taq I restriction-modification system.

    PubMed Central

    Slatko, B E; Benner, J S; Jager-Quinton, T; Moran, L S; Simcox, T G; Van Cott, E M; Wilson, G G

    1987-01-01

    The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene. Images PMID:2827113

  20. Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37.

    PubMed Central

    Coleman, T; Grass, S; Munson, R

    1991-01-01

    Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin. Images PMID:1673447

  1. Preparation of North American type II PRRSV infectious clone expressing green fluorescent protein.

    PubMed

    Wang, Liyue; Zhang, Kao; Lin, Hongyu; Li, Wenyan; Wen, Jiexia; Zhang, Jianlou; Zhang, Yonghong; Li, Xiujin; Zhong, Fei

    2014-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid by Spe I and Xho I sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesized in vitro and amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV. PMID:24895571

  2. Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

    PubMed Central

    Cheng, Yuqiang; Sun, Yingjie; Wang, Hengan; Shi, Shuduan; Yan, Yaxian; Li, Jing

    2015-01-01

    Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-κB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation. PMID:25269719

  3. Cloning, expression and purification of the factor H binding protein and its interaction with factor H

    PubMed Central

    Yarian, Fatemeh; Bandehpour, Mojgan; Seyed, Negar; Kazemi, Bahram

    2016-01-01

    Background and Objective: Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein. Materials and Methods: A 820 base pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting. Results and Conclusions: SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein. PMID:27092222

  4. Molecular cloning and functional expression of a brain-specific somatostatin receptor.

    PubMed Central

    Bruno, J F; Xu, Y; Song, J; Berelowitz, M

    1992-01-01

    The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor. Images PMID:1360663

  5. Genome-wide gene expression and DNA methylation differences in abnormally cloned and normally natural mating piglets.

    PubMed

    Zou, C; Fu, Y; Li, C; Liu, H; Li, G; Li, J; Zhang, H; Wu, Y; Li, C

    2016-08-01

    Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer. PMID:27028246

  6. Cloning

    MedlinePlus

    ... mammals. These twins are produced when a fertilized egg splits, creating two or more embryos that carry ... of the donor animal's somatic cell into an egg cell, or oocyte, that has had its own ...

  7. Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci.

    PubMed Central

    Courtney, H S; Li, Y; Dale, J B; Hasty, D L

    1994-01-01

    Lipoteichoic acid and several streptococcal proteins have been reported to bind fibronectin (Fn) or fibrinogen (Fgn), which may serve as host receptors. We searched for such proteins by screening a library of genes from M type 5 group A streptococci cloned into Escherichia coli. Lysates of clones were probed with biotinylated Fn and biotinylated Fgn. One clone expressed a 54-kDa protein that reacted with Fn and Fgn. The protein, termed FBP54, was purified and used to immunize rabbits. Anti-FBP54 serum reacted with purified, recombinant FBP54 and with a protein of similar electrophoretic mobility in extracts of M type 5, 6, and 24 streptococci. Anti-FBP54 serum also reacted with 5 of 15 strains of intact, live streptococci, suggesting that FBP54 may be a surface antigen. Southern blot analysis confirmed that the gene is found in group A streptococci but not in Staphylococcus aureus or E. coli. The cloned gene was sequenced and contained an open reading frame encoding a protein with a calculated molecular weight of 54,186. Partial amino acid sequencing of purified FBP54 confirmed that this open reading frame encoded the protein. As determined by utilizing fusion proteins containing truncated forms of FBP54, the primary Fn/Fgn-binding domain appears to be contained in residues 1 to 89. These data suggest that FBP54 may be a surface protein of streptococci that reacts with both Fn and Fgn and therefore may participate in the adhesion of group A streptococci to host cells. Images PMID:8063411

  8. [Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli].

    PubMed

    Lavrov, K V; Ianenko, A S

    2013-10-01

    The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases. PMID:25474901

  9. [Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli].

    PubMed

    2013-10-01

    The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases. PMID:25508680

  10. Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

    PubMed Central

    Ghasemi, Amir; Ghafourian, Sobhan; Vafaei, Sedighe; Mohebi, Reza; Farzi, Maryam; Taherikalani, Morovat; Sadeghifard, Nourkhoda

    2015-01-01

    Objectives In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. Methods Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured. Results Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system. Conclusion These results indicate that this expression system was appropriate for the production of thermostable α-amylase. PMID:26835242

  11. Advanced lymphoblastic clones detection in T-cell leukemia.

    PubMed

    Minervina, A A; Komkov, A Y; Mamedov, I Z; Lebedev, Y B

    2016-03-01

    T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the lymphocyte precursors that suffered malignant transformation arresting the lymphoid cell differentiation. Clinical studies revealed monoor, more rarely, oligoclonal nature of the disease. A precise identification of malignant clone markers is both the crucial stage of early diagnostics and the essential prognostic factor for therapeutic treatment. Here we present an improved system for unbiased detection of lymphoblastic clones in bone marrow aspirates of T-ALL patients. The system based on multiplex PCR of rearranged T-cell receptor locus (TRB) and straightforward sequencing of the resulted PCR fragments. Testing of the system on genomic DNA from Jurkat cell line and four clinical bone marrow aspirates revealed a set of unique TRB rearrangements that precisely characterize each of tested samples. Therefore, the outcome of the system produces highly informative molecular genetic markers for further monitoring of minimal residual disease in T-ALL patients. PMID:27193704

  12. RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida.

    PubMed

    Lee, M D; Henk, A D

    1997-03-01

    The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional beta-galactosidase as determined by o-nitrophenyl-beta-D-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida. PMID:9100336

  13. Cloning and expression of a rat brain. alpha. sub 2B -adrenergic receptor

    SciTech Connect

    Flordellis, C.S.; Handy, D.E.; Bresnahan, M.R.; Zannis, V.I.; Gavras, H. )

    1991-02-01

    The authors isolated a cDNA clone (RB{alpha}{sub 2B}) and its homologous gene (GR{alpha}{sub 2B}) encoding an {alpha}{sub 2B}-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor ({alpha}{sub 2}-C4) and divergent from the rat kidney nonglycosylated {alpha}{sub 2B} subtype (RNG{alpha}{sub 2}). Transient expression of RB{alpha}{sub 2B} in COS-7 cells resulted in high-affinity saturable binding for ({sup 3}H)rauwolscine and a high receptor number in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine {gt} yohimbine {gt} prazosin {gt} oxymetazoline, with a prazosin-to-oxymetazoline K{sub i} ratio of 0.34. This profile is characteristic of the {alpha}{sub 2B}-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major and two minor mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR{alpha}{sub 2B} may be transcriptionally active. These findings show that rat brain expresses an {alpha}{sub 2B}-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated {alpha}{sub 2B} subtype. Thus the rat expresses at least two divergent {alpha}{sub 2B}-adrenergic receptors.

  14. Cloning, expression, and functional characterization of the rat Pax6 5a orthologous splicing variant.

    PubMed

    Wei, Fei; Li, Min; Cheng, Sai-Yu; Wen, Liang; Liu, Ming-Hua; Shuai, Jie

    2014-08-15

    Pax6 functions as a pleiotropic regulator in eye development and neurogenesis. Its splice variant Pax6 5a has been cloned in many vertebrate species including human and mouse, but never in rat. This study focused on the cloning and characterization of the Pax6 5a orthologous splicing variant in rat. It was cloned from Sprague-Dawley rats 10 days post coitum (E10) by RT-PCR and was sequenced for comparison with Pax6 sequences in the GenBank by BLAST. The rat Pax6 5a was revealed to contain an additional 42 bp insertion at the paired domain. At the nucleotide level, the rat Pax6 5a coding sequence (1,311 bp) had a higher degree of homology to the mouse (96% identical) than to the human (93% identical) sequence. At the amino acid (aa) level, rat PAX6 5a shares 99.8% identity with the mouse sequence and 99.5% with the human sequence. The splice variant is preferentially expressed in the rat E10 embryonic headfolds and not in the trunk of neurula. Its effects on the proliferation of rat mesenchymal stem cells (rMSCs) were preliminarily evaluated by the MTT assay. Both pLEGFP-Pax6 5a-transfected cells and pLEGFP-Pax6-transfected cells exhibited a similar growth curve (P>0.05), suggesting that the Pax6 5a has a similar effect on the proliferation of rMSCs as Pax6. PMID:24952136

  15. Re-Cloning the N27 Dopamine Cell Line to Improve a Cell Culture Model of Parkinson's Disease

    PubMed Central

    Symmes, Breanna; Freed, Curt R.

    2016-01-01

    Parkinson’s disease is characterized by the death of dopaminergic neurons in the substantia nigra. To understand the molecular mechanisms of the disease, an in vitro model is important. In the 1990s, we used the SV40 large T antigen to immortalize dopaminergic neurons derived from Embryonic Day 14 rat mesencephalon. We selected a clone for its high expression of dopaminergic neuron markers such as tyrosine hydroxylase (TH), and we named it 1RB3AN27 (N27). Because the original N27 cell line has been passaged many times, the line has become a mixture of cell types with highly variable expression of TH. In the current study, we have performed multiple rounds of clonal cultures and have identified a dopaminergic cell clone expressing high levels of TH and the dopamine transporter (DAT). We have named this new clone N27-A. Nearly 100% of N27-A cells express TH, DAT and Tuj1. Western blots have confirmed that N27-A cells have three to four times the levels of TH and DAT compared to the previous mixed population in N27. Further analysis has shown that the new clone expresses the dopamine neuron transcription factors Nurr1, En1, FoxA2 and Pitx3. The N27-A cells express the vesicular monoamine transporter (VMAT2), but do not express dopamine-beta-hydroxylase (DβH), the enzyme responsible for converting dopamine to norepinephrine. Functional analysis has shown that N27-A cells are more sensitive than N27 cells to neurotoxins taken up by the dopamine transporter such as 6-hydroxydopamine and 1-methyl-4-phenylpyridine (MPP+). The DAT inhibitor nomifensine can block MPP+ induced toxicity. The non-selective toxic effects of hydrogen peroxide were similar in both cell lines. The N27-A cells show dopamine release under basal and depolarization conditions. We conclude that the new N27-A clone of the immortalized rat dopaminergic cell line N27 should provide an improved in vitro model for Parkinson’s disease research. PMID:27512998

  16. Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

    PubMed Central

    Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua

    2013-01-01

    Abstract The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro–cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT. PMID:24033142

  17. Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase.

    PubMed Central

    Wu, L W; Baylink, D J; Lau, K H

    1996-01-01

    Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single 'PTP catalytic domain'. The PTP catalytic domain shows 45-50% sequence identity with the catalytic domain of human HPTP beta and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST-cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc m

  18. Molecular cloning and expression of bovine kappa-casein in Escherichia coli

    SciTech Connect

    Kang, Y.C.; Richardson, T.

    1988-01-01

    A cDNA library was constructed using poly(A)/sup +/RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using /sup 32/P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein.

  19. Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

    SciTech Connect

    Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano; Macone, Alberto; Boffi, Alberto; Ilari, Andrea

    2008-04-01

    The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.

  20. [Stem cells and therapeutic cloning, medical perspectives under discussion].

    PubMed

    Manuel, Catherine; Lafon, Claude; Hairion, Dominique; Antoniotti, Stéphanie

    2004-03-13

    Innovative biotechnical progress over the past few years regards stem cells and therapeutic cloning, which open promising medical horizons for many presently incurable diseases. THE CURRENT DEBATE: The research work in France has been stalled because of the prohibitions listed in the so-called "bioethical" laws of 1994. The ongoing revision of these laws is based on a certain number of ethical questions and launches a disputable parlementary debate. Other than reproductive cloning and research on the embryo, the possibilities provided by stem cells and therapeutic cloning should be emphasized and the different positions advanced specified, showing an evolution in the laws in France. ABUSIVE LEGISLATIVE PROHIBITIONS: The proposed law, which maintains the prohibition for research on the embryo, with a 5-Year dispensation, and which explicitly prohibits therapeutic cloning, is not in keeping with the widening of in this field expected by research teams. Many scientists and physicians, supported by patients' associations, are aware of the importance of therapeutic progress attached to such research. They should not be stalled in their studies by the prohibitions maintained in the new law. PMID:15041874

  1. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    SciTech Connect

    Xu, H. Howard . E-mail: hxu3@calstatela.edu; Real, Lilian; Bailey, Melissa Wu

    2006-11-03

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

  2. Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP.

    PubMed

    García-Vivas, Jessica; López-Camarillo, César; Azuara-Liceaga, Elisa; Orozco, Esther; Marchat, Laurence A

    2005-07-01

    In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively. PMID:15955317

  3. Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis).

    PubMed

    Zhang, Liyan; Liu, Wanjun; Shao, Changwei; Zhang, Ning; Li, Hailong; Liu, Kun; Dong, Zhongdian; Qi, Qian; Zhao, Wen; Chen, Songlin

    2014-12-01

    piwi is an important regulator gene in germ cell division during spermatogenesis. piwi homologous genes are involved in gametogenesis and germline specification, and knocking down these genes could affect germ cell meiotic progression. To understand the function of piwi-related genes in spermatogenesis, we cloned a Piwi-subfamily member (piwil2 gene) from the gonad of Cynoglossus semilaevis. The full-length of piwil2 cDNA was 3314bp, including a 3162bp open reading frame (ORF), a 60bp 5'-UTR, along with a 92bp 3'-UTR, and encoded a predicted protein of 1053 amino acid residues. Phylogenetic analysis showed that the PIWIL2 putative protein belonged to the Argonaute protein family, and Piwi-subfamily, with typical PAZ and Piwi domains. Ultrathin sections of different gonadal stages, and real-time quantitative PCR showed that the relative expression of the piwil2 gene couldn't be detected until day 95 (95days) larvae, when germ cell divided rapidly in C. semilaevis. The piwil2 transcripts were more abundant in gonads of males and neo-males than in females, and weakly expressed in other tissues and organs. Compared with the relative expression of piwil2 in gonads, the CpG methylation levels were significantly higher in females. Chromosomal fluorescence in situ hybridization (FISH) showed that the piwil2 gene was located on the Z sex chromosome of C. semilaevis. These results suggest that piwil2 plays an important role in spermatogenesis of C. semilaevis. PMID:24794875

  4. Molecular cloning, chromosomal location, and expression analysis of porcine CD14.

    PubMed

    Sanz, Gema; Pérez, Eva; Jiménez-Marín, Angeles; Mompart, Florence; Morera, Luis; Barbancho, Manuel; Llanes, Diego; Garrido, Juan J

    2007-01-01

    CD14 is a membrane-associated glycosylphosphatidylinositol (GPI)-anchored protein that binds lipopolysaccharide (LPS) of Gram-negative bacteria and enables LPS-dependent responses in a variety of cells. In this study a cDNA containing the porcine CD14 coding sequence has been cloned and its complete sequence determined. The amino acid sequence deduced from pig CD14 cDNA encodes a 373 amino acid polypeptide that exhibits 75%, 72%, 69%, 66%, 57% and 56% similarity to CD14 from cow, horse, human, rabbit, mouse and rat, respectively. Structural analysis showed that the porcine CD14 is a membrane glycoprotein with a GPI-anchor site and an extracellular domain containing 11 leucine-rich repeats. In addition, the LPS-binding regions identified in the human CD14 are highly conserved in the N-terminal domain of the porcine sequence. Fluorescence in situ hybridization was used to locate the CD14 gene on the pig chromosome 2, band q28. Expression analysis revealed that porcine CD14 transcripts were detected in all tissues and cells examined, suggesting that the expression of porcine CD14 gene is not restricted to myeloid cell lineage. Finally, we report that LPS stimulation significantly up-regulated CD14 gene expression in porcine alveolar macrophages. PMID:17169425

  5. Cloning and expression of pinB gene from Triticum monococum seeds.

    PubMed

    Mohammadi, M; Kane, V; Ng, A; Zaidi, M A; Altosaar, I; Ni, F; Tanchak, M

    2003-01-01

    Puroindolines (PIN) are low molecular weight, cysteine-rich, endosperm-specific, basic proteins with a unique tryptophan-rich domain found in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) as well as other members of Triticaceae. PINs appear to be involved in both flour softness as well as resistance against fungal diseases. These proteins are known to be the major components of 'friabilin' associated with the surface of water washed starch grains and possess lipid binding properties. Structural characterization of puroindolines from Triticum monococum was initiated by amplifying and subsequently cloning the corresponding pin gene into an expression vector, known as pET-32a(+). The protein contains five tryptophanin domains and ten cysteine residues. The pinB gene was fused with the 109aa Trx.Tag thioredoxin for a high-level expression. The cloning sites used for producing fusion proteins also contained cleavable His.Tag and S.tag sequences for detection and purification. After transformation of competent Origami cells, fusion protein expression was detected by growing a transformant in LB medium in the presence of 0.1 mM IPTG at room temperature for 6 hrs on a shaker. Both soluble and insoluble fusion proteins were extracted from Origami cells after sonication. Ni-NTA column (Qiagen) was used to extract and purify these fractions. Following an overnight digestion of the recombinant protein with enterokinase at room temperature, the corresponding fractions were electrophoresed in polyacrylamide gel, electroblotted onto a nitrocellulose membrane and cross-reacted with the anti-friabilin monoclonal antibody. We found that the recombinant PINB protein had a molecular weight of 16 kDa whereas TrxB was 21 kDa. Fusion protein ran at 34 kDa. PINB protein from wheat was shown to be immunologically related to a homologue, tryptophanin, in oat seed. Further study is currently underway to characterize these proteins structurally using NMR. PMID:15151287

  6. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates. PMID:26577600

  7. Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene.

    PubMed

    Montpetit, Jonatan; Vivancos, Julien; Mitani-Ueno, Namiki; Yamaji, Naoki; Rémus-Borel, Wilfried; Belzile, François; Ma, Jian Feng; Bélanger, Richard R

    2012-05-01

    Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake. PMID:22351076

  8. Construction and characterization of a full-length infectious simian T-cell lymphotropic virus type 3 molecular clone.

    PubMed

    Chevalier, Sébastien Alain; Walic, Marine; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Gessain, Antoine; Mahieux, Renaud

    2007-06-01

    Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo. PMID:17428869

  9. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

    2015-09-01

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  10. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    SciTech Connect

    Woon, J. S. K. Murad, A. M. A. Abu Bakar, F. D.

    2015-09-25

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.