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Sample records for cell clones expressing

  1. Expression cloning of human B cell immunoglobulins.

    PubMed

    Wardemann, Hedda; Kofer, Juliane

    2013-01-01

    The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

  2. Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells

    PubMed Central

    1983-01-01

    We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined. PMID:6220105

  3. Expression of cloned immunoglobulin genes introduced into mouse L cells.

    PubMed Central

    Gilles, S D; Tonegawa, S

    1983-01-01

    Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells. Images PMID:6316279

  4. Faithful expression of imprinted genes in donor cells of SCNT cloned pigs.

    PubMed

    Wang, Dongxu; Yuan, Lin; Sui, Tingting; Song, Yuning; Lv, Qingyan; Wang, Anfeng; Li, Zhanjun; Lai, Liangxue

    2015-07-22

    To understand if the genomic imprinting status of the donor cells is altered during the process of SCNT (somatic cell nuclear transfer), cloned pigs were produced by SCNT using PEF (porcine embryonic fibroblast) and P-PEF (parthenogenetic-PEF) cells as donors. Then, the gene expression and methylation patterns of H19, IGF2, NNAT and MEST were compared between PEF vs. C-PEF (cloned-PEF), P-PEF vs. CP-PEF (cloned-P-PEF), respectively. Taken together, the results revealed that there was no significant difference in the expression of imprinted genes and conserved genomic imprints between the donor and cloned cells.

  5. Heterogeneity of Functional Properties of Clone 66 Murine Breast Cancer Cells Expressing Various Stem Cell Phenotypes

    PubMed Central

    Mukhopadhyay, Partha; Farrell, Tracy; Sharma, Gayatri; McGuire, Timothy R.; O’Kane, Barbara; Sharp, J. Graham

    2013-01-01

    Introduction Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Methods Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. Results The proportion of cells expressing CD44highCD24low/neg, side population (SP) cells, ALDH1+, CD49fhigh, CD133high, and CD34high differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1+, CD34low, and CD49fhigh suggested properties of transit amplifying cells. Colony formation was higher from ALDH1− and non-SP cells than ALDH1+ and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than “non-stem” cells. Fewer SP cells were needed to form tumors than ALDH1+ cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. Conclusions These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells. PMID:24265713

  6. Alloreactive T cell clones.

    PubMed

    Fitch, F W

    1984-01-01

    T cell clones are useful models for studying lymphocyte function both at the level of the individual cell and in interacting systems. Murine cytolytic and non- cytolyic T cell clones have been obtained with relative ease, and the particular procedure used to derive and maintain T cell clones may influence profoundly the characteristics of the resulting cells. The method of choice depends on the specific question to be asked. Although some clones have characteristics that would have been expected on the basis of results observed with bulk cell populations, other clones have rather unexpected properties. Although most T cell clones appear to be either cytolytic or non-cytolytic, this distinction is not always absolute. A high proportion of both cytolytic and non-cytolytic T cell clones have dual reactivity. This is true for cells which by other criteria appear to be true clones. The frequency of such cells is high enough to suggest that most if not all T cells may have reactivity for more than one antigenic determinant or that antigenic determinants recognized by T cells are shared widely and unexpectedly. It is not clear whether one or two different antigen receptors account for such dual reactivity. The nature of the T cell receptor for antigen remains obscure. T cell clones, because of their homogeneous nature, should make it easier to answer these important immunological questions. Although it remains to be determined how many distinct molecules account for the numerous biological activities found in the culture supernatants from antigen-stimulated T cell clones, it is clear that these factors influence several different types of cells that are involved directly and indirectly in immune responses. IL-2 stimulates both cytolytic and non-cytolytic T cells to proliferate. BCSF causes polyclonal activation of B cells, and there may be other factors which influence B cell responses to antigenic stimulation. IL-3 apparently stimulates maturation of immature T cells

  7. 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

  8. 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.

  9. Cloning and expression of Xenopus Prickle, an orthologue of a Drosophila planar cell polarity gene.

    PubMed

    Wallingford, John B; Goto, Toshiyasu; Keller, Ray; Harland, Richard M

    2002-08-01

    We have cloned Xenopus orthologues of the Drosophila planar cell polarity (PCP) gene Prickle. Xenopus Prickle (XPk) is expressed in tissues at the dorsal midline during gastrulation and early neurulation. XPk is later expressed in a segmental pattern in the presomitic mesoderm and then in recently formed somites. XPk is also expressed in the tailbud, pronephric duct, retina, and the otic vesicle. The complex expression pattern of XPk suggests that PCP signaling is used in a diverse array of developmental processes in vertebrate embryos.

  10. Cloning and expression of a cDNA for the T-cell-activating protein TAP.

    PubMed Central

    Reiser, H; Coligan, J; Palmer, E; Benacerraf, B; Rock, K L

    1988-01-01

    The T-cell-activating protein TAP is a murine phosphatidylinositol-anchored glycoprotein whose expression is controlled by the Ly-6 locus. Previous studies have suggested an important role for this protein in physiological T-cell activation. Using oligonucleotide probes, we have now isolated a cDNA clone whose predicted sequence would encode a protein with an NH2-terminal sequence identical to that of the TAP molecule. Further analysis of the predicted protein sequence revealed a cysteine-rich protein with a hydrophobic domain at the COOH terminus and without N-linked glycosylation sites--all features consistent with our previous analysis of the TAP protein. In Southern blot analysis, the Ly-6.2 cDNA clone detects a multigene family and a restriction fragment length polymorphism that maps precisely to the Ly-6 locus. Expression of the cDNA clone in COS cells demonstrates that it codes for TAP and clarifies the relationship between the epitopes recognized by various alpha Ly-6 monoclonal antibodies. Finally, we have studied the expression of Ly-6 mRNA in a variety of cell lineages. Ly-6 transcripts were detected in all organs examined, including spleen, kidney, lung, brain, and heart. This demonstrates that the Ly-6 locus is transcriptionally active in a wide range of organs and suggests that the role of TAP or TAP-like proteins might extend to other tissues. Images PMID:2895473

  11. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    PubMed

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  12. Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration.

    PubMed

    Lee, J S; Ray, R; Chien, C B

    2001-06-01

    We report the cloning and expression patterns of three novel zebrafish Roundabout homologs. The Roundabout (robo) gene encodes a transmembrane receptor that is essential for axon guidance in Drosophila and Robo family members have been implicated in cell migration. Analysis of extracellular domains and conserved cytoplasmic motifs shows that zebrafish Robo1 and Robo2 are orthologs of mammalian Robo1 and Robo2, respectively, while zebrafish Robo3 is likely to be an ortholog of mouse Rig-1. The three zebrafish robos are expressed in distinct but overlapping patterns during embryogenesis. They are highly expressed in the developing nervous system, including the olfactory system, visual system, hindbrain, cranial ganglia, spinal cord, and posterior lateral line primordium. They are also expressed in several nonneuronal tissues, including somites and fin buds. The timing and patterns of expression suggest roles for zebrafish robos in axon guidance and cell migration. Wiley-Liss, Inc.

  13. Molecular cloning, expression, and regulation of the ovalbumin gene in pigeon oviduct epithelial cells.

    PubMed

    Zhang, H; Lu, L Z; Chen, L; Tao, Z R; Chen, F; Zhong, S L; Liu, Y L; Tian, Y; Yan, P S

    2014-01-10

    The full-length pigeon ovalbumin (OVA) gene cDNA was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends. A 386-amino acid protein was predicted for the obtained sequence, which had 67% identity with the chicken protein. Similar to chicken OVA, the pigeon OVA gene is a non-inhibitory serine protease inhibitor. Quantitative PCR analysis revealed that pigeon OVA mRNA was highly expressed in the oviduct, and trace amounts were detected in other tissues. During the reproductive cycle, pigeon oviduct OVA mRNA expression reached its peak during the egg-laying stage, decreased with brooding, and then increased again during the squab-feeding period. Moreover, the relative OVA expression level in pigeon oviduct epithelial cells could be upregulated by a constant concentration of steroid hormones.

  14. 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.

  15. 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.

  16. Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

    PubMed Central

    Reed, K E; Westphale, E M; Larson, D M; Wang, H Z; Veenstra, R D; Beyer, E C

    1993-01-01

    Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins. Images PMID:7680674

  17. Characterisation of connexin expression and electrophysiological properties in stable clones of the HL-1 myocyte cell line.

    PubMed

    Dias, Priyanthi; Desplantez, Thomas; El-Harasis, Majd A; Chowdhury, Rasheda A; Ullrich, Nina D; Cabestrero de Diego, Alberto; Peters, Nicholas S; Severs, Nicholas J; MacLeod, Kenneth T; Dupont, Emmanuel

    2014-01-01

    The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼ 80%) compared to inhibition of the sodium current by lidocaine (∼ 20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.

  18. Molecular cloning, expression and characterization of programmed cell death 10 from sheep (Ovis aries).

    PubMed

    Yang, Yong-Jie; Liu, Zeng-Shan; Lu, Shi-Ying; Li, Chuang; Hu, Pan; Li, Yan-Song; Liu, Nan-Nan; Tang, Feng; Xu, Yun-Ming; Zhang, Jun-Hui; Li, Zhao-Hui; Feng, Xiao-Li; Zhou, Yu; Ren, Hong-Lin

    2015-03-01

    Programmed cell death 10 (PDCD10) is a highly conserved adaptor protein. Its mutations result in cerebral cavernous malformations (CCMs). In this study, PDCD10 cDNA from the buffy coat of Small Tail Han sheep (Ovis aries) was cloned from a suppression subtractive hybridization cDNA library, named OaPDCD10. The full-length cDNA of OaPDCD10 was 1343bp with a 639bp open reading frame (ORF) encoding 212 amino acid residues. Tissue distribution of OaPDCD10 mRNA determined that it was ubiquitously expressed in all tested tissue samples, and the highest expression was observed in the heart. The differential expression of OaPDCD10 between infected sheep (challenged with Brucella melitensis) and vaccinated sheep (vaccinated with Brucella suis S2) was also investigated. The results revealed that, compared to the control group, the expression of OaPDCD10 from infected and vaccinated sheep was both significantly up-regulated (p<0.05). Moreover, the expression levels of OaPDCD10 from the vaccinated sheep were significantly higher than the infected sheep (p<0.05) after 30days post-inoculation. The recombinant OaPDCD10 (rOaPDCD10) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rOaPDCD10 protein was demonstrated to induce apoptosis and promote cell proliferation. Our studies are intended to discover potential diagnostic biomarkers of brucellosis to discern infected sheep from vaccinated sheep, and OaPDCD10 could be considered as a potential diagnostic biomarker of brucellosis.

  19. Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA.

    PubMed

    De Marzí, Mauricio C; Fernández, Marisa M; Sundberg, Eric J; Molinero, Luciana; Zwirner, Norberto W; Llera, Andrea S; Mariuzza, Roy A; Malchiodi, Emilio L

    2004-10-01

    Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.

  20. Molecular cloning and expression in mammalian cells of ricin B chain

    SciTech Connect

    Chang, M.

    1987-01-01

    In these studies, the cDNA encoding the B chain of ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with {sup 35}S-methionine and {sup 35}S-cysteine and demonstrating secretion of a protein with a Mr of 30-32,000 which was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B chain antibody. The amount of recombinant B chain secreted by the COS-M6 cells was determined by radioimmunoassay to be 1-10 ng/ml of media. Virtually all the recombinant B chain formed active ricin when mixed with native A chain; it could also bind as effectively as native B chain to the galactose-containing glycoprotein, asialofetuin. These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function.

  1. Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope

    PubMed Central

    1996-01-01

    Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535- 551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO

  2. 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

  3. 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.

  4. High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

    PubMed

    DiMaio, D; Corbin, V; Sibley, E; Maniatis, T

    1984-02-01

    A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

  5. Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

    PubMed

    Mima, H; Morikawa, H; Fukuda, K; Kato, S; Shoda, T; Mori, K

    1997-12-11

    Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

  6. Restoration of Mitochondrial Gene Expression Using a Cloned Human Gene in Chinese Hamster Lung Cell Mutant

    PubMed Central

    Sherif, Zaki A; Broome, Carolyn W

    2015-01-01

    Background Gal−32 is a Chinese hamster lung cell nuclear mutant that is unable to grow in galactose due to a defect in mitochondrial protein synthesis. Since the product of the Gal−32 gene was unknown, it was imperative to use phenotypic complementation to clone a human gene that corrected the Gal−32 mutation. Results Recessive Gal−32 cells were co-transformed with pSV2-neo plasmid DNA and recombinant DNA from a human genomic library containing the dominant human Gal+ gene and a chloramphenicol-resistance (camr) gene present in the pSV13 vector. Primary transformants were selected by growth in galactose and the neomycin analog G418. In order to rescue the human Gal+ gene, a genomic library was constructed with primary transformant DNA and the pCV108 cosmid vector. The camr gene was used to identify clones with the nearby human sequences. DNA from two camr, Alu-hybridizing clones was able to transform the recessive Gal−32 cells to the Gal+ phenotype and to restore mitochondrial protein synthesis. Conclusion These data demonstrate the isolation of two pCV108-transformant recombinant clones containing a human gene that complements the Chinese hamster Gal−32 mutation and restores galactose metabolism. PMID:26052559

  7. 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.

  8. Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans.

    PubMed Central

    Birse, C E; Irwin, M Y; Fonzi, W A; Sypherd, P S

    1993-01-01

    The gene ECE1 (extent of cell elongation 1) was isolated by differential hybridization screening of a Candida albicans cDNA library by using probes derived from populations of yeast cells or hyphae. Expression of this gene was not detected when C. albicans grew as a budding yeast cell but was observed within 30 min after cells had been induced to form hyphae. In all strains tested, regardless of the induction signal, ECE1 expression correlated with the extent of cell elongation. The genomic version of ECE1 was cloned and sequenced. The deduced 271-amino-acid polypeptide consisted of eight tandem repeats of a degenerate 34-amino-acid sequence which contained no discernible homology with other known sequences. An ECE1 null mutant displayed no morphological alterations, which demonstrated that ECE1 is not essential for cell elongation or hypha formation despite the strict morphological association of its expression. Images PMID:8359888

  9. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells.

    PubMed

    Diepenbruck, Carolin; Klinger, Matthias; Urbig, Thomas; Baeuerle, Patrick; Neef, Rüdiger

    2013-06-01

    Selection of lead candidates in drug discovery is a complex and time-consuming process. Here, we describe an approach that allows prediction of the productivity and quality of recombinant proteins by stable producer cell clones with the help of transient transfection studies. This is exemplified for three distinct bispecific T cell engager (BiTE(®))-a new class of single-chain antibody-based therapeutics showing very promising results in the treatment of cancer. BiTE(®) titers of transiently transfected HEK cells showed a striking correlation with titers of selected stable CHO cell clones. Likewise, the percentage of the monomeric BiTE(®) fraction in cell culture supernatants correlated well between transiently expressing HEK and stably expressing CHO cell clones. This validates the use of transient transfection studies for the selection of biopharmaceutical lead candidates with desired pharmaceutical properties.

  10. Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

    PubMed

    Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

    1986-05-01

    Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

  11. Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

    PubMed Central

    Dron, M; Modjtahedi, N; Brison, O; Tovey, M G

    1986-01-01

    Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells. Images PMID:3785169

  12. Expression Cloning and Production of Human Heavy-Chain-Only Antibodies from Murine Transgenic Plasma Cells

    PubMed Central

    Drabek, Dubravka; Janssens, Rick; de Boer, Ernie; Rademaker, Rik; Kloess, Johannes; Skehel, John; Grosveld, Frank

    2016-01-01

    Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH–VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs. PMID:28066429

  13. Functional expression in primate cells of cloned DNA coding for the hemagglutinin surface glycoprotein of influenza virus.

    PubMed Central

    Sveda, M M; Lai, C J

    1981-01-01

    We have used simian virus 40 (SV40) DNA as a vector for expression of functional activity of a cloned influenza viral DNA segment in primate cells. Cloned full-length DNA sequences coding for the hemagglutinin of influenza A virus (Udorn/72/[H3N2]) were inserted into the late region of a viable deletion mutant of SV40, and the hybrid DNA was propagated in the presence of an early SV40 mutant (tsA28) helper. Infection of primate cells with the hybrid virus produced a polypeptide similar in molecular size to the hemagglutinin of influenza virus, as shown by immunoprecipitation and gel electrophoresis. The polypeptide was glycosylated, as shown by incorporation of radioactive sugars. The putative hemagglutinin exhibited functional activity, as shown by agglutination of erythrocytes. In addition, an indirect immunofluorescence assay showed that the hemagglutinin polypeptide of the hybrid virus could be detected on the surface of infected cells. Images PMID:6272305

  14. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    PubMed Central

    Zhu, Ge-Jian; Yu, Ying-Nian; Li, Xin; Qian, Yu-Li

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography (HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild typeCYP2C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 ± 0.109 μmol•min-1 ·g-1 S9 protein or 8.62 ± 2.02 mol•min-1 ·mol-1 CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL-CYP2C9, efficiently expressing the protein of CYP2C9, was established. PMID:11925616

  15. 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.

  16. High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells.

    PubMed Central

    Boulerice, F; Bour, S; Geleziunas, R; Lvovich, A; Wainberg, M A

    1990-01-01

    Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone. Images PMID:1690823

  17. 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.

  18. Regulation of cytokine expression by an autoreactive B cell clone derived from MRL/MP-lpr/lpr mice

    PubMed Central

    Iwasaki, T; Hamano, T; Fujimoto, J; Ogata, A; Kakishita, E

    1998-01-01

    The B cell line, MRL159.5, was established by somatic hybridization between splenic MRL/MP-lpr/lpr (lpr) mice B cells and 2.52M, a hypoxanthine-aminopterine-thymidine (HAT) medium-sensitive B cell line mutant. It possessed a receptor molecule for mouse erythrocytes treated with bromelain (Br-MRBC) on its surface, likely to be an autoreactive B cell clone specific for Br-MRBC as detected by rosette-forming assay with Br-MRBC. MRL159.5 spontaneously produced IL-6 and secreted IgM, and was induced to augment IgM secretion when treated with Br-MRBC or IL-6. Triggering of CD40 led to an augmentation of IgM secretion as well as IL-6 expression. Blocking the binding of IL-6 to its cellular receptor through the use of inhibitory antibodies inhibited CD40-induced IgM secretion, suggesting a possible autocrine role of IL-6 for CD40-induced differentiation of this B cell hybridoma. Addition of IL-4 or Br-MRBC augmented IL-6 expression as well as IgM secretion by CD40-activated MRL159.5 cells. CD40 also augmented tumour necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in decreased IL-10 expression. Furthermore, under conditions where IL-6 expression was augmented, IL-6Rα (gp80) expression was down-regulated, suggesting a negative feedback mechanism of an IL-6 autocrine loop in this hybridoma. These results demonstrate a role by which T cell-dependent activation through CD40 regulates an IL-6 autocrine loop, controlling differentiation of autoreactive B cells in autoimmune disease. PMID:9764595

  19. [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.

  20. Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

    PubMed Central

    Muñoz, Rosario; García, José L.; Carrascosa, Alfonso V.; Gonzalez, Ramon

    2004-01-01

    Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy. PMID:15128507

  1. Cloning of the authentic bovine gene encoding pepsinogen a and its expression in microbial cells.

    PubMed

    Muñoz, Rosario; García, José L; Carrascosa, Alfonso V; Gonzalez, Ramon

    2004-05-01

    Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with alpha-, beta-, or kappa-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.

  2. Expression of the fetal Alz-50 clone 1 protein induces apoptotic cell death

    SciTech Connect

    Strachan, Gordon D.; Ostrow, Liya Avshalumov; Jordan-Sciutto, Kelly L. . E-mail: Jordan@path.dental.upenn.edu

    2005-10-21

    The fetal Alz-50 clone 1 (FAC1) protein exhibits altered expression patterns in neurodegenerative disease. Though it has been shown to bind DNA in a site-specific, phosphorylation-dependent manner, its cellular function remains unknown. Here, we demonstrate that overexpression of FAC1 in PT67 fibroblasts induces nuclear condensation and cleavage of caspase 3 to its active form indicating induction of apoptosis. The amino-terminal domain of FAC1 is necessary and sufficient to induce both nuclear condensation and activation of caspase 3. Disruption of FAC1 interaction with a known binding partner, kelch-like ECH-associated protein 1 (Keap1), enhances activation of caspase 3. Keap1 is known to block activation of the antioxidant response gene products by direct interaction with the transcriptional activator, Nrf2. Disruption of the Keap1:Nrf2 interaction enhances FAC1 induction of apoptosis. These findings suggest a role for FAC1 in apoptosis following release of Nrf2 from Keap1 in response to oxidative stress.

  3. Molecular cloning, expression, and regulation of estrogen receptors in pigeon oviduct epithelial cells.

    PubMed

    Zhang, H; Chen, F; Li, G L; Ding, Y Y; Tao, Z R; Li, J J; Zhong, S L; Lu, L Z

    2014-03-17

    Estrogen regulates reproductive behavior and drives the proliferation and differentiation of several cell types. These physiological functions of estrogen are mediated by estrogen receptors (ERs), and each ER isoform plays a distinct role. To clarify the molecular mechanism of estrogen action and to evaluate the effect of ERs on the secretion of ovalbumin (OVA) in pigeon oviduct epithelial cells (POECs), we determined the complete coding sequences encoding ER alpha (ERα) and ER beta (ERβ) in pigeons. The abundance of pigeon ERα and ERβ mRNA was detected using quantitative polymerase chain reaction. These results revealed that pigeon ERα is highly expressed in the oviduct, while pigeon ERb is highly expressed in the ovary and kidney. We hypothesize that ERα mRNA predominates over that of ERβ in the oviduct. The expression of ERα can be down-regulated by 17β-estradiol, and the knockdown of ERα promoted OVA mRNA expression in cultured POECs, indicating that ERα may play an important role in OVA secretion.

  4. Molecular cloning of cDNA for rat argininosuccinate lyase and its expression in rat hepatoma cell lines.

    PubMed Central

    Lambert, M A; Simard, L R; Ray, P N; McInnes, R R

    1986-01-01

    Using antibody and plaque hybridization screening, we isolated rat argininosuccinate lyase (AS lyase) cDNA clones from a liver cDNA library prepared in the phage expression vector lambda gt11. Five overlapping cDNAs covering 1.7 kilobases of the estimated 2.0-kilobase AS lyase mRNA were characterized and confirmed as AS lyase sequences by hybrid selection. We examined the differential expression of AS lyase in rat liver and four rat hepatoma cell lines (7800C1, H4, HTC, and MH1C1). These cells exhibited a 60-fold range of AS lyase enzyme activity, with a direct correlation between activity, amount of AS lyase immunoreactive protein, and quantity of specific AS lyase mRNA. These observations suggest that the differences in AS lyase expression between rat liver and the hepatoma cell lines result from variations in AS lyase transcriptional activity or alterations in nuclear processing of AS lyase RNA. Images PMID:3785176

  5. [Cloning of 5', 3' flanking sequence of ovine BLG and regulating the expression of GFP in mammary gland cell line].

    PubMed

    Liu, Ming-Jun; Li, Wen-Rong; Wu, Jian; Huang, Jun-Cheng; Guo, Zhi-Qin; Qu, Xin-Yong; Paul, Kroon

    2002-01-01

    5' and 3' flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently. By partially sequencing, the sequences of BLG 5' and 3' flanking were the same as that of publication completely. The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2 kb 5' flanking with 2.1 kb 3' flanking. In order to assess the efficiency of BLG regulatory elements, green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47). Through observation under UV microscope and detection by fluorometer, it is demonstrated that the GFP has been successfully expressed in TD47 cell line. By virtue of direct observation and quantitative analysis, the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.

  6. 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

  7. Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells.

    PubMed

    Han, B H; Park, D J; Lim, R W; Im, J H; Kim, H D

    1998-05-19

    We report the molecular cloning of a novel guanylate-binding protein (GBP), termed mouse GBP3 (mGBP3) in Friend virus-induced mouse erythroid progenitor (FVA) cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGK(S/T) and DXXG, but lack the third element, (N/T)KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides (GTP, GDP and GMP). Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K(m) and Vmax values of 77 +/- 4 microM and 21 +/- 0.5 pmol min-1 microgram-1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylation/methylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number U44731.

  8. Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage.

    PubMed

    Yoshizaki, G; Sakatani, S; Tominaga, H; Takeuchi, T

    2000-04-01

    The origin of germ cells and the molecular mechanisms of primordial germ cell (PGC) determination in teleosts are unclear. Vasa is a member of the DEAD protein family and plays an indispensable role in germ cell determination in Drosophila and Xenopus species. In this study, we isolated and characterized a rainbow trout vasa cDNA as a first step towards understanding the molecular mechanisms of PGC determination and development and to develop a molecular marker to identify the PGCs in rainbow trout. Cloning of vasa cDNA was performed by degenerate- and RACE-PCR. The predicted amino acid sequence of rainbow trout Vasa contained eight consensus sequences for the DEAD protein family and five arginine-glycine-glycine repeats, a common character of known Vasa homologues. Overall amino acid similarity to the Vasa of Drosophila was 79.2%. Whole-mount in situ hybridization of eyed stage embryos (eighty somite stage) revealed that signals were localized to the putative PGCs. In adult rainbow trout tissues, both ovaries and testes contained large amounts of vasa gene transcripts. A reverse transcription-polymerase chain reaction analysis of unfertilized eggs proved that trout vasa is a maternal factor. Although we have not determined whether rainbow trout vasa functions as a germ cell determinant, its limited expression in the germ cell lineage proved that rainbow trout vasa can be used as a marker molecule for PGCs. This marker will make it possible to identify the PGCs or presumptive PGCs in early trout embryos whose germ cells can not be distinguished by morphological characteristics.

  9. Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.

    PubMed

    Uchida, D; Kawamata, H; Omotehara, F; Miwa, Y; Hino, S; Begum, N M; Yoshida, H; Sato, M

    2000-06-01

    We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.

  10. 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.

  11. 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 Central

    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.

    2015-01-01

    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 1H-15N 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

  12. 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

  13. Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase.

    PubMed

    Ito, Yasuko; Tomita, Toshio; Roy, Narayan; Nakano, Akito; Sugawara-Tomita, Noriko; Watanabe, Seiji; Okai, Naoko; Abe, Naoki; Kamio, Yoshiyuki

    2003-12-01

    We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.

  14. 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

  15. Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

    PubMed Central

    Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

    2015-01-01

    Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

  16. Functional consequences of anti-sense RNA-mediated inhibition of CD8 surface expression in a human T cell clone

    PubMed Central

    1988-01-01

    An experimental approach for defining the function of CD8 has been developed by linking anti-sense RNA mutagenesis and T cell cloning technologies. We have transfected an anti-sense CD8 episomal expression vector into a CD8+ nontransformed human T cell clone that is specific for the human class I alloantigen HLA-B35. Expression of CD8 on this T cell clone, JH.ARL.1, was selectively and efficiently inhibited. Stimulation of this CD8- variant with specific alloantigen resulted in a marked loss of a number of functional responses, including cytotoxicity, proliferation, IL-2 secretion, and IL-2-R expression. However, these same functional responses could be elicited with stimuli that do not require antigen recognition to activate the T cell (anti- CD3 mAbs, PHA). The results of our study support the hypothesis that CD8 is required for recognition of class I MHC alloantigens that results in activation of T cell functional responses. PMID:2459296

  17. High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system.

    PubMed

    Hou, Jeff Jia Cheng; Hughes, Ben S; Smede, Matthew; Leung, Kar Man; Levine, Kara; Rigby, Susan; Gray, Peter P; Munro, Trent P

    2014-05-25

    Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

  18. 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.

  19. 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 ...

  20. Method for cloning lymphoblastoid cells

    SciTech Connect

    Hammerling, U.; Kosinski, S.

    1989-02-14

    A method is described for increasing cloning frequency of human lymphocyte or lumphoblastoid cells which have been transformed with Epstein Barr virus comprising growing the transformed cells in a semi-solid agarose medium. A lower and an upper layer of agarose are used, the lower layer comprising fibroblasts suspended in the agarose layer and the upper layer comprising irradiated fibroblasts and the transformed cells suspended in the agarose layer wherein the upper agarose layer is added after the lower layer has gelled.

  1. Expression Cloning of the High Affinity Choline Transporter

    DTIC Science & Technology

    1993-05-05

    clones. It encodes a GABA transporter that we found to be localized to the glial cells of the purely cholinergic electromotor nucleus of Torpedo. In a...expression cloning approach employing frog oocytes and mRNA from Torpedo C.B. Gundersen electromotor nucleus to isolate a choline transporter cDNA...The rationale for this is that the electromotor neurons should harbor one of the highest abundances of choline transporter mRNA in the animal kingdom

  2. Cloning, Expression, Invasion, and Immunological Reactivity of a Mammalian Cell Entry Protein Encoded by the mce1 Operon of Nocardia farcinica

    PubMed Central

    Ji, Xingzhao; Tan, Xiaoluo; Hou, Xuexin; Si, Chenchen; Xu, Shuai; Tang, Lu; Yuan, Xiuqin; Li, Zhenjun

    2017-01-01

    Bacterial mammalian cell entry (Mce) proteins have been implicated in pathogen invasion of mammalian host cells. The aim of this study was to examine the invasion-conferring ability of mce1E operon-encoded proteins, in vivo expression of Mce1E in cells from infected mice and rabbits, and Mce1E immunogenicity. Nocardia farcinica mce1E was cloned into pet30a(+) vectors, expressed in Escherichia coli, and purified. Invasion assays, transmission electron microscopy (TEM), immunoblots, and enzyme-linked immunosorbent assay (ELISA) detection of cytokines were conducted. TEM confirmed the invasion of HeLa cells by Mce1E-coated beads. The antigenicity of E. coli-expressed recombinant Mce1E was confirmed in immunoblots with sera from N. farcinica-infected mouse and rabbit sera. Co-incubation of Mce1E with splenocytes of N. farcinica-infected mice demonstrated upregulation of interferon (IFN-γ), but not interleukin (IL)-4 or IL-10, in the cultural supernatant. These findings demonstrate that Mce1E may facilitate N. farcinica interactions with and invasion of mammalian cells. Notably, Mce1E are expressed and elicited antibody responses in mice and rabbits during infection. Besides, it may play a role in cell-mediated immune reactions and cause host inflammation responses to N. farcinica infection. PMID:28275374

  3. Expression systems for cloned xenobiotic transporters

    SciTech Connect

    Pritchard, John B.

    2005-05-01

    One challenge of modern biology is to be able to match genes and their encoded proteins with events at the molecular, cellular, tissue, and organism levels, and thus, provide a multi-level understanding of gene function and dysfunction. How well this can be done for xenobiotic transporters depends on a knowledge of the genes expressed in the tissue, the cellular locations of the gene products (do they function for uptake or efflux?), and our ability to match substrates with transporters using information obtained from cloned transporters functioning in heterologous expression systems. Clearly, making a rational choice of expression system to use for the characterization and study of cloned xenobiotic transporters is a critical part of study design. This choice requires well-defined goals, as well as an understanding of the strengths and weaknesses of candidate expression systems.

  4. Cloning an expressed gene shared by the human sex chromosomes

    SciTech Connect

    Darling, S.M.; Banting, G.S.; Pym, B.; Wolfe, J.; Goodfellow, P.N.

    1986-01-01

    The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage lambdagt11 expression system in Escherichia coli and immunoscreening techniques, the authors have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. The authors conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical.

  5. Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr.

    PubMed

    Jahns, R; Borgese, F; Lindenthal, S; Straub, A; Motais, R; Fiévet, B

    1996-06-01

    Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

  6. Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2)

    PubMed Central

    1992-01-01

    This study was designed to identify the target molecules of the natural killer (NK) cell-mediated recognition of normal allogeneic target cells. As previously shown, the gene(s) governing the first NK-defined allospecificity (specificity 1) were found to be localized in the major histocompatibility complex region between BF gene and HLA-A. In addition, the analysis of a previously described family revealed that a donor (donor 81) was heterozygous for three distinct NK-defined allospecificities (specificities 1, 2, and 5). HLA variants were derived from the B-Epstein-Barr virus cell line of donor 81 by gamma irradiation followed by negative selection using monoclonal antibodies specific for the appropriate HLA allele. Several variants were derived that lacked one or more class I antigen expressions. These variants were analyzed for the susceptibility to lysis by NK clones recognizing different allospecificities. The loss of HLA-A did not modify the phenotype (i.e., "resistance to lysis"). On the other hand, a variant lacking expression of all class I antigens became susceptible to lysis by all alloreactive clones. Variants characterized by the selective loss of class I antigens coded for by the maternal chromosome became susceptible to lysis by anti-2-specific clones. Conversely, variants selectively lacking class I antigens coded for by paternal chromosome became susceptible to lysis by anti-1 and anti-5 clones (but not by anti-2 clones). Since the Cw3 allele was lost in the variant that acquired susceptibility to lysis by anti-2 clones and, in informative families, it was found to cosegregate with the character "resistance to lysis" by anti-2 clones, we analyzed whether Cw3 could represent the element conferring selective resistance to lysis by anti-2 clones. To this end, murine P815 cells transfected with HLA Cw3 (or with other HLA class I genes) were used as target cells in a cytolytic assay in which effector cells were represented by alloreactive NK clones

  7. Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Kolodrubetz, D; Dailey, T; Ebersole, J; Kraig, E

    1989-01-01

    The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of juvenile periodontitis. To initiate a genetic analysis of the role of this protein in disease, we have cloned the leukotoxin gene in Escherichia coli. Recombinant colonies carrying toxin gene sequences were isolated by screening a genomic A. actinomycetemcomitans library with a DNA probe for the leukotoxin gene from a related bacterium, Pasteurella haemolytica. To demonstrate that the cloned A. actinomycetemcomitans DNA contained a functional leukotoxin gene, protein extracts of E. coli containing the A. actinomycetemcomitans clone were tested directly for leukotoxic activity against human cell lines in chromium release assays. A construct containing the entire cloned region produced a functional toxin. No cytotoxicity was seen when extracts from cells containing plasmids with deletions in the putative coding region were used. Furthermore, the toxin produced by the cloned gene has the same target cell specificity as the leukotoxin extracted directly from A. actinomycetemcomitans. These results indicate that sequences encoding a functional leukotoxin have been cloned and are expressed in E. coli. Southern blot analysis of DNA from leukotoxin-producing (Lkt+) and non-leukotoxin-producing (Lkt-) strains indicated that the Lkt- strain also contained a copy of the gene. Images PMID:2707855

  8. Cell wall invertase in developing rice caryopsis: molecular cloning of OsCIN1 and analysis of its expression in relation to its role in grain filling.

    PubMed

    Hirose, Tatsuro; Takano, Makoto; Terao, Tomio

    2002-04-01

    To establish the significance of cell wall invertase in grain filling of rice (Oryza sativa L.), we cloned a cDNA for a cell wall invertase from developing grains of rice. The cDNA, designated OsCIN1, contains an open reading frame of 1731 bp encoding a polypeptide of 577 amino acid residues. The deduced amino acid sequence showed typical features of the cell wall invertases, including a beta-fructosidase motif and a cysteine catalytic site, and shared 78.6 and 73.7% identity with maize cell wall invertases, Incw1 and Incw2, respectively. OsCIN1 is expressed in roots, in sink- and source-leaves, and in panicles. During the course of grain filling in the caryopses, OsCIN1 transcript is detectable only in the very early stage of their development, 1-4 d after flowering, when the cell wall invertase activity is the highest and the increase in caryopsis length is rapid. In situ localization of the mRNA revealed that OsCIN1 is expressed preferentially in the vascular parenchyma of the dorsal vein, integument and its surrounding cells, and is expressed weakly in the nucellar projection and nucellar epidermis. These results suggest that, during the early stage of caryopsis development, OsCIN1 is important for supplying a carbon source to developing filial tissues by cleaving unloaded sucrose in the apoplast.

  9. Cloning of Plasmodium falciparum by single-cell sorting.

    PubMed

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples.

  10. 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.

  11. Defective Chromatin Structure in Somatic Cell Cloned Mouse Embryos*

    PubMed Central

    Zhang, Miao; Wang, Fengchao; Kou, Zhaohui; Zhang, Yu; Gao, Shaorong

    2009-01-01

    Epigenetic reprogramming plays a central role in the development of cloned embryos generated by somatic cell nuclear transfer, and it is believed that aberrant reprogramming leads to the abnormal development of most cloned embryos. Recent studies show that trimethylation of H3K27 (H3K27me3) contributes to the maintenance of embryonic stem cell pluripotency because the differentiation genes are always occupied by nucleosomes trimethylated at H3K27, which represses gene expression. Here, we provide evidence that differential H3K27me3 modification exists between normal fertilization-produced blastocysts and somatic cell nuclear transfer cloned blastocysts; H3K27me3 was specifically found in cells of the inner cell mass (ICM) of normal blastocysts, whereas there was no modification of H3K27me3 in the ICM of cloned blastocysts. Subsequently, we demonstrated that the differentiation-related genes, which are marked by H3K27me3 in embryonic stem cells, were expressed at significantly higher levels in cloned embryos than in normal embryos. The polycomb repressive complex 2 (PRC2) component genes (Eed, Ezh2, and Suz12), which are responsible for the generation of H3K27me3, were expressed at lower levels in the cloned embryos. Our results suggest that reduced expression of PRC2 component genes in cloned embryos results in defective modification of H3K27me3 to the differentiation-related genes in pluripotent ICM cells. This results in premature expression of developmental genes and death of somatic cloned embryos shortly after implantation. Taken together, these studies suggest that H3K27me3 might be an important epigenetic marker with which to evaluate the developmental potential of cloned embryos. PMID:19602512

  12. Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

    DTIC Science & Technology

    2011-07-01

    Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries Daniel Lindner The Cleveland Clinic Cleveland, OH 44195 We have...MDS cell pools. This validation will ensure that the insert causes the desired phenotype. Myelodysplastic syndrome , lentivirus, cDNA libraries...A., I. Martin-Padura, P. Mancuso, P. Marighetti, C. Rabascio, G. Pruneri, L. D. Shultz, and F. Bertolini. 2008. Human acute leukemia cells injected

  13. 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.

  14. Liver-intestine cadherin: molecular cloning and characterization of a novel Ca(2+)-dependent cell adhesion molecule expressed in liver and intestine

    PubMed Central

    1994-01-01

    A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI- cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine. PMID:8207063

  15. Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA.

    PubMed

    Roth, M G; Compans, R W; Giusti, L; Davis, A R; Nayak, D P; Gething, M J; Sambrook, J

    1983-06-01

    Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

  16. Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

    PubMed

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM.

  17. Post-translational folding of the influenza C virus glycoprotein HEF: defective processing in cells expressing the cloned gene.

    PubMed

    Szepanski, S; Veit, M; Pleschka, S; Klenk, H D; Schmidt, M F; Herrler, G

    1994-05-01

    The post-translational processing of the influenza C virus glycoprotein HEF was analysed. In cells infected with influenza C virus, HEF protein is synthesized as a glycosylated 80K polypeptide. A post-translational conformational rearrangement involving the formation of intramolecular disulphide bonds results in a decrease in its electrophoretic mobility. Therefore, SDS-PAGE under non-reducing conditions suggests an Mr of about 100K, whereas under reducing conditions an 80K protein is observed which is in accordance with the sequence data. The 100K form was detected 10 min after synthesis of HEF, and transport to the cell surface took about 60 min. This result indicates that the conformational change presumably occurs in the endoplasmic reticulum. A difference in post-translational processing was observed when the HEF gene was expressed in the absence of other influenza C virus genes. In cells infected with recombinant simian virus 40, the 80K precursor was synthesized, but this protein was neither converted to the 100K form nor transported to the cell surface. Deletion of the short cytoplasmic tail of HEF (Arg-Thr-Lys) or replacement of the two basic amino acids by hydrophobic (Ile) or acidic residues (Glu) resulted in HEF protein which was partially converted to the 100K form. Influenza C virus glycoprotein obtained after transient expression of the HEF gene using the vaccinia virus system was completely converted to the 100K form. However, in neither expression system was HEF transported to the cell surface. The possibility is discussed that the interaction of HEF with another viral protein is required for the post-translational folding and transport of this glycoprotein. The M protein of influenza C virus is suggested as a candidate for the chaperone which might interact with the cytoplasmic tail of HEF.

  18. 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.

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

    PubMed

    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

    2014-05-01

    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.

  20. 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

  1. Food-grade cloning and expression system for Lactococcus lactis.

    PubMed Central

    Platteeuw, C; van Alen-Boerrigter, I; van Schalkwijk, S; de Vos, W M

    1996-01-01

    A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis. The expression vectors were equipped with the controlled and strong lacA promoter of the lactococcal lactose operon. In addition, the transcriptional terminator of the aminopeptidase N gene, pepN, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA. The small, 0.3-kb lacF gene encoding the soluble carrier enzyme IIALac was used as a dominant selection marker in the plasmid-free L. lactis strain NZ3000 carrying an in-frame deletion of the chromosomal lacF gene. Lactose-utilizing transformants were easily selected on lactose indicator plates at high frequencies and showed a copy number of approximately 50 plasmids per cell. All vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some instability when their host was grown on glucose-containing medium. The application potentials of the expression vectors carrying the lacF marker were determined by cloning of the promoterless Escherichia coli gusA reporter gene under control of the lacA promoter followed by analysis of its expression. While in one of the vectors this resulted in a promoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was observed. PMID:8975595

  2. 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.

  3. Cloning

    MedlinePlus

    ... that have been cloned from somatic cells include: cat, deer, dog, horse, mule, ox, rabbit and rat. ... with cell division. In other mammals, such as cats, rabbits and mice, the two spindle proteins are ...

  4. Cloning and characterization of 5E6(Ly-49C), a receptor molecule expressed on a subset of murine natural killer cells

    PubMed Central

    1995-01-01

    5E6 is a cell surface molecule expressed on a subpopulation of murine natural killer (NK) cells that are involved in the specific rejection of H-2d or H-2f (hemopoietic histocompatibility determinant 2) bone marrow cell grafts. Here, we isolated and cloned the gene encoding 5E6 and determined the nucleotide sequence of the cDNA. 5E6 is nearly identical to Ly-49C; the deduced amino acid sequence reveals a polypeptide of 266 amino acids with a molecular weight of 31,284 that contains multiple cysteine residues to explain its disulfide-linked homodimer structure and five potential N-linked glycosylation sites. 5E6 is a type II integral membrane protein with an extracellular carbohydrate recognition domain characteristic of C-type (Ca(2+)- dependent) animal lectins. Chromosomal mapping indicates that 5E6 is located within the NK gene complex on chromosome 6. The sequence of 5E6 mRNA and the degree of glycosylation of 5E6 protein are under genetic control. Immunoprecipitation before removal of N-linked sugars reveals different size molecules. There are several nucleotide differences among BALB/c, B6, and NZB mRNAs; however, none of them would be expected to affect N-glycosylation. Of particular interest are two findings: (a) BALB/c, B6, and (BALB/c x B6)F1 5E6 reduced molecules are approximately 65, 54, and 54 kD, and (b) the cDNA sequence of (BALB/c x B6)F1 is identical to B6. Thus, there appears to be allelic exclusion of 5E6 expression that may be related to the ability of F1 hybrid mice to reject parental H-2d bone marrow cell grafts. PMID:7629496

  5. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

    PubMed

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

    2016-10-01

    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  6. Generation of cloned transgenic cats expressing red fluorescence protein.

    PubMed

    Yin, Xi Jun; Lee, Hyo Sang; Yu, Xian Feng; Choi, Eugene; Koo, Bon Chul; Kwon, Mo Sun; Lee, Young S; Cho, Su Jin; Jin, Guang Zhen; Kim, Lyoung Hyo; Shin, Hyoung Doo; Kim, Teoan; Kim, Nam Hyung; Kong, Il Keun

    2008-03-01

    A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

  7. Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases.

    PubMed

    Liu, Benye; Falkenstein-Paul, Hildegard; Schmidt, Werner; Beerhues, Ludger

    2003-06-01

    Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.

  8. Continuous expression and replication of the hepatitis delta virus genome in Hep G2 hepatoblastoma cells transfected with cloned viral DNA.

    PubMed Central

    Chen, P J; Kuo, M Y; Chen, M L; Tu, S J; Chiu, M N; Wu, H L; Hsu, H C; Chen, D S

    1990-01-01

    To establish stable cell clones allowing continuous replication of hepatitis delta virus (HDV), Hep G2, a hepatoblastoma cell line containing no hepatitis B virus (HBV) DNA sequences, was transfected with a recombinant plasmid containing a tandem trimer of HDV cDNA (driven by the simian virus 40 late promoter) and a neomycin-resistance gene. After selection with the neomycin analogue G418, at least two of the resistant clones were shown to have intact delta antigen by specific immunoblotting, and the delta antigen was located in the cell nucleus by immunofluorescence. Transfected cloned viral DNAs were found to be integrated into cell chromosomes. Replication of the HDV genome was demonstrated by the presence of not only genomic and antigenomic HDV RNAs but also HDV RNAs in multimeric and circular forms. In addition, a 0.8-kilobase antigenomic RNA containing a poly(A) tail and encoding the delta-antigen open reading frame was documented. Continuous replication and transcription of the HDV genome was thus achieved in these transfected cell lines. The results confirmed that replication of HDV was unassisted by HBV. Stable passage of such cell lines strongly suggests that HDV lacks direct cytopathicity in hepatocytes. These clones should be useful in studying the details of the HDV life cycle and the relationship between HDV and its helper virus, HBV. Images PMID:2164671

  9. Transfer and expression of three cloned human non-HLA-A,B,C class I major histocompatibility complex genes in mutant lymphoblastoid cells.

    PubMed Central

    Shimizu, Y; Geraghty, D E; Koller, B H; Orr, H T; DeMars, R

    1988-01-01

    The HLA-A, -B, and -C class I human histocompatibility antigens and the genes that encode them have been isolated and characterized. Apparently complete class I non-HLA-A, B, C genes have been identified on HindIII-generated 5.4-kilobase (kb), 6.0-kb, and 6.2-kb DNA fragments derived from lymphoblastoid cell line (LCL) 721. We studied the expressibility of these genes by subcloning them into the nonintegrating pHeBo vector and transferring the chimeric plasmids into mutant LCL 721.221. This mutant was derived from LCL 721 by means of immunoselections following gamma-ray mutagenesis that eliminated expressions of the HLA-A, -B, and -C alpha chains. The HLA-A, B, C-null phenotype of mutant 721.221 made it possible to monitor the expression of class I genes transferred into it by assaying cell surface binding of monoclonal antibodies BBM.1 and W6/32, which recognize beta 2-microglobulin and HLA class I alpha-chain epitopes, respectively. Increased binding of BBM.1 and W6/32 was clearly observed in transferents containing the class I gene of the 6.0-kb DNA fragment but not in transferents containing the class I genes of the 5.4- and 6.2-kb DNA fragments. However, one-dimensional gel electrophoresis of BBM.1 and W6/32 immunoprecipitates made with [35S]methionine-labeled cell lysates showed that transfer of each non-HLA-A, B, C class I gene into 721.221 resulted in the appearance of an alpha chain that coprecipitated with beta 2-microglobulin. The three previously unreported alpha chains differed from each other in size and were smaller than HLA-A, -B, and -C alpha chains. These observations clearly show that these three cloned, nonallelic, non-HLA-A, B, C class I genes encode alpha chains that can be expressed in human cells. Images PMID:3257565

  10. Aberrant gene expression in organs of bovine clones that die within two days after birth.

    PubMed

    Li, Shijie; Li, Yanxin; Du, Weihua; Zhang, Lei; Yu, Shuyang; Dai, Yunping; Zhao, Chunjiang; Li, Ning

    2005-02-01

    Cloning by somatic nuclear transfer is an inefficient process in which some of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we used real-time quantitative reverse transcription-polymerase chain reaction to examine expression patterns of eight developmentally important genes (PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF) in six organs (heart, liver, spleen, lung, kidney, and brain) of both cloned bovines that died soon after birth (n=9) and normal control calves produced by artificial insemination. In somatic cloning of cattle, fibroblasts have often been used for doner nuclei, and the effect of the age of the fibroblast donor cells on gene expression profiles was investigated. Aberrant expressions of seven genes were found in these clones. The majority of aberrantly expressed genes were common in clones derived from adult fibroblast (AF) and in clones derived from fetal fibroblast (FF) compared to controls, whereas some genes were dysregulated either in AF cell-derived or in FF cell-derived clones. For the studied genes, kidney was the organ least affected by gene dysregulation, and heart was the organ most affected, in which five genes were aberrant. Most dysregulations (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and Hsp70.1 were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation. Our results suggest that the aberrant gene expression occurred in most tissues of cloned bovines that died soon after birth. For each specific gene, aberrant expression resulting from nuclear transfer was tissue-specific. Because these genes play important roles in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

  11. Expression of the nfa1 gene cloned from pathogenic Naegleria fowleri in nonpathogenic N. gruberi enhances cytotoxicity against CHO target cells in vitro.

    PubMed

    Jeong, Seok-Ryoul; Lee, Sang-Chul; Song, Kyoung-Ju; Park, Sun; Kim, Kyongmin; Kwon, Myung-Hee; Im, Kyung-Il; Shin, Ho-Joon

    2005-07-01

    The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5' upstream regions, the nfa1 open reading frame, and 3' downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

  12. Notch signalling inhibits the adipogenic differentiation of single-cell-derived mesenchymal stem cell clones isolated from human adipose tissue.

    PubMed

    Osathanon, Thanaphum; Subbalekha, Keskanya; Sastravaha, Panunn; Pavasant, Prasit

    2012-01-01

    ADSCs (adipose-derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue-derived single-cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose-derived single-cell-clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft-tissue augmentation application.

  13. Endangered wolves cloned from adult somatic cells.

    PubMed

    Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hwang, Woo Suk; Hossein, Mohammad Shamim; Kim, Joung Joo; Shin, Nam Shik; Kang, Sung Keun; Lee, Byeong Chun

    2007-01-01

    Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

  14. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC).

    PubMed

    Camilo, Cesar M; Polikarpov, Igor

    2014-07-01

    Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coli, when compared to His-tag alone.

  15. Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos.

    PubMed

    Saini, M; Selokar, N L; Agrawal, H; Singla, S K; Chauhan, M S; Manik, R S; Palta, P

    2016-04-01

    We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

  16. Dogs cloned from adult somatic cells.

    PubMed

    Lee, Byeong Chun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hossein, M Shamim; Shamim, M Hossein; Kim, Jung Ju; Kang, Sung Keun; Schatten, Gerald; Hwang, Woo Suk

    2005-08-04

    Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

  17. Mouse ornithine decarboxylase gene: cloning, structure, and expression.

    PubMed Central

    Brabant, M; McConlogue, L; van Daalen Wetters, T; Coffino, P

    1988-01-01

    We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression. Images PMID:3353375

  18. 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.

  19. Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

    PubMed Central

    Tse, C M; Ma, A I; Yang, V W; Watson, A J; Levine, S; Montrose, M H; Potter, J; Sardet, C; Pouyssegur, J; Donowitz, M

    1991-01-01

    A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger. Images PMID:1712287

  20. Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

    PubMed

    Lace, Michael J; Anson, James R; Klussmann, Jens P; Wang, Dong Hong; Smith, Elaine M; Haugen, Thomas H; Turek, Lubomir P

    2011-02-01

    Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

  1. Cloning of Mammary Stem Cells

    DTIC Science & Technology

    2001-11-01

    these parity-induced cells do represent a totipotent mammary stem cell population per se, but these cells might support stem cell maintenance as... Stem Cells PRINCIPAL INVESTIGATOR: Dr. Kay-Uwe Wagner CONTRACTING ORGANIZATION: University of Nebraska Medical Center Omaha, Nebraska 68198-6810 REPORT...Mammary Stem Cells DAMD17-00-1-0641 6. AUTHOR(S) Dr. Kay-Uwe Wagner 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT

  2. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    PubMed

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  3. Cloning and expression of bovine glucose transporter GLUT12.

    PubMed

    Miller, Peter J; Finucane, Kiera A; Hughes, Megan; Zhao, Feng-Qi

    2005-11-01

    GLUT12 is a new member of facilitative glucose transporters. It was originally cloned from a human breast cancer cell line and its expression has been detected in rat mammary gland. Glucose transport across the plasma membrane of mammary epithelial cells is a rate-limiting factor in milk production. To examine GLUT12's expression and facilitate the study of GLUT12's potential role in supporting milk synthesis in lactating bovine mammary gland, we cloned bovine GLUT12 and examined its distribution of mRNA expression in bovine tissues. The full-length mRNA of bGLUT12 is 2,423 base pairs long and is predicted to encode a protein of 621 amino acids with a molecular weight of approximately 67 kDa. The deduced amino acid sequence of bovine GLUT12 is 87% and 82% identical to the sequences of human and mouse GLUT12. The sequence of bGLUT12 contains several characteristically conserved sugar transporter family signatures. Analysis of current bovine genomic data indicates that bovine GLUT12 gene consists of five exons. The major in vitro transcription and translation product of bovine GLUT12 cDNA migrated at an apparent molecular weight of 41 kDa. In the presence of canine microsomal membranes, the translation product increased to 43 kDa, suggesting glycosylation. GLUT12 mRNA was found in all bovine tissues examined, but most abundant in bovine spleen and skeletal muscle, at intermediate levels in bovine kidney, testes, and mammary gland, and at lower levels in bovine liver, lung and intestine. Immunofluorescence staining showed that, in the presence of insulin, bGLUT12 is mainly distributed in the cytoplasm of the transiently transfected MAC-T bovine mammary epithelial cells.

  4. Human somatic cell nuclear transfer and cloning.

    PubMed

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6.

  5. A transgenic-cloned pig model expressing non-fluorescent modified Plum

    PubMed Central

    NAGAYA, Masaki; WATANABE, Masahito; KOBAYASHI, Mirina; NAKANO, Kazuaki; ARAI, Yoshikazu; ASANO, Yoshinori; TAKEISHI, Toki; UMEKI, Ikuma; FUKUDA, Tooru; YASHIMA, Sayaka; TAKAYANAGI, Shuko; WATANABE, Nobuyuki; ONODERA, Masafumi; MATSUNARI, Hitomi; UMEYAMA, Kazuhiro; NAGASHIMA, Hiroshi

    2016-01-01

    Genetically modified pigs that express fluorescent proteins such as green and red fluorescent proteins have become indispensable biomedical research tools in recent years. Cell or tissue transplantation studies using fluorescent markers should be conducted, wherein the xeno-antigenicity of the fluorescent proteins does not affect engraftment or graft survival. Thus, we aimed to create a transgenic (Tg)-cloned pig that was immunologically tolerant to fluorescent protein antigens. In the present study, we generated a Tg-cloned pig harboring a derivative of Plum modified by a single amino acid substitution in the chromophore. The cells and tissues of this Tg-cloned pig expressing the modified Plum (mPlum) did not fluoresce. However, western blot and immunohistochemistry analyses clearly showed that the mPlum had the same antigenicity as Plum. Thus, we have obtained primary proof of principle for creating a cloned pig that is immunologically tolerant to fluorescent protein antigens. PMID:27396383

  6. 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

  7. Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones.

    PubMed

    Drakulic, D; Krstic, A; Stevanovic, M

    2012-05-15

    SOX2, a universal marker of pluripotent stem cells, is a transcription factor that helps control embryonic development in vertebrates; its expression persists in neural stem/progenitor cells into adulthood. Considering the critical role of the SOX2 transcription factor in the regulation of genes required for self-renewal and pluripotency of stem cells, we developed and characterized SOX2-overexpressing NT2/D1 cell clones. Using Southern blot and semi-quantitative RT-PCR, we confirmed integration and expression of exogenous SOX2 in three NT2/D1 cell clones. Overexpression of the SOX2 gene was detected in two of these clones. SOX2 overexpression in NT2/D1 cell clones resulted in altered expression of key pluripotency genes OCT4 and NANOG. Furthermore, SOX2-overexpressing NT2/D1 cell clones entered into retinoic acid-dependent neural differentiation, even when there was elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of neural markers (neuroD1 and synaptophysin) was higher in induced cell clones than in induced parental cells. The cell clone with SOX2 overexpression had an approximately 1.3-fold higher growth rate compared to parental cells. SOX2 overexpression did not increase the population of cells undergoing apoptosis. Taken together, we developed two SOX2-overexpressing cell clones, with constitutive SOX2 expression after three weeks of retinoic acid treatment. SOX2 overexpression resulted in altered expression of pluripotency-related genes, increased proliferation, and altered expression of neural markers after three weeks of retinoic acid treatment.

  8. Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

    PubMed

    Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

    2014-09-01

    An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones.

  9. 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

  10. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-10-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

  11. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed Central

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  12. [Construction of directional T vector for gene cloning and expression].

    PubMed

    Zhong, Xing; Zhai, Chao; Chen, Liang; Yu, Xiaolan; Jiang, Sijing; Yan, Hong; Yang, Dengxiang; Ma, Lixin

    2013-04-01

    Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression.

  13. Cloning and detecting signature microRNAs from mammalian cells.

    PubMed

    Sun, Guihua; Li, Haitang; Rossi, John J

    2007-01-01

    MicroRNAs (miRNAs) are about 19- to 24-nucleotides long noncoding regulatory small RNAs that could silence target gene expression through base pairing to the complementary sequences in the 3' untranslated region (3'UTR) of targeted genes. They are evolutionally conserved and play an important regulatory role in embryogenesis, cell differentiation, and proliferation. They are also involved in pathogenesis and progression of some human diseases. There are about 1000 human miRNAs predicted today, and it is estimated that they could target about 30% of all human transcripts. Profiling the miRNAs that are expressed in the experimental cells became an important issue as different cells express different signature miRNAs or express the same miRNAs at different level. Small RNA cloning is a reliable way to characterize those tissue- or cell-specific signature miRNAs. This chapter describes a relatively nonlaborious polyadenylation-mediated complementary DNA (cDNA) cloning method that will identify most of the small RNAs expressed in the cells of interest. This procedure can also be used to verify bioinformatic predictions of miRNAs/small interfering RNAs (siRNAs) as well as to identify new miRNAs/siRNAs.

  14. Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

    DTIC Science & Technology

    2013-01-01

    Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries PRINCIPAL INVESTIGATOR: Daniel J. Lindner, M.D., Ph.D...YYYY) 2013 2. REPORT TYPE Final 3. DATES COVERED (From - To) 201 31 2012 4. TITLE AND SUBTITLE Complementation of Myelodysplastic Syndrome Clones...vitro and engrafted in the marrow of SG3, but not NSG mice. 15. SUBJECT TERMS Myelodysplastic syndrome , lentivirus, cDNA libraries

  15. Cloning and expression of recombinant, functional ricin B chain

    SciTech Connect

    Chang, M.S.; Russell, D.W.; Uhr, J.W.; Vitetta, E.S.

    1987-08-01

    The cDNA encoding the B chain of the plant toxin ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with (/sup 35/S)methionine and (/sup 35/S)-cysteine and demonstrating the secretion of a protein with a M/sub r/ of 30,000-32,000 that was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B-chain antibody and the amount of recombinant B chain secreted by the COS-M6 cells was determined by a radioimmunoassay. Virtually all of the recombinant B chain formed active ricin when mixed with native A chain; it could also bind to the galactose-containing glycoprotein asialofetuin as effectively as native B chain.These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function

  16. Cloning and expression of genes encoding Haemophilus somnus antigens.

    PubMed Central

    Corbeil, L B; Chikami, G; Yarnall, M; Smith, J; Guiney, D G

    1988-01-01

    A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease. Images PMID:2843469

  17. 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.

  18. 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

  19. Cloning and expression of pigeon IFN-γ gene.

    PubMed

    Fringuelli, Elena; Urbanelli, Lorena; Tharuni, Omar; Proietti, Patrizia Casagrande; Bietta, Annalisa; Davidson, Irit; Franciosini, Maria Pia

    2010-12-01

    This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.

  20. Cloning, expression and biological activity of equine interleukin (IL)-5.

    PubMed

    Cunningham, F M; Vandergrifft, E; Bailey, S R; Sepulveda, M F; Goode, N T; Horohov, D W

    2003-09-15

    The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.

  1. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

    PubMed

    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 and 12.8% 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

  2. Cloning of a brain-type isoform of human Rab GDI and its expression in human neuroblastoma cell lines and tumor specimens.

    PubMed

    Nishimura, N; Goji, J; Nakamura, H; Orita, S; Takai, Y; Sano, K

    1995-11-15

    Rab proteins, a family of Ras-related small GTP-binding proteins, play a key role in regulating intracellular vesicle trafficking. Rab GDP dissociation inhibitor (GDI3) forms a soluble complex with Rab proteins and thereby prevents the exchange of GDP for GTP. Recently, two isoforms of Rab GDI cDNA were isolated from rats and mice. In this study, we have isolated a brain-type isoform of human Rab GDI cDNA and examined its expression in neuroblastoma. We tentatively designate it as human Rab GDI alpha (hu GDI alpha) and another human Rab GDI, as human Rab GDI beta (hu GDI beta). Hu GDI alpha cDNA encodes a protein of 447 amino acids with a deduced molecular weight of 50,200. Northern blot analysis revealed that hu GDI alpha gene is expressed abundantly in the brain but much less in other tissues, while hu GDI beta gene is ubiquitously expressed. All human neuroblastoma cell lines and tumor specimens examined express hu GDI alpha gene to various extents, while a human T cell leukemia cell line, MOLT3, does not. The levels of both hu GDI alpha and beta mRNA were constant in a human neuroblastoma cell line, NB1, during its neuronal differentiation, while Rab3A and neurofilament-L gene expression and the number of neurosecretory granules were elevated at this condition. These results suggest that hu GDI alpha gene expression is not related to the differentiation state of neuronal cells.

  3. Cloning and expression of Ca2+-activated chloride channel from rat brain.

    PubMed

    Jeong, Sang Min; Park, Hye-Kyung; Yoon, In-Soo; Lee, Jun-Ho; Kim, Jong-Hoon; Jang, Choon-Gon; Lee, C Justin; Nah, Seung-Yeol

    2005-08-26

    To clone the gene product responsible for the calcium-activated chloride channel (CLCA) in rat brain cerebrum, we performed a reverse transcription-PCR (RT-PCR) with gene-specific primers of a rat EST clone. We successfully cloned a rat brain CLCA (rbCLCA). The full-length cDNA is 2895 bp long and codes for a 902 amino acid protein. The clone consists of four transmembrane domains and shows a 79.1% of significant homology with previously reported mouse smooth muscle chloride channel sequence. We also performed RT-PCR using single neuron and glia, and various tissues to determine the tissue expression of rbCLCA. We found that rbCLCA was expressed in both neuron and glia. In peripheral organs, rbCLCA showed the predominant expressions in cerebrum, cerebellum, kidney, small intestine, and stomach but not in heart, large intestine, liver, lung, and spleen. Whole-cell patch clamp studies in HEK293 cells transfected with the clone identified a niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride current but we could not observe this chloride current in mock-transfected cells. The identification of genes belonging to the CLCA family from rat brain and its functional expression will help to evaluate its physiological role in brain as anion channel.

  4. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    PubMed

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  5. Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning.

    PubMed

    Gillet, L; Daix, V; Donofrio, G; Wagner, M; Koszinowski, U H; China, B; Ackermann, M; Markine-Goriaynoff, N; Vanderplasschen, A

    2005-04-01

    Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.

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

    SciTech Connect

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. ); McPherson, J.P. ); Evans, G.A. )

    1992-02-15

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

  7. Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.

    PubMed

    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

    2013-10-01

    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.

  8. Molecular cloning of a new secreted sulfated mucin-like protein with a C-type lectin domain that is expressed in lymphoblastic cells.

    PubMed

    Bannwarth, S; Giordanengo, V; Lesimple, J; Lefebvre, J C

    1998-01-23

    We have previously demonstrated hyposialylation of the two major CD45 and leukosialin (CD43) molecules at the surface of latently human immunodeficiency virus type 1-infected CEM T cells (CEMLAI/NP), (Lefebvre, J. C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P., Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265-274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609-1617). Searching to clarify mechanism(s) of hyposialylation, we observed two sulfated secreted glycoproteins (molecular mass approximately 47 and approximately 40 kDa) (P47 and P40), which were differentially sulfated and/or differentially secreted in the culture supernatants of CEMLAI/NP cells when compared with parental CEM cells. A hybridoma clone (7H1) resulting from the fusion between CEMLAI/NP and human embryonic fibroblasts MRC5 cells produced very large amounts of P47 that was purified using Jacalin lectin (specific for O-glycans) and microsequenced. Cloning of P47 was achieved using a CEMLAI/NP cDNA library screened with a degenerate oligonucleotide probe based on its NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking alpha-helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal portion is rich in glutamic acids with a predicted pI of 3.9. In addition, a hinge region with numerous condensed potential sites for O-glycan side chains, which are also the most likely sulfation sites, is located between the RGD and leucine zipper domains. Transcripts were detected in lymphoid tissues (notably bone marrow) and abundantly in T and B lymphoblastoid but very faintly in monocytoid cell lines.

  9. Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

    PubMed

    Sonoda, Masatoshi; Ide, Hiroaki; Nakayama, Shinya; Sasaki, Asako; Kitazaki, Shinei; Sato, Takahide; Nakagawa, Hiroki

    2003-04-01

    The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

  10. Expression of structurally diverse Qa-2-encoded molecules on the surface of cloned cytotoxic T lymphocytes

    PubMed Central

    1984-01-01

    Extracts of 125I-labeled cloned murine cytotoxic T lymphocytes (CTL) were immunoprecipitated with alloantisera to the cloned CTL and rabbit antisera to beta-2 microglobulin. Polyacrylamide gel electrophoresis (PAGE) of the specific precipitates revealed, as expected, 125I-labeled components that corresponded to products of class I genes of the major histocompatibility complex (MHC). However, additional class I gene products of relatively low apparent molecular weight (Mr) were also observed. Similar analyses of spleen cells from a variety of MHC- congenic mouse strains suggested that the class I molecules of relatively low Mr are encoded in the Qa-2 region of the MHC, and this was confirmed by immunoprecipitation with a monoclonal antibody to Qa- 2. Surprisingly, however, the cell surface Qa-2 molecules of different CTL clones differed in Mr, in isoelectric focusing (IEF) pattern, and in the number of distinguishable molecules expressed per clone: some clones seemed to express only a single Qa-2-encoded molecule while others expressed two distinct ones. Treatment of the immunoprecipitated Qa-2 with endoglycosidase F (Endo F) resulted in a decrease in Mr of approximately 5,000-6,000, corresponding to the expected loss of N- linked oligosaccharides, but the decrease did not eliminate structural variability among the clones. Structural diversity of the Qa-2-encoded molecules expressed on CTL could arise because CTL clones differ (a) in the particular Qa-2 genes they express, (b) in the way they splice Qa-2 gene transcripts or, perhaps, (c) in Endo F-resistant oligosaccharides on their Qa-2 molecules. PMID:6333483

  11. Melanoma antigen expression and metastatic ability of mutant B16 melanoma clones.

    PubMed

    Nozue, M; Sakiyama, H; Tsuchiya, K; Hirabayashi, Y; Taniguchi, M

    1988-11-15

    The biological functions of murine melanoma-associated antigens recognized by monoclonal antibodies (MAbs) (M562, M622 and M2590) were examined by using mutant clones which differed in their degree of expression of these antigens. Four clones of high expressors of 3 types of antigen (MEA group), 5 clones of low or non-expressors of M562- and M622-recognizing antigens (MEB group) and 4 clones of non-expressor of GM3 recognized by M2590 (MEC group) were used. Attachment of these clones to components of extracellular matrix was different between the groups. Two clones of the MEA group showed the highest ability to adhere to laminin and type-IV collagen, whereas the clones of the MEB and MEC groups significantly lost their ability to attach to laminin and type-IV collagen. In experimental lung metastasis, metastasizing ability of MEA-group cells was higher than that of MEB- and MEC-group cells. Our results suggest that these antigens play some functional role in metastasis mediated by increasing capacity for attachment to laminin and type-IV collagen.

  12. Cloning and expression of human tyrosine aminotransferase cDNA.

    PubMed

    Séralini, G E; Luu-Thé, V; Labrie, F

    1995-01-02

    Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.

  13. Cloning and expression analysis of the murine lymphotoxin beta gene.

    PubMed Central

    Pokholok, D K; Maroulakou, I G; Kuprash, D V; Alimzhanov, M B; Kozlov, S V; Novobrantseva, T I; Turetskaya, R L; Green, J E; Nedospasov, S A

    1995-01-01

    Tumor necrosis factor alpha (TNF-alpha) and soluble lymphotoxin (LT) (also called LT-alpha or TNF-beta) are cytokines with similar biological activities that are encoded by related and closely linked genes. TNF-alpha, a mediator of the inflammatory response, exists in soluble and transmembrane forms. LT-alpha can be secreted or retained at the cell surface by binding to a 33-kDa transmembrane subunit, LT-beta. The recently cloned human LT-beta gene encodes another TNF family member and is linked to the TNF/LT locus within the major histocompatibility complex locus. The cell surface LT is a heterotrimer consisting of LT-alpha and LT-beta, whose physiological function is not yet clearly defined. We now report the sequence analysis of the genomic region and cDNA of murine LT-beta gene, which is closely associated with the TNF-alpha and LT-alpha genes within the murine major histocompatibility complex locus. Unlike the TNF-alpha, LT-alpha, and human LT-beta genes, which contain four exons, the murine LT-beta contains three exons and encodes a 244-amino acid polypeptide with a 66-amino acid insert that is absent from the human homologue. In situ hybridization demonstrates constitutive expression of LT-beta in lymphoid and hematopoietic tissues. LT-beta transcription is maximal in the thymic medulla and in splenic white pulp. LT-beta mRNA is also detected in the skin and in specific regions of the brain. The LT-beta promoter region contains putative Ets-binding sites, suggesting that the expression of LT-beta may be regulated in part by Ets transcription factors whose pattern of lymphoid expression overlaps that of LT-beta. Images Fig. 3 Fig. 4 PMID:7846035

  14. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    SciTech Connect

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  15. Cell phoney: human cloning after Quintavalle.

    PubMed

    Morgan, Derek; Ford, Mary

    2004-12-01

    Reproductive cloning has thrown up new scientific possibilities, ethical conundrums, and legal challenges. An initial question, considered by the English courts in 2003, was whether the technique presently available, that of cell nucleus replacement, falls outside the provisions of the Human Fertilisation and Embryology Act 1990. If it does, the creation and use, including use in research protocols, of human embryos would be unregulated, disclosing a need to consider remedial legislation. The resolution by the courts of this legal question dramatically engages them in a resolution of fundamental ethical dilemmas, and discloses the possibilities and limitation of negotiating science policy through the processes of litigation.

  16. A High Efficiency Cloning and Expression System for Proteomic Analysis

    DTIC Science & Technology

    2006-03-19

    Research, Rockville, MD, USA 0 U.a . The recent description of the complete genomes of the two most pathogenic species of Brucella Received: Januarp9...level high-efficiency cloning and expression system (HECES) that Accepted: March 19, 2006 allow expression of large numbers of Brucella proteins based...method will allow selection of new reagents for diagnosis of bru- cellosis and development of vaccine against Brucella , an important zoonotic disease and

  17. Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats.

    PubMed

    Meng, Li; Jia, Ruo-Xin; Sun, Yan-Yan; Wang, Zi-Yu; Wan, Yong-Jie; Zhang, Yan-Li; Zhong, Bu-Shuai; Wang, Feng

    2014-02-01

    Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung development. Our objective was to investigate molecular background of lung developmental problems in transgenic (random insertion of exogenous DNA) cloned goats. We examined expression of 15 genes involved in growth regulation, imprinting, and epigenetic transcription in lung tissue of deceased transgenic cloned and normal goats of various ages. Compared with normal goats of the same age from conventional reproduction, expression of 13 genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, CDKNIC, PCAF, MeCP2, HDAC1, and Dnmt3b) decreased in transgenic cloned goats that died at or shortly after birth; Expression of eight genes (FGF10, PDGFR, RABP, VEGF, PCAF, HDAC1, MeCP2, and Dnmt3b) decreased in fetal death of transgenic cloned goats. Expression of two epigenetic transcription genes (PCAF and Dnmt3b) decreased in disease death of transgenic cloned goats (1-4 months old). Disruptions in gene expression might be associated with the high neonatal mortality in transgenic cloned animals. These findings have implications in understanding the low efficiency of transgenic cloning.

  18. Immunogenicity of recombinant Mycobacterium bovis bacille Calmette-Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens.

    PubMed

    Mohamud, Rohimah; Azlan, Maryam; Yero, Daniel; Alvarez, Nadine; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd-Nor

    2013-01-01

    Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.

  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. 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.

  1. Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

    PubMed Central

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2014-01-01

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation. PMID:20085739

  2. Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

    PubMed

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-08

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  3. Aberrant Expression of TIMP-2 and PBEF Genes in the Placentae of Cloned Mice Due to Epigenetic Reprogramming Error

    PubMed Central

    Kim, Hong Rye; Lee, Jae Eun; Oqani, Reza Kheirkhahi; Kim, So Yeon; Wakayama, Teruhiko; Li, Chong; Sa, Su Jin; Woo, Je Seok; Jin, Dong Il

    2016-01-01

    Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant

  4. 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

  5. Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos.

    PubMed

    Hao, Lanping; Midic, Uros; Garriga, Judith; Latham, Keith E

    2014-01-15

    Cumulus oophorus cells play an essential role in oocyte development. They are also widely employed as donor cells for cloning by somatic cell nuclear transfer. Our previous studies revealed that Cbx4 mRNA was overexpressed in cloned two-cell embryos. These data indicated that CBX4 may regulate normal cumulus cell differentiation and that its overexpression in clones could contribute to aberrant gene regulation. We used siRNA-mediated knockdown of Cbx4 to assess its role in determining cumulus cell phenotype and compared the effects of this knockdown to published data for aberrant gene regulation in cloned embryos. We observed widespread effects on the expression of genes related to diverse processes in cultured cumulus cells, including cell assembly/proliferation and DNA replication/repair, endocrine function, carbohydrate and lipid metabolism, inflammation, and cell morphology, with apparent effects of CBX4 in promoting cumulus cell proliferation and survival and inhibiting differentiation. Overall, the data implicate CBX4 as a key component in the pathway integrating endocrine signals, intraovarian paracrine factors, and oocyte-derived factors in the control of cumulus cell functions. We also observed altered expression of 25 cumulus cell markers of oocyte quality, indicating an important role of CBX4 in production of high quality oocytes. Finally, we found that about one-quarter of the genes showing aberrant transcription in cloned embryos are sensitive to Cbx4 knockdown in cumulus cells, consistent with a role for aberrant Cbx4 regulation in elaborating abnormal cloned embryo characteristics.

  6. 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.

  7. Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones.

    PubMed

    Iacopino, Fortunata; Angelucci, Cristiana; Piacentini, Roberto; Biamonte, Filippo; Mangiola, Annunziato; Maira, Giulio; Grassi, Claudio; Sica, Gigliola

    2014-01-01

    Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca(2+) signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca(2+)-channels but they exhibited increased intracellular Ca(2+) levels in response to ATP. These Ca(2+) signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca(2+)-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

  8. 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).

  9. Cloning and expression of the complement receptor glycoprotein C from Herpesvirus simiae (herpes B virus): protection from complement-mediated cell lysis.

    PubMed

    Huemer, Hartwig P; Wechselberger, Christian; Bennett, Alice M; Falke, Dietrich; Harrington, Lesley

    2003-05-01

    Simian herpes B virus (SHBV) is the herpes simplex virus (HSV) homologue for the species MACACA: Unlike in its natural host, and unlike other animal herpesviruses, SHBV causes high mortality in accidentally infected humans. SHBV-infected cells, like those infected with HSV-1 and equine herpesvirus types 1 and 4, express complement C3 receptor activity. To study immunoregulatory functions involved in susceptibility/resistance against interspecies transmission, the SHBV glycoprotein C (gC(SHBV)) gene (encoding 467 aa) was isolated. Sequence analysis revealed amino acid identity with gC proteins from HSV-2 (46.9 %), HSV-1 (44.5 %) and pseudorabies virus (21.2 %). Highly conserved cysteine residues were also noted. Similar to gC(HSV-2), gC(SHBV) is less glycosylated than gC(HSV-1), resulting in a molecular mass of 65 kDa if expressed in replication-deficient vaccinia virus Ankara. Stable transfectants expressing full-length gC(SHBV) on the cell surface induced C3 receptor activity and were substantially protected from complement-mediated lysis; no protection was observed with control constructs. This suggests that expression of the gC homologues on infected cell surfaces might also contribute to the survival of infected cells in addition to decreased virion inactivation. Interestingly, soluble gC(SHBV) isolated from protein-free culture supernatants did not interfere with the binding of the alternative complement pathway activator properdin to C3b, which is similar to our findings with gC(HSV-2) and could be attributed to major differences in the amino-terminal portion of the protein with extended deletions in both gC(SHBV) and gC(HSV-2). Binding of recombinant gC(SHBV) to polysulphates was observed. This, together with the heparin-sensitivity of the gC(SHBV)-C3 interaction on the infected cell surface, suggests a role in adherence to heparan sulphate, similar to the gC proteins of other herpesviruses.

  10. 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.

  11. An epitope tagged mammalian/prokaryotic expression vector with positive selection of cloned inserts.

    PubMed

    Schneider, S; Georgiev, O; Buchert, M; Adams, M T; Moelling, K; Hovens, C M

    1997-09-15

    A dual eukaryotic/prokaryotic expression vector has been developed which combines the features of positive selection for cloned inserts along with the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells as well as high level induced expression in E. coli cells harbouring T7 RNA polymerase. This vector, pZilch, has two MCSs flanking a mutant E. coli phenylalanyl-tRNA synthetase gene, pheS, which when expressed in combination with the phenylalanine analog p-CI-Phe, results in termination of host cell protein synthesis. Cloning of inserts using unique sites in the flanking MCS regions results in loss of the pZilch pheS allele and hence permits growth of colonies harbouring recombinants on p-Cl-Phe plates. Additional features of the vector include an optimal Kozak consensus sequence for high level eukaryotic cell expression and an efficient prokaryotic translation initiation site in frame and downstream from the eukaryotic initiation site. Recombinant proteins can be produced with an N-terminal FLAG epitope which can be removed via a specific protease cleavage site. Flanking T7 and SP6 RNA polymerase promoter sites permit in vitro transcription and translation of cloned inserts. A derivative of the vector has also been constructed enabling nuclear accumulation of the tagged proteins via an SV40 nuclear localisation signal upstream of the 5' MCS.

  12. 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

  13. Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression.

    PubMed

    Mourtada-Maarabouni, Mirna; Kirkham, Lucy; Farzaneh, Farzin; Williams, Gwyn T

    2004-12-16

    Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways independently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone' by ultraviolet radiation, and by the anticancer drug cisplatin. In all cases, colony-forming ability is protected, indicating that fau affects the critical events prior to commitment to cell death. Overexpression of fau in the sense orientation induces cell death, which is inhibited both by Bcl-2 and by inhibition of caspases, in line with its proposed role in apoptosis.

  14. Analysis of imprinted messenger RNA expression in deceased transgenic cloned goats.

    PubMed

    Jia, R X; Zhou, Z R; Zhang, G M; Wang, L Z; Fan, Y X; Wan, Y J; Zhang, Y L; Wang, Z Y; Wang, F

    2016-01-29

    Genomic imprinting is an important epigenetic mechanism that has vital effects on fetal growth and development. We observed the differences in four tissues (heart, spleen, liver, and kidney) from dead transgenic cloned goats using hematoxylin and eosin (H&E) staining. Eight imprinted genes in the tissues of dead transgenic cloned and normal goats were analyzed using reverse transcription polymerase chain reaction. H&E staining results from the abortion group indicated the lack of obvious morphological changes in heart and spleen tissues, while inflammatory cell infiltration and glomerular nephritis characteristics were observed in liver and kidney tissues, respectively. Compared to the control group, CDKN1C, H19, IGF2R, and SNRPN were significantly (P < 0.05) overexpressed in the heart tissue of the abortion group, while XIST was significantly reduced. In the liver tissues, CDKN1C and DLK1 expression decreased, while GNAS, H19, IGF2R, PEG3, and XIST expression increased significantly. In the spleen tissues, DLK1 expression increased, while GNAS, H19, IGF2R, PEG3, SNRPN, and XIST expression decreased. In the kidney tissues, CDKN1C, DLK1, GNAS, IGF2R, and PEG3 expression increased, while H19 and XIST expression decreased. The overall expression of imprinted genes was abnormal in different tissues of transgenic cloned goats, and the degree of abnormal genomic imprinting was more severe in the abortion group compared to the death and control groups. These results suggest that abnormal expression of imprinted genes may cause developmental defects in transgenic cloned goats. Moreover, abnormal epigenetic modifications may affect the reprogramming of transgenic donor cells.

  15. T-cell clones in human trichinellosis: Evidence for a mixed Th1/Th2 response.

    PubMed

    Della Bella, C; Benagiano, M; De Gennaro, M; Gomez-Morales, M A; Ludovisi, A; D'Elios, S; Luchi, S; Pozio, E; D'Elios, M M; Bruschi, F

    2017-03-01

    In humans, studies on the cellular immune response against Trichinella are scarce. Aim of this study was to characterize the cytokine profile of T cells specific for Trichinella britovi in trichinellosis patients. Peripheral blood mononuclear cells (PBMC) were obtained from five patients involved in a trichinellosis outbreak caused by T. britovi, which occurred in 2013 in Tuscany (Italy). All the patients resulted positive for Trichinella-specific IgG, IgE and presented eosinophilia. T cells were investigated for their proliferation to excretory/secretory antigens from Trichinella spiralis muscle larvae (TsES) and for their cytokine profile. A total of 284 CD4+ and 42 CD8+ T-cell clones were obtained from the TsES-specific T-cell lines from PBMC. All T-cell clones proliferated in response to mitogen. Of the 284 CD4+ T-cell clones generated from TsES-specific T-cell lines, 135 (47%) proliferated significantly to TsES; 26% CD8+ T-cell clones showed proliferation to TsES. In the series of the 135 TsES-specific CD4+ clones, 51% expressed a Th2 profile, 30% a Th0 and 19% Th1. In the series of the 11 TsES-specific CD8+ T-cell clones, 18% were Tc2, 45% Tc0 and 36% Tc1. In human trichinellosis, the cellular immune response is, during the chronic phase, mixed Th1/Th2.

  16. 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.

  17. Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

    DTIC Science & Technology

    2012-07-01

    were validated; they induced myeloid colonies in vitro and engrafted in the marrow of SG3, but not NSG mice. Myelodysplastic syndrome , lentivirus...cDNA libraries, complementation The Cleveland Clinic Foundation Cleveland, OH 44195 Complementation of Myelodysplastic Syndrome Clones with...Martin-Padura, P. Mancuso, P. Marighetti, C. Rabascio, G. Pruneri, L. D. Shultz, and F. Bertolini. 2008. Human acute leukemia cells injected in NOD

  18. Cloning and expression of the human vasoactive intestinal peptide receptor.

    PubMed Central

    Sreedharan, S P; Robichon, A; Peterson, K E; Goetzl, E J

    1991-01-01

    Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands. Images PMID:1675791

  19. Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2015-01-01

    Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable. PMID:25561925

  20. 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.

  1. Cloning and prokaryotic expression of the porcine lipasin gene.

    PubMed

    Li, M M; Geng, J; Guo, Y J; Jiao, X Q; Lu, W F; Zhu, H S; Wang, Y Y; Yang, G Y

    2015-11-23

    Lipasin has recently been demonstrated to be involved in lipid metabolism. In this study, two specific primers were used to amplify the lipasin open reading frame from porcine liver tissue. The polymerase chain reaction product was cloned to a pGEM®-T Easy Vector, digested by SalI and NotI, and sequenced. The lipasin fragment was then cloned to a pET21(b) vector and digested by the same restriction enzyme. The recombinant plasmid was transferred to Escherichia coli (BL21), and the lipasin protein was induced with isopropyl-β-D-thiogalactopyranoside. The protein obtained was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. A pET-lipasin prokaryotic recombinant expression vector was successfully constructed, and a 25.2-kDa protein was obtained. This study provides a basis for further research on the biological function of porcine lipasin.

  2. Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization.

    PubMed

    Sidhu, Kuldip S; Tuch, Bernard E

    2006-02-01

    Here we describe the first report of three human embryonic stem cell (hESC) clones, hES 3.1, 3.2, and 3.3, derived from the parent line hES3 by sorting of single-cell preparations by flow cytometry. The viability of single-cell preparations before and after cell sorting remained >98%. The hESC were selected by size gating and forward-angle light scatter and were dispersed directly as single cell/ well into 96-well plates containing human fetal fibroblasts as feeder layers. Single stem cell dispersion into 96-well plates was confirmed by using cells from a hES3 line that constitutively expressed green fluorescence protein (eGFP) under similar conditions of flow cytometry. Three clones were obtained from the parent line hES3 -- hES3.1, 3.2, and 3.3 -- and they have been in continuous culture for more than 1 year. The cloning efficiency was less than <0.5%. These hESC clones show normal stem cell characteristics, such as undifferentiated growth, high nucleocytoplasmic ratio, the same karyotype as that of the parent line (46 XX), stem cell surface markers (i.e., SSEA3, SSEA4, OCT4, TRA-1-60, and TRA-1-81), and gene expression for pluripotency (Oct-4 and nanog). They all formed embryoid bodies in suspension cultures, and after seeding in culture plates they showed pluripotency in vitro by forming cell lineages derived from all three germ layers as indicated by expression of the ectodermal marker nestin, the mesodermal marker renin, and the endodermal markers alpha-fetoprotein and GATA6. All clones showed normal expression of alkaline phosphatase activity, a marker of in vitro pluripotency. When hESC clones (1-2 x 10(6) total) were injected into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice under the kidney capsule, all formed teratomas within 6-8 weeks. Analysis of the stem cell surface marker TRA-1-160 by flow cytometry showed nonsignificant (p < 0.05) differences between the clones and the parent line. The clones also differed in their expression

  3. Clones of ectopic stem cells in the regeneration of muscle defects in vivo.

    PubMed

    Yang, Rujing; Chen, Mo; Lee, Chang Hun; Yoon, Richard; Lal, Shan; Mao, Jeremy J

    2010-10-20

    Little is known about whether clones of ectopic, non-muscle stem cells contribute to muscle regeneration. Stem/progenitor cells that are isolated for experimental research or therapeutics are typically heterogeneous. Non-myogenic lineages in a heterogeneous population conceptually may compromise tissue repair. In this study, we discovered that clones of mononucleated stem cells of human tooth pulp fused into multinucleated myotubes that robustly expressed myosin heavy chain in vitro with or without co-culture with mouse skeletal myoblasts (C2C12 cells). Cloned cells were sustainably Oct4+, Nanog+ and Stro1+. The fusion indices of myogenic clones were approximately 16-17 folds greater than their parent, heterogeneous stem cells. Upon infusion into cardio-toxin induced tibialis anterior muscle defects, undifferentiated clonal progenies not only engrafted and colonized host muscle, but also expressed human dystrophin and myosin heavy chain more efficaciously than their parent heterogeneous stem cell populations. Strikingly, clonal progenies yielded ∼9 times more human myosin heavy chain mRNA in regenerating muscles than those infused with their parent, heterogeneous stem cells. The number of human dystrophin positive cells in regenerating muscles infused with clonal progenies was more than ∼3 times greater than muscles infused with heterogeneous stem cells from which clonal progenies were derived. These findings suggest the therapeutic potential of ectopic myogenic clones in muscle regeneration.

  4. Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2.

    PubMed

    Iversen, Annette; Cazzamali, Giuseppe; Williamson, Michael; Hauser, Frank; Grimmelikhuijzen, Cornelis J P

    2002-12-13

    The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.

  5. Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone.

    PubMed

    Chevalier, Sébastien Alain; Ko, Nga Ling; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Kehn, Kylene; Brady, John N; Kashanchi, Fatah; Gessain, Antoine; Mahieux, Renaud

    2008-07-01

    We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

  6. Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

    PubMed

    Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

    2011-04-15

    A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning.

  7. 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.

  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. 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.

  11. 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.

  12. Evaluation of cloned cells, animal model, and ATRA sensitivity of human testicular yolk sac tumor

    PubMed Central

    2012-01-01

    The testicular yolk sac tumor (TYST) is the most common neoplasm originated from germ cells differentiated abnormally, a major part of pediatric malignant testicular tumors. The present study aimed at developing and validating the in vitro and vivo models of TYST and evaluating the sensitivity of TYST to treatments, by cloning human TYST cells and investigating the histology, ultra-structure, growth kinetics and expression of specific proteins of cloned cells. We found biological characteristics of cloned TYST cells were similar to the yolk sac tumor and differentiated from the columnar to glandular-like or goblet cells-like cells. Chromosomes for tumor identification in each passage met nature of the primary tumor. TYST cells were more sensitive to all-trans-retinoic acid which had significantly inhibitory effects on cell proliferation. Cisplatin induced apoptosis of TYST cells through the activation of p53 expression and down-regulation of Bcl- expression. Thus, we believe that cloned TYST cells and the animal model developed here are useful to understand the molecular mechanism of TYST cells and develop potential therapies for human TYST. PMID:22410253

  13. Cloning and expression profiling of testis-expressed piRNA-like RNAs

    PubMed Central

    Ro, Seungil; Park, Chanjae; Song, Rui; Nguyen, Dan; Jin, Jingling; Sanders, Kenton M.; McCarrey, John R.; Yan, Wei

    2007-01-01

    Using a novel small RNA cloning method, we identified 630 piRNA-like RNAs (pilRNAs) from the mouse testis, and 498 of them are novel. These pilRNA genes were mapped to all chromosomes as 71 clusters, and the majority of them (∼84%) are derived from intergenic, intronic, and exonic sequences. One of the structural characteristics for pilRNAs is that a single locus can encode numerous homologous pilRNAs with overlapping sequences. Hundreds or even thousands of pilRNAs from a single pilRNA gene cluster are all produced from a single long transcript. Expression profiling for 64 pilRNAs revealed that ∼14% of all the pilRNAs analyzed displayed a ubiquitous expression pattern, although the majority of (∼86%) pilRNAs were preferentially or exclusively expressed in meiotic and haploid male germ cells of the testis. Our semiquantitative analyses also suggest that the testis is the organ with the highest expression of pilRNAs both in number and in abundance. The large number, high abundance, unique genomic locations, and biogenesis all suggest that pilRNAs have important regulatory roles not only in spermatogenesis but also in other biological processes. PMID:17698640

  14. Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M.

    PubMed Central

    Malik, N; Kallestad, J C; Gunderson, N L; Austin, S D; Neubauer, M G; Ochs, V; Marquardt, H; Zarling, J M; Shoyab, M; Wei, C M

    1989-01-01

    Oncostatin M is a polypeptide of Mr approximately 28,000 that acts as a growth regulator for many cultured mammalian cells. We report the cDNA and genomic cloning, sequence analysis, and functional expression in heterologous cells of oncostatin M. cDNA clones were isolated from mRNA of U937 cells that had been induced to differentiate into macrophagelike cells by treatment with phorbol 12-myristate 13-acetate, and a genomic clone was also isolated from human brain DNA. Sequence analysis of these clones established the 1,814-base-pair cDNA sequence as well as exon boundaries. This sequence predicted that oncostatin M is synthesized as a 252-amino-acid polypeptide, with a 25-residue hydrophobic sequence resembling a signal peptide at the N terminus. The predicted oncostatin M amino acid sequence shared no homology with other known proteins, but the sequence of the 3' noncoding region of the cDNA contained an A + T-rich stretch with sequence motifs found in the 3' untranslated regions of many cytokine and lymphokine cDNAs. Oncostatin M mRNA of approximately 2 kilobase pairs was detected in phorbol 12-myristate 13-acetate-treated U937 cells and in activated human T cells. Transfection of cDNA encoding the oncostatin M precursor into COS cells resulted in the secretion of proteins with the structural and functional properties of oncostatin M. The unique amino acid sequence, expression by lymphoid cells, and growth-regulatory activities of oncostatin M suggest that it is a novel cytokine. Images PMID:2779549

  15. Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

    PubMed

    Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A

    2013-01-30

    Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT.

  16. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    DTIC Science & Technology

    2011-09-01

    Bacillus Anthracis in Escherichia Coli . Biochem Biophys Res Commun 2001, 283 (2), 308–15. 15. Ivins, B. E .; Welkos, S. L. Cloning and Expression of the...Buffer 4 and bovine serum albumin (BSA) at 37 ºC for at least 2 h. The enzymes were heat -inactivated for 20 min at 65 ºC. The pET-22b(+) vector was...to the previous reaction. The phosphatase was heat -inactivated for 20 min at 65 ºC prior to ligation. For each ligation, T4 DNA ligase and its

  17. Cloning, expression, and bioinformatics analysis of the sheep CARP gene.

    PubMed

    Ma, Guoda; Wang, Haiyang; Li, You; Cui, Lili; Cui, Yudong; Li, Qingzhang; Li, Keshen; Zhao, Bin

    2013-06-01

    The cardiac ankyrin repeat protein (CARP) is a multifunctional protein that is expressed specifically in mammalian cardiac muscle and plays important roles in stress responses, transcriptional regulation, myofibrillar assembly, and the development of cardiac and skeletal muscle. In this study, the sheep homolog of the CARP gene was cloned and characterized. The coding region of the gene consists of 960 bp and encodes 319 amino acids with molecular weight 36.2 KD. Bioinformatics analysis demonstrated that the 3' untranslated region (3'-UTR) of the gene contains many AU-rich elements that are associated with mRNA stability and a potential regulatory site for miRNA binding. The protein was predicted to contain 14 potential phosphorylation sites and an O-GlcNAc glycosylation site and to be expressed in both the nucleus and cytoplasm. The evolutionary analysis revealed that the sheep CARP exhibited a high level of homology with the mammalian counterparts; however, the protein exhibited an increased evolutionary distance from the chicken, frog, and fish homologs. RT-PCR revealed that in addition to its high mRNA expression level in cardiac muscle, trace amounts of the sheep CARP mRNA were expressed in the skeletal muscle, stomach, and small intestine. However, western blot analysis demonstrated that the CARP protein was expressed only in cardiac muscle. The coding sequence was cloned into the pET30a-TEV-LIC vector, and the soluble CARP-MBP (maltose-binding protein) fusion protein was expressed in a prokaryotic host and purified by affinity chromatography. Our data provide the basis for future studies of the structure and function of sheep CARP.

  18. Inhibition of murine nephritogenic effector T cells by a clone-specific suppressor factor.

    PubMed Central

    Meyers, C M; Kelly, C J

    1994-01-01

    We have used a murine model of organ-specific autoimmunity to characterize therapeutic modalities capable of down-regulating the cellular limb of the autoimmune response. Murine interstitial nephritis is an autoimmune disease mediated by tubular antigen-specific CD8+ nephritogenic effector T cells which are delayed-type hypersensitivity (DTH) reactive and cytotoxic to renal epithelial cells. Previous studies have demonstrated that disease can be suppressed with experimentally induced populations of T cells (Ts1 and Ts2 cells) obtained after injection of tubular antigen-coupled splenocytes into syngeneic mice. As the target of Ts2 is the CD8+ effector T cell, we have evaluated its effects on nephritogenic effector T cell clones isolated from diseased animals. Our studies demonstrate that soluble proteins expressed by Ts2 cells (TsF2) specifically abrogate the DTH, cytotoxic, and nephritogenic potential of M52 cells, although T cell receptor and IL-2 receptor expression are unchanged in these unresponsive M52 clones. TsF2-induced inhibition is dependent on new mRNA and protein synthesis. In a cytotoxic clone, M52.26, exposure to TsF2 induces expression of TGF-beta 1 which is, in turn, required for inhibition of cytotoxicity and nephritogenicity. Our studies are consistent with TGF-beta 1 behaving, at least in some T cells, as a nonspecific final effector of clone-specific suppression. Images PMID:7962556

  19. [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.

  20. Identification of putative human T cell receptor delta complementary DNA clones

    SciTech Connect

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  1. 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.

  2. 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

  3. Ultra-sensitive detection of rare T cell clones.

    PubMed

    Robins, Harlan; Desmarais, Cindy; Matthis, Jessica; Livingston, Robert; Andriesen, Jessica; Reijonen, Helena; Carlson, Christopher; Nepom, Gerold; Yee, Cassian; Cerosaletti, Karen

    2012-01-31

    Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay. T cell clones, each with one fixed productive TCR rearrangement, are doped into complex blood cell samples. TCRs from a total of eleven samples are sequenced, with the doped T cell clones ranging from 10% of the total sample to 0.001% (one cell in 100,000). The assay is able to detect even the rarest clones. The precision of the assay is demonstrated across five orders of magnitude. The accuracy for each clone is within an overall factor of three across the 100,000 fold dynamic range. Additionally, the assay is shown to be highly repeatable.

  4. Recent advancements in cloning by somatic cell nuclear transfer

    PubMed Central

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

  5. Recent advancements in cloning by somatic cell nuclear transfer.

    PubMed

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  6. Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

    PubMed

    Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

    2011-02-01

    Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

  7. Cloning and expression of a novel neuropeptide Y receptor.

    PubMed

    Weinberg, D H; Sirinathsinghji, D J; Tan, C P; Shiao, L L; Morin, N; Rigby, M R; Heavens, R H; Rapoport, D R; Bayne, M L; Cascieri, M A; Strader, C D; Linemeyer, D L; MacNeil, D J

    1996-07-12

    The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

  8. A high efficiency cloning and expression system for proteomic analysis.

    PubMed

    Ding, Xuan Z; Paulsen, Ian T; Bhattacharjee, Apurba K; Nikolich, Mikeljon P; Myers, Gary; Hoover, David L

    2006-07-01

    The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are produced with high efficiency in a microarray format conducive to analysis of their sero-reactivity against serum from immunized animals. This method is applicable at either small or large scale of protein processing. While it does not require robotics, the format is amenable to robotic implementation for all aspects of the process and subsequent analysis of protein characteristics. This method will allow selection of new reagents for diagnosis of brucellosis and development of vaccine against Brucella, an important zoonotic disease and biothreat agent.

  9. Sex-reversed somatic cell cloning in the mouse.

    PubMed

    Inoue, Kimiko; Ogonuki, Narumi; Mekada, Kazuyuki; Yoshiki, Atsushi; Sado, Takashi; Ogura, Atsuo

    2009-10-01

    Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.

  10. 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.

  11. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation.

    PubMed Central

    Miki, T; Fleming, T P; Crescenzi, M; Molloy, C J; Blam, S B; Reynolds, S H; Aaronson, S A

    1991-01-01

    We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest. Images PMID:2052597

  12. Comparative expression analysis of resistant and susceptible Populus clones inoculated with Septoria musiva.

    PubMed

    Liang, Haiying; Staton, Margaret; Xu, Yi; Xu, Tao; Leboldus, Jared

    2014-06-01

    Septoria musiva is a major pathogen of Populus and can cause leaf spots and stem cankers in susceptible clones. In order to investigate defense mechanisms of Populus in response to S. musiva, differential gene expression in leaf tissues of two resistant (DN34, P. deltoides×nigra; NM6, P. nigra×maximowiczii) and two susceptible clones (DN164, P. deltoides×nigra; NC11505, P. maximowiczii×trichocarpa) was analyzed by RNA-Seq. Of the 511 million reads obtained, 78% and 0.01% were successfully aligned to the genomes of P. trichocarpa and S. musiva, respectively. Functional annotation of differentially expressed genes based on comparisons between resistant and susceptible clones revealed that there were significant differences in the expression of genes involved in disease/stress resistance and oxidation-reduction in mock-inoculated leaves. Four days post inoculation with S. musiva, 36 differentially expressed genes were found to be regulated in the same direction in both resistant clones. The 22 up-regulated loci in resistant clones included genes involved in protein fate, cell wall structure, and responsiveness to various biotic and abiotic stresses. In particular, Potri.008G187100 locus encodes a putative multi antimicrobial extrusion protein and Potri.006G272600 encodes a family1 glycosyltransferase required for pathogen resistance. The differentially expressed loci with increased expression in the susceptible clones corresponded to NB-ARC domain-containing disease resistance protein, phospholipase A 2A, MutT/nudix family protein, and an elicitor-activated gene 3-1 product. The results from this study indicate that strong defense mechanisms involved in oxidation-reduction, protein fate, secondary metabolism, and accumulation of defense-related gene products may contribute to Septoria resistance in DN34 and NM6, while increased expression of hypersensitive response-loci, particularly those encoding NB-ARC domain-containing disease resistance proteins, may

  13. Molecular cloning and expression of rat prostaglandin E receptor EP2 subtype.

    PubMed

    Sando, T; Usui, T; Tanaka, I; Mori, K; Sasaki, Y; Fukuda, Y; Namba, T; Sugimoto, Y; Ichikawa, A; Narumiya, S

    1994-05-16

    A cDNA clone encoding the rat prostaglandin (PG) E receptor EP2 subtype was cloned from a rat lung cDNA library. It encodes 488 amino acid residues with putative seven-transmembrane domains. Specific binding of [3H]PGE2 was found in COS-7 cells transfected with the cDNA and was displaced with unlabeled prostaglandins in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the significant expression being observed in the thymus, lung, spleen, heart stomach, and pancreas.

  14. Characterization of rat T-cell clones with bacterial specificity.

    PubMed Central

    Eastcott, J W; Yamashita, K; Taubman, M A; Smith, D J

    1990-01-01

    We have isolated 10 rat T-cell clones from the spleen or lymph nodes of seven different donors. These rats were immunized with 2-5 x 10(8) killed Actinobacillus actinomycetemcomitans (Aa) bacteria, injected either subcutaneously (s.c.) in complete Freund's adjuvant or intraperitoneally (i.p.) in saline. Clones studied to date have demonstrated a T-helper (Th) phenotype W3/13+, W3/25+, OX8- and OX22-. Clones were not stimulated in vitro by purified Aa-lipopolysaccharide (LPS) or heterologous Gram-negative bacteria, but proliferated when stimulated by bacteria representative of each of the three serological groups of Actinobacillus, indicating specificity for an Actinobacillus-common antigen other than LPS. One clone (A4) proliferated vigorously when stimulated with concanavalin A (Con A) in vitro, produced interleukin-2 (IL-2) and was provisionally classified as a Th1 type. This appears to be one of the few Th1-type rat clones reported. All other clones tested did not produce IL-2, exhibited B-cell help to some extent, did not induce delayed-type hypersensitivity (DTH) when injected into the footpads of naive rats along with the specific antigen, and were classified as Th2 type. Adoptive transfer of 10(6) cells of one Th2-type Aa-specific clone into syngeneic recipients resulted in a specific splenocyte in vitro response to Aa 12-14 weeks after cell transfer, indicating survival of cloned cells in recipient animals. The use of such clones in studies of experimental periodontal disease is discussed. PMID:1698711

  15. Molecular cloning, characterization, and expression of wheat cystatins.

    PubMed

    Kuroda, M; Kiyosaki, T; Matsumoto, I; Misaka, T; Arai, S; Abe, K

    2001-01-01

    We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47-68% amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63-67% similalities to one another while 93% of amino acids were identical between WC1 and WC3. This suggested that WCI, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.

  16. In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors

    PubMed Central

    1991-01-01

    Purified CD3-4- thymocytes were obtained by depletion of CD3+ and CD4+ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20- 40% of thymic CD16+ cells coexpressed CD1, while approximately half were cyCD3+. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3-4- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3-4- or CD3-4-16- thymocytes in the presence of irradiated H9 cells resulted in large proportions (approximately 50%) of CD16+ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16+ cyCD3+; CD16+ cyCD3-; CD16- cyCD3+; and CD16- cyCD3-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-gamma and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16+ cells expressed transcripts for CD16 and for CD3 epsilon (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long, 1988. J. Immunol. 140:1685.) and zeta chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Lond.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3- CD16+ NK cells may arise from immature thymocytes. PMID:1711562

  17. Differentially expressed microRNAs and affected signaling pathways in placentae of transgenic cloned cattle.

    PubMed

    Liu, Feng-Jun; Jin, Li-Jun; Ma, Xue-Gang; Zhang, Yu-Ling; Zhai, Xiao-Wei; Chen, Jun-Jie; Yang, Xue-Yi

    2014-07-15

    Placental deficiencies are related to the developmental abnormalities of transgenic cattle produced by somatic cell nuclear transfer, but the concrete molecular mechanism is not very clear. Studies have shown that placental development can be regulated by microRNAs (miRNAs) in normal pregnancy. Thus, this study screened differentially expressed miRNAs by the next-generation sequencing technology to reveal the relationship between miRNAs expression and aberrant development of placentae produced by the transgenic-clone technology. Expressions of miRNAs and mRNAs in different placentae were compared, the placentae derived from one natural pregnancy counterpart (PNC), one natural pregnancy of a cloned offspring as a mother (PCM), and two transgenic (human beta-defensin-3) cloned pregnancy: one offspring was alive after birth (POL) and the other offspring was dead in 2 days after birth (POD). Further, signaling pathway analysis was conducted. The results indicated that 694 miRNAs were differentially expressed in four placental samples, such as miR-210, miR-155, miR-21, miR-128, miR-183, and miR-145. Signaling pathway analysis revealed that compared with PNC, significantly upregulated pathways in POL, POD, and PCM mainly included focal adhesion, extracellular matrix-receptor interaction, pathways in cancer, regulation of actin cytoskeleton, endosytosis, and adherens junction, and significantly downregulated pathways mainly included malaria, nucleotide binding oligomerization domain-like receptor signaling, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway. In conclusion, this study confirmed alterations of the expression profile of miRNAs and signaling pathways in placentae from transgenic (hBD-3) cloned cattle (PTCC), which could lead to the morphologic and histologic deficiencies of PTCC. This information would be useful for the relative research in future.

  18. Arginine kinase from Litopenaeus vannamei: cloning, expression and catalytic properties.

    PubMed

    Yao, Cui-Luan; Ji, Pei-Feng; Kong, Peng; Wang, Zhi-Yong; Xiang, Jian-Hai

    2009-03-01

    Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the full-length cDNA of AK was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Escherichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps.

  19. A macrophage receptor for apolipoprotein B48: Cloning, expression, and atherosclerosis

    PubMed Central

    Brown, Matthew L.; Ramprasad, M. P.; Umeda, Patrick K.; Tanaka, Akira; Kobayashi, Yasushi; Watanabe, Teruo; Shimoyamada, Hiroaki; Kuo, Wen-Lin; Li, Ran; Song, Ruiling; Bradley, William A.; Gianturco, Sandra H.

    2000-01-01

    We have cloned a human macrophage receptor that binds to apolipoprotein (apo)B48 of dietary triglyceride (TG)-rich lipoproteins. TG-rich lipoprotein uptake by the apoB48R rapidly converts macrophages and apoB48R-transfected Chinese hamster ovary cells in vitro into lipid-filled foam cells, as seen in atherosclerotic lesions. The apoB48R cDNA (3,744 bp) encodes a protein with no known homologs. Its ≈3.8-kb mRNA is expressed primarily by reticuloendothelial cells: monocytes, macrophages, and endothelial cells. Immunohistochemistry shows the apoB48R is in human atherosclerotic lesion foam cells. Normally, the apoB48R may provide essential lipids to reticuloendothelial cells. If overwhelmed, foam cell formation, endothelial dysfunction, and atherothrombogenesis may ensue, a mechanism for cardiovascular disease risk of elevated TG. PMID:10852956

  20. Identification of a unique B-cell-stimulating factor produced by a cloned dendritic cell.

    PubMed Central

    Clayberger, C; DeKruyff, R H; Fay, R; Cantor, H

    1985-01-01

    We describe a cloned dendritic cell, clone Den-1, which is a potent accessory cell for some B-cell responses. Clone Den-1 produces a unique lymphokine that induces polyclonal B-cell proliferation in the absence of other costimulators. This clone or factors produced by it also stimulate purified B cells to develop plaque-forming cell responses to type 2 antigens. The effect of this factor(s) on various B-cell populations and its relationship to previously described B-cell-stimulating factors is discussed. Images PMID:3871522

  1. The immunogenicity of L1210 lymphoma clones correlates with their ability to function as antigen-presenting cells.

    PubMed

    Cycon, Kelly A; Clements, James L; Holtz, Renae; Fuji, Hiroshi; Murphy, Shawn P

    2009-09-01

    Major histocompatibility complex class II (MHCII) antigen expression is directly correlated with immunogenicity, and inversely correlated with tumorigenicity, in clones of the L1210 murine B lymphoma. Moreover, loss of MHCII expression on human diffuse large B-cell lymphoma is associated with dramatic decreases in patient survival. Thus, the role that MHCII antigens play in the progression of B-cell lymphomas is clinically important. In this study, we investigated the basis for the immunogenicity of MHCII(+) L1210 clones. Immunogenic, but not tumorigenic L1210 clones stimulated the proliferation of naïve T cells and their interleukin (IL)-2 production, which indicates that the immunogenic clones can function as antigen-presenting cells (APCs). However, subclonal variants of the immunogenic L1210 clones, which form tumours slowly in mice, could not activate T cells. The costimulatory molecules B7-1, B7-2 and CD40 were expressed on the immunogenic L1210 clones, but not the tumorigenic clones. Importantly, the tumour-forming subclonal variants expressed MHCII and B7-1, but lacked B7-2 and CD40. These results suggest that MHCII and B7-1 expression on L1210 cells is insufficient to activate naïve T cells, and, furthermore, loss of B7-2 and/or CD40 expression contributes to the decreased immunogenicity of L1210 subclones. Blocking B7-1 or B7-2 function on immunogenic L1210 cells reduced their capacity to activate naïve T cells. Furthermore, incubation of immunogenic L1210 cells with CD40 antibodies significantly enhanced APC function. Therefore, the immunogenicity of L1210 cells directly correlates (i) with their ability to stimulate naïve T cells, and (ii) with the concomitant expression of MHCII, B7-1, B7-2, and CD40.

  2. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

    PubMed Central

    Lin, C S; Aebersold, R H; Kent, S B; Varma, M; Leavitt, J

    1988-01-01

    The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors. Images PMID:3211125

  3. Somatic cell nuclear transfer cloning: practical applications and current legislation.

    PubMed

    Niemann, H; Lucas-Hahn, A

    2012-08-01

    Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

  4. 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.

  5. 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.

  6. Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis.

    PubMed

    Fang, Chee-Mun; Zainuddin, Zainul F; Musa, Mustaffa; Thong, Kwai-Lin

    2006-06-01

    Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.

  7. Cloning and expression of cDNA for a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase from the CEM T-cell line.

    PubMed

    Giordanengo, V; Bannwarth, S; Laffont, C; Van Miegem, V; Harduin-Lepers, A; Delannoy, P; Lefebvre, J C

    1997-07-15

    Complementary DNA encoding a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA library using a 23-base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(beta1-3)GalNAc alpha2,3-sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% similarity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo-bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772-5776]. In previous studies, we have shown hyposialylation of O-glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-cell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.

  8. Islamic perspective on human cloning and stem cell research.

    PubMed

    Larijani, B; Zahedi, F

    2004-12-01

    Recent advances in the field of cloning and stem cell research have introduced new hope for treatment of serious diseases. But this promise has been accompanied by enormous questions. Currently, cloning is a matter of public discussion. It is rare that a field of science causes debate and challenge not only among scientists but also among ethicists, religious scholars, governments, and politicians. One important concern is religious arguments. Various religions have different attitudes toward the morality of these subjects; even within a particular religious tradition there is a diversity of opinions. The following article briefly reviews Islamic perspectives about reproductive/therapeutic cloning and stem cell research. The majority of Muslim jurists distinguish between reproductive and therapeutic cloning. The moral status of the human embryo, the most sensitive and disputed point in this debate, is also discussed according to Holy Quran teachings.

  9. Immortalization and Characterization of Mouse Temporomandibular Joint Disc Cell Clones with Capacity for Multi-lineage Differentiation

    PubMed Central

    Park, Young; Hosomichi, Jun; Ge, Chunxi; Xu, Jinping; Franceschi, Renny; Kapila, Sunil

    2015-01-01

    Objective Despite the importance of TMJ disc in normal function and disease, studying the responses of its cells has been complicated by the lack of adequate characterization of the cell subtypes. The purpose of our investigation was to immortalize, clone, characterize and determine the multi-lineage potential of mouse TMJ disc cells. Design Cells from 12-week-old female mice were cultured and immortalized by stable transfection with human telomerase reverse transcriptase. The immortalized cell clones were phenotyped for fibroblast- or chondrocyte-like characteristics and ability to undergo adipocytic, osteoblastic and chondrocytic differentiation. Results Of 36 isolated clones, four demonstrated successful immortalization and maintenance of stable protein expression for up to 50 passages. Two clones each were initially characterized as fibroblast-like and chondrocyte-like on the basis of cell morphology and growth rate. Further the chondrocyte-like clones had higher mRNA expression levels of cartilage oligomeric matrix protein (>3.5-fold), collagen × (>11-fold), collagen II expression (2-fold) and collagen II:I ratio than the fibroblast-like clones. In contrast, the fibroblast-like clones had higher mRNA expression level of vimentin (>1.5-fold), and fibroblastic specific protein 1 (>2.5-fold) than he chondrocyte-like clones. Both cell types retained multi-lineage potential as demonstrated by their capacity to undergo robust adipogenic, osteogenic and chondrogenic differentiation. Conclusions These studies are the first to immortalize TMJ disc cells and characterize chondrocyte-like and fibroblast-like clones with retained multi-differentiation potential that would be a valuable resource in studies to dissect the behavior of specific cell types in health and disease and for tissue engineering. PMID:25887369

  10. Molecular cloning, genomic organization and cell-binding characteristics of mouse Spalpha.

    PubMed

    Gebe, J A; Llewellyn, M; Hoggatt, H; Aruffo, A

    2000-01-01

    Several group B scavenger receptor cysteine-rich (SRCR) proteins have been shown to function as modulators in the immune response. Recently, we reported the cloning of a new member of this family, human Spalpha (hSpalpha). Herein we report the cloning and characterization of the mouse homologue of hSpalpha. Like its human counterpart, mouse Spalpha (mSpalpha), is a secreted protein containing three SRCR domains. Most lymphoid tissues express RNA transcripts encoding mSpalpha. Characterization of a genomic clone encoding the mature mSpalpha protein showed that each of the SRCR domains of mSpalpha is encoded by a single exon. Comparison of the sequence of mSPalpha with those of other published proteins indicates that it is the same as the recently reported protein named AIM (apoptosis inhibitor expressed by macrophages). Cell-binding studies with a mSpalpha immunoglobulin (mSpalpha-Rgamma) fusion protein indicated that mSpalpha is capable of binding to spleen-derived CD19+ B cells and minimally to peritoneal cavity-derived CD19+ B cells but not to peripheral blood-derived B cells. Spleen-derived CD3+ T cells also bound mSpalpha-Rgamma; however, no binding was observed to either peripheral blood mononuclear cells or peritoneal cavity-derived CD3+ T cells. The mSpalpha-Rgamma fusion protein was also shown to bind to the mouse cell lines WEHI3 (monocytic) and EL-4 (thymoma, T cell). The cloning of cDNA and genomic clones encoding mSpalpha and the identification of cells expressing a putative mSpalpha receptor(s) should facilitate in vivo studies designed to investigate the function of Spalpha in the immune compartment.

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

    PubMed Central

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

    1985-01-01

    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. Images PMID:3905610

  12. [Cloning, expression and charaterization of chalcone synthase from Saussurea medusa].

    PubMed

    Xia, Fang; Li, Houhua; Fu, Chunxiang; Yu, Zhenzhen; Xu, Yanjun; Zhao, Dexiu

    2011-09-01

    A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1313 bp, containing an open reading frame (1170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

  13. Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

    PubMed

    Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

    2012-01-01

    Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

  14. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    PubMed

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  15. Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1.

    PubMed Central

    Bogdan, J A; Adams-Burton, C; Pedicord, D L; Sukovich, D A; Benfield, P A; Corjay, M H; Stoltenborg, J K; Dicker, I B

    1998-01-01

    The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact. PMID:9820826

  16. Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

    PubMed Central

    Harlow, E; Williamson, N M; Ralston, R; Helfman, D M; Adams, T E

    1985-01-01

    Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules. Images PMID:3894933

  17. Global gene expression profiles reveal significant nuclear reprogramming by the blastocyst stage after cloning.

    PubMed

    Smith, Sadie L; Everts, Robin E; Tian, X Cindy; Du, Fuliang; Sung, Li-Ying; Rodriguez-Zas, Sandra L; Jeong, Byeong-Seon; Renard, Jean-Paul; Lewin, Harris A; Yang, Xiangzhong

    2005-12-06

    Nuclear transfer (NT) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. Here, we compared global gene expression profiles of individual bovine NT blastocysts with their somatic donor cells and fertilized control embryos using cDNA microarray technology. The NT embryos' gene expression profiles were drastically different from those of their donor cells and closely resembled those of the naturally fertilized embryos. Our findings demonstrate that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, problems may occur during redifferentiation for tissue genesis and organogenesis, and small reprogramming errors may be magnified downstream in development.

  18. A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

    PubMed Central

    Rayner, J R; Gonda, T J

    1994-01-01

    cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes. Images PMID:8289827

  19. Molecular cloning and expression of the mouse ornithine decarboxylase gene.

    PubMed Central

    McConlogue, L; Gupta, M; Wu, L; Coffino, P

    1984-01-01

    We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue. Images PMID:6582509

  20. Establishment of bipotent progenitor cell clone from rat skeletal muscle.

    PubMed

    Murakami, Yousuke; Yada, Erica; Nakano, Shin-ichi; Miyagoe-Suzuki, Yuko; Hosoyama, Tohru; Matsuwaki, Takashi; Yamanouchi, Keitaro; Nishihara, Masugi

    2011-12-01

    The present study describes the isolation, cloning and characterization of adipogenic progenitor cells from rat skeletal muscle. Among the obtained 10 clones, the most highly adipogenic progenitor, 2G11 cells, were further characterized. In addition to their adipogenicity, 2G11 cells retain myogenic potential as revealed by formation of multinucleated myotubes when co-cultured with myoblasts. 2G11 cells were resistant to an inhibitory effect of basic fibroblast growth factor on adipogenesis, while adipogenesis of widely used preadipogenic cell line, 3T3-L1 cells, was suppressed almost completely by the same treatment. In vivo transplantation experiments revealed that 2G11 cells are able to possess both adipogenicity and myogenicity in vivo. These results indicate the presence of bipotent progenitor cells in rat skeletal muscle, and suggest that such cells may contribute to ectopic fat formation in skeletal muscle.

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

    PubMed Central

    OKAYAMA, Hiroto

    2012-01-01

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

  2. 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. 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.

  4. Establishment of mouse-immortalized hybrid clones expressing characteristics of differentiated neurons derived from the cerebellar and brain stem regions.

    PubMed

    Satoh, J; Gallyas, F; Endoh, M; Yamamura, T; Kunishita, T; Kobayashi, T; Tabira, T

    1992-09-01

    Two clonal immortalized neurons designated CL8c4.7 and CL8a5.2 were established by somatic cell fusion between a hypoxanthine phosphoribosyltransferase-(HPRT-) deficient neuroblastoma N18TG2 and newborn mouse cerebellar/brain stem neurons. In the serum-containing medium without extra differentiating agents, both clones exhibited a morphology of differentiated neurons. They contained high levels of glutamate but no gamma-aminobutyric acid (GABA). The CL8a5.2 clone synthesized choline acetyltransferase and serotonin. In immunocytochemical studies, both clones expressed 200 kD neurofilament protein, neuron-specific enolase, microtubule-associated protein 2 (MAP2), tau protein, neuronal cell adhesion molecule (N-CAM), HNK-1, Thy-1.2, saxitoxin-binding sodium channel protein, and glutamate. Synaptophysin immunoreactivity was identified in the neuritic terminals of CL8c4.7 cells. Most of these antigens were barely detectable on N18TG2 cells. Electrophysiologically, both clones generated action potentials in response to electrical stimuli. The hybrid clones that express characteristics of differentiated neurons derived from the cerebellar and brain stem regions might be invaluable for the study of the molecular basis of neuronal differentiation and degeneration in these regions.

  5. 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…

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    PubMed

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  12. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    PubMed

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  13. Gene expression in cotton (Gossypium hirsutum L.) fiber: cloning of the mRNAs.

    PubMed

    John, M E; Crow, L J

    1992-07-01

    Cotton, an important natural fiber, is a differentiated epidermal cell. The number of genes that are active in fiber cells is similar to those in leaf, ovule, or root tissues. Through differential screening of a fiber cDNA library, we isolated five cDNA clones that are preferentially expressed in fiber. One of the cDNA clones, pCKE6, corresponded to an abundant mRNA in fiber. Transcripts for E6 were detected throughout the development of the fiber. Immunoprecipitation of in vitro translation products and Western blot analysis of fiber proteins showed two polypeptides in the range of 30-32 kDa as the products of E6 mRNA. Sequence analysis and hybrid-selected RNA translation also suggest that E6 mRNAs encode two polypeptides. Concentrations of E6 mRNA and protein are highest during the late primary cell wall and early secondary cell wall synthesis stages. Sequence comparison of E6 with other known eukaryotic and prokaryotic genes reveals no significant homology (GenBank; December 1991). E6 or a homologous gene(s) is conserved in several members of Malvaceae as well as in one other fiber-producing plant, kapok, but is not found in several other plants examined or in Acetobacter xylinum. A genomic clone corresponding to pCKE6 was isolated, and the promoter element of the E6 gene was shown to direct the expression of a carrot extensin mRNA in a tissue-specific and developmentally regulated fashion in transgenic cotton plants.

  14. Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen, using linkage of cotransfected markers.

    PubMed

    Bill, J; Palmer, E; Jones, C

    1987-09-01

    We report the molecular cloning of a human gene MER-2 located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2 gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO X human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2 gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2 expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2 expression.

  15. 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

  16. 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

  17. Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system.

    PubMed Central

    Piccioli, P; Ruberti, F; Biocca, S; Di Luzio, A; Werge, T M; Bradbury, A; Cattaneo, A

    1991-01-01

    We present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles. Images PMID:1712102

  18. 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.

  19. 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.

  20. 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.

  1. Human cloning, stem cell research. An Islamic perspective.

    PubMed

    Al-Aqeel, Aida I

    2009-12-01

    The rapidly changing technologies that involve human subjects raise complex ethical, legal, social, and religious issues. Recent advances in the field of cloning and stem cell research have introduced new hopes for the treatment of serious diseases. But this promise has raised many complex questions. This field causes debate and challenge, not only among scientists but also among ethicists, religious scholars, governments, and politicians. There is no consensus on the morality of human cloning, even within specific religious traditions. In countries in which religion has a strong influence on political decision making, the moral status of the human embryo is at the center of the debate. Because of the inevitable consequences of reproductive cloning, it is prohibited in Islam. However, stem cell research for therapeutic purposes is permissible with full consideration, and all possible precautions in the pre-ensoulment stages of early fetus development, if the source is legitimate.

  2. 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.

  3. Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

    PubMed

    Lipman, M L; Panda, D; Bennett, H P; Henderson, J E; Shane, E; Shen, Y; Goltzman, D; Karaplis, A C

    1998-05-29

    Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

  4. Human pyridoxal phosphatase. Molecular cloning, functional expression, and tissue distribution.

    PubMed

    Jang, Young Min; Kim, Dae Won; Kang, Tae-Cheon; Won, Moo Ho; Baek, Nam-In; Moon, Byung Jo; Choi, Soo Young; Kwon, Oh-Shin

    2003-12-12

    Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 microM and 1.52 s(-1), respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

  5. Cloning, expression, functional validation and modeling of cinnamyl alcohol dehydrogenase isolated from xylem of Leucaena leucocephala.

    PubMed

    Pandey, Brijesh; Pandey, Veda Prakash; Dwivedi, Upendra Nath

    2011-10-01

    A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.

  6. 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.

  7. Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

    PubMed

    Feng, Q L; Ladd, T R; Tomkins, B L; Sundaram, M; Sohi, S S; Retnakaran, A; Davey, K G; Palli, S R

    1999-02-25

    We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

  8. Single Cell Clones Purified from Human Parotid Glands Display Features of Multipotent Epitheliomesenchymal Stem Cells

    PubMed Central

    Yi, TacGhee; Lee, Songyi; Choi, Nahyun; Shin, Hyun-Soo; Kim, Junghee; Lim, Jae-Yol

    2016-01-01

    A better understanding of the biology of tissue-resident stem cell populations is essential to development of therapeutic strategies for regeneration of damaged tissue. Here, we describe the isolation of glandular stem cells (GSCs) from a small biopsy specimen from human parotid glands. Single colony-forming unit-derived clonal cells were isolated through a modified subfractionation culture method, and their stem cell properties were examined. The isolated clonal cells exhibited both epithelial and mesenchymal stem cell (MSC)-like features, including differentiation potential and marker expression. The cells transiently displayed salivary progenitor phenotypes during salivary epithelial differentiation, suggesting that they may be putative multipotent GSCs rather than progenitor cells. Both epithelial and mesenchymal-expressing putative GSCs, LGR5+CD90+ cells, were found in vivo, mostly in inter-secretory units of human salivary glands. Following in vivo transplantation into irradiated salivary glands of mice, these cells were found to be engrafted around the secretory complexes, where they contributed to restoration of radiation-induced salivary hypofunction. These results showed that multipotent epitheliomesenchymal GSCs are present in glandular mesenchyme, and that isolation of homogenous GSC clones from human salivary glands may promote the precise understanding of biological function of bona fide GSCs, enabling their therapeutic application for salivary gland regeneration. PMID:27824146

  9. Cloning of ES cells and mice by nuclear transfer.

    PubMed

    Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

    2009-01-01

    We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

  10. Expression cloning of the murine interferon gamma receptor cDNA.

    PubMed

    Munro, S; Maniatis, T

    1989-12-01

    A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.

  11. Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

    PubMed Central

    Pilbrough, Warren; Munro, Trent P.; Gray, Peter

    2009-01-01

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise

  12. Molecular cloning and expression of rat liver aminopeptidase B.

    PubMed

    Fukasawa, K M; Fukasawa, K; Kanai, M; Fujii, S; Harada, M

    1996-11-29

    We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

  13. Evolution of multiple cell clones over a 29-year period of a CLL patient

    PubMed Central

    Zhao, Zhikun; Goldin, Lynn; Liu, Shiping; Wu, Liang; Zhou, Weiyin; Lou, Hong; Yu, Qichao; Tsang, Shirley X.; Jiang, Miaomiao; Li, Fuqiang; McMaster, MaryLou; Li, Yang; Lin, Xinxin; Wang, Zhifeng; Xu, Liqin; Marti, Gerald; Li, Guibo; Wu, Kui; Yeager, Meredith; Yang, Huanming; Xu, Xun; Chanock, Stephen J.; Li, Bo; Hou, Yong; Caporaso, Neil; Dean, Michael

    2016-01-01

    Chronic lymphocytic leukaemia (CLL) is a frequent B-cell malignancy, characterized by recurrent somatic chromosome alterations and a low level of point mutations. Here we present single-nucleotide polymorphism microarray analyses of a single CLL patient over 29 years of observation and treatment, and transcriptome and whole-genome sequencing at selected time points. We identify chromosome alterations 13q14−, 6q− and 12q+ in early cell clones, elimination of clonal populations following therapy, and subsequent appearance of a clone containing trisomy 12 and chromosome 10 copy-neutral loss of heterogeneity that marks a major population dominant at death. Serial single-cell RNA sequencing reveals an expression pattern with high FOS, JUN and KLF4 at disease acceleration, which resolves following therapy, but reoccurs following relapse and death. Transcriptome evolution indicates complex changes in expression occur over time. In conclusion, CLL can evolve gradually during indolent phases, and undergo rapid changes following therapy. PMID:27982015

  14. Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties.

    PubMed

    Joo, Seong Soo; Ryu, In Wang; Park, Ji-Kook; Yoo, Yeong Min; Lee, Dong-Hyun; Hwang, Kwang Woo; Choi, Hyoung-Tae; Lim, Chang-Jin; Lee, Do Ik; Kim, Kyunghoon

    2008-02-29

    Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.

  15. 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

  16. 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.

  17. Cloning, expression and purification of the SRCR domains of glycoprotein 340.

    PubMed

    Purushotham, Sangeetha; Deivanayagam, Champion

    2013-08-01

    Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340's domains and oral streptococcal AgI/II domains, we undertook to express human gp340's first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

  18. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

    PubMed

    Katsuyama, M; Nishigaki, N; Sugimoto, Y; Morimoto, K; Negishi, M; Narumiya, S; Ichikawa, A

    1995-09-25

    A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 > iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

  19. Murine muscle-specific enolase: cDNA cloning, sequence, and developmental expression.

    PubMed Central

    Lamandé, N; Mazo, A M; Lucas, M; Montarras, D; Pinset, C; Gros, F; Legault-Demare, L; Lazar, M

    1989-01-01

    In vertebrates, the glycolytic enzyme enolase (EC 4.2.1.11) is present as homodimers and heterodimers formed from three distinct subunits of identical molecular weight, alpha, beta, and gamma. We report the cloning and sequencing of a cDNA encoding the beta subunit of murine muscle-specific enolase. The corresponding amino acid sequence shows greater than 80% homology with the beta subunit from chicken obtained by protein sequencing and with alpha and gamma subunits from rat and mouse deduced from cloned cDNAs. In contrast, there is no homology between the 3' untranslated regions of mouse alpha, beta, and gamma enolase mRNAs, which also differ greatly in length. The short 3' untranslated region of beta enolase mRNA accounts for its distinct length, 1600 bases. It is known that a progressive transition from alpha alpha to beta beta enolase occurs in developing skeletal muscle. We show that this transition mainly results from a differential regulation of alpha and beta mRNA levels. Analysis of myogenic cell lines shows that beta enolase gene is expressed at the myoblast stage. Moreover, transfection of premyogenic C3H10T1/2 cells with MyoD1 cDNA shows that the initial expression of beta transcripts occurs during the very first steps of the myogenic pathway, suggesting that it could be a marker event of myogenic lineage determination. Images PMID:2734297

  20. A new set of useful cloning and expression vectors derived from pBlueScript.

    PubMed

    Mayer, M P

    1995-09-22

    A new set of cloning vectors derived from pBlueScript (Stratagene, La Jolla, CA, USA) is presented. The ampicillin-resistance-encoding gene (ApR) of pBlueScript has been replaced by genes encoding resistance to either kanamycin (KmR) or tetracycline (TcR). The origin of DNA replication (ori), conferring to pBlueScript a very high-copy-number (500-700 copies/chromosome), has been replaced by the pBR322 ori (15-20 copies/chromosome) or the P15A ori (10-12 copies/chromosome) [Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989]. Therefore, eight new vectors with different drug selection markers and low, medium or high plasmid copy-number were created which are compatible with each other (ColE1 ori and P15A ori) and can be selected to replace one another. These vectors were further modified by the insertion of an expression cassette based on the promoter and AraC repressor/activator of the ara operon, which allows high-level expression, extremely tight regulation and very inexpensive induction. High-level expression of one or two genes within the same cell is demonstrated.

  1. New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally

    PubMed Central

    1989-01-01

    We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM- 2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation. PMID:2466037

  2. [Stem cells--cloning, plasticity, bioethic].

    PubMed

    Pflegerl, Pamina; Keller, Thomas; Hantusch, Brigitte; Hoffmann, Thomas Sören; Kenner, Lukas

    2008-01-01

    Stem cells with certain characteristics have become promising tools for molecular medicine. They have the potential to self-regenerate and to differentiate into specific tissues. Besides their great potential, embryonic stem cells (ESC) run the risk of enhanced tumorigenesis. The use of human embryonic stem cells (hESC) is ethically problematic because their isolation involves the destruction of human embryos. Recently developed methods generate are able to pluripotent stem cells from fibroblasts. Alternatives for ESC are adult stem cells (ASC) derived from bone marrow, cord blood, amniotic fluid and other tissues. The following article is on the basis of testimony of Lukas Kenner for the German Bundestag about the use of ESC for research, therapy and drug development. Ethical aspects are taken into consideration.

  3. International policy failures: cloning and stem-cell research.

    PubMed

    Tauer, Carol A

    In late 2003, two international bodies were unable to resolve disagreements that involved bioethical issues. First, the United Nations General Assembly failed to pass a treaty on reproductive cloning because of insistence by some countries that the treaty include a ban on cloning for research. In view of the importance of enacting prohibition of reproductive cloning, the two issues should be separated and each argued on its own merits. Relevant objections to separation of the two issues can be refuted. Second, the European Union (EU) failed to agree on conditions for funding stem-cell research because of the diversity of views and policies of the countries of the EU. Because a stalemate was reached, funding decisions in the next programme cycle will be made on an ad hoc basis. Scientists will not have information they need to plan research programmes, suggesting that clear guidelines, even if restrictive, are preferable to vague unpublicised criteria.

  4. Cloning of the rat ecotropic retroviral receptor and studies of its expression in intestinal tissues

    SciTech Connect

    Puppi, M.; Henning, S.J.

    1995-05-01

    A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5{prime} end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5{prime} untranslated region and a 3{prime} poly(A)tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y{sup +} cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium. 37 refs., 4 figs.

  5. Effect of culture medium type on canine adipose-derived mesenchymal stem cells and developmental competence of interspecies cloned embryos.

    PubMed

    Kim, Geon A; Oh, Hyun Ju; Lee, Tae Hee; Lee, Ji Hyun; Oh, Sang Hwan; Lee, Ju Hyun; Kim, Jin Wook; Kim, Se Woon; Lee, Byeong Chun

    2014-01-15

    Canine adipose-derived mesenchymal stem cells (ASCs) are promising as donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that different cell cultures possess different capacities to support pre-implantation development of SCNT embryos. The aim of this study is to investigate whether two culture medium (RCMEP, Dulbecco's modified Eagle's medium [DMEM]) affect gene expression of ASCs, subsequent development of interspecies SCNT (iSCNT) and gene expression of cloned embryos. The RCMEP-cultured cells contained significantly greater amounts of SOX2, NANOG, OCT4, DNMT1, and MeCP2 than DMEM-cultured cells (P < 0.05). In iSCNT, the use of DMEM medium for culturing cells resulted in similar development to the blastocyst stage than those derived from RCMEP cultured cells (4.5% and 3.2%, respectively; P > 0.05). The expression of all transcripts except for DNMT1 in cloned blastocysts from RCMEP cultured cells followed those of cloned blastocysts derived from DMEM cultured cells. The alteration of gene expression in ASCs by culture medium was not manifested in the iSCNT embryos derived from these cells. Although the culture medium can induce changes of gene expression by ASCs, such alterations in donor cells did not affect the developmental competence or gene expression patterns of iSCNT embryos.

  6. 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.

  7. 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

    Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua; Liu, Dewu; Wu, Zhenfang

    2013-10-01

    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.

  8. [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.

  9. Expression of cloned α6* nicotinic acetylcholine receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Lindstrom, Jon

    2015-09-01

    Nicotinic acetylcholine receptors (AChRs) are ACh-gated ion channels formed from five homologous subunits in subtypes defined by their subunit composition and stoichiometry. Some subtypes readily produce functional AChRs in Xenopus oocytes and transfected cell lines. α6β2β3* AChRs (subtypes formed from these subunits and perhaps others) are not easily expressed. This may be because the types of neurons in which they are expressed (typically dopaminergic neurons) have unique chaperones for assembling α6β2β3* AChRs, especially in the presence of the other AChR subtypes. Because these relatively minor brain AChR subtypes are of major importance in addiction to nicotine, it is important for drug development as well as investigation of their functional properties to be able to efficiently express human α6β2β3* AChRs. We review the issues and progress in expressing α6* AChRs. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  10. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.

    PubMed

    Cases, S; Novak, S; Zheng, Y W; Myers, H M; Lear, S R; Sande, E; Welch, C B; Lusis, A J; Spencer, T A; Krause, B R; Erickson, S K; Farese, R V

    1998-10-09

    The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a approximately 46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3beta-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

  11. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

    SciTech Connect

    Hao, Z.; Chen, S.; Wilson, D.B.

    1999-11-01

    An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake system that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.

  12. Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

    DTIC Science & Technology

    2005-01-01

    with Brucella melitensis WR201(16MDeltapurEK), immunized intramuscularly with dialyzed cell lysate of Infect. Immun. 67 (1999) 5877-5884. B. melitensis ...bacterioferritin gene of Brucella melitensis 16M strain, FEBS Lett. 361 (2-3) (1995) 238-242. serum and derived IgG had strong reaction to the Bru- [7] L.E. Lindler...detection by the antiserum. The pro- nucleotide sequence, and expression of the Brucella melitensis tein samples were prepared by treatment of WRR51 omp31

  13. Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.

    PubMed

    Yang, Feikun; Hao, Ru; Kessler, Barbara; Brem, Gottfried; Wolf, Eckhard; Zakhartchenko, Valeri

    2007-01-01

    The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned

  14. Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli.

    PubMed Central

    Heinzen, R A; Frazier, M E; Mallavia, L P

    1992-01-01

    A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia. Images PMID:1500190

  15. 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.

  16. [Cloning, expression and characterization of a new hybrid AMP gene of Hex-Mag].

    PubMed

    Li, Gui-ping; Chen, Yi-ben

    2007-02-01

    To enhance the antibacterial ability of Magaininl-12, its N side was joined with an alkaline peptide named Hexapeptide( RRWQWR), which would make Magaininl-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris, a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin( 1-12). Synthesized through gene splicing by overlap extension, the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation, the hybrid antibacterial peptide was expressed on high level. The new peptide, which has a weight of 2.3kDa, could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay, Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.

  17. Cloning of the rat ecotropic retroviral receptor and studies of its expression in intestinal tissues.

    PubMed

    Puppi, M; Henning, S J

    1995-05-01

    A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal tissues. The cDNA for rat EcoR was cloned by screening a size-selected rat intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5' end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5' untranslated region and a 3' poly(A) tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y+ cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium.

  18. 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.

  19. Molecular Cloning, Characterization, and Expression of the M Antigen of Histoplasma capsulatum

    PubMed Central

    Zancopé-Oliveira, Rosely M.; Reiss, Errol; Lott, Timothy J.; Mayer, Leonard W.; Deepe, George S.

    1999-01-01

    The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2,187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity. PMID:10085041

  20. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    PubMed

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  1. T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells

    PubMed Central

    Witkover, Aviva; Tanaka, Yuetsu; Fields, Paul; Bangham, Charles R. M.

    2016-01-01

    There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease. PMID:27893842

  2. T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells.

    PubMed

    Rowan, Aileen G; Witkover, Aviva; Melamed, Anat; Tanaka, Yuetsu; Cook, Lucy B M; Fields, Paul; Taylor, Graham P; Bangham, Charles R M

    2016-11-01

    There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease.

  3. SLiCE: a novel bacterial cell extract-based DNA cloning method.

    PubMed

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-04-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

  4. Promethean medicine: spirituality, stem cells, and cloning.

    PubMed

    Sulmasy, Daniel P

    2006-12-01

    Every ethos implies a mythos. That is, every ethical system depends upon some fundamental story disclosing its assumptions about human nature, freedom, good and evil, and the workings of the universe. A romanticized version of the myth of Prometheus, who stole fire from the gods and was punished by being chained to a rock and having his liver plucked out by vultures, seems to under-gird much of contemporary healthcare. Christianity offers a different view--one in which the universe is not a zero sum game and human beings do not need to steal fire because God has already freely given them all the fire they need in Christ and in his spirit. A critical virtue for physicians, taught by Christianity, is sagacious engagement--the ability to engage the world practically, discerning what can and should be changed and what should be accepted as unchangeable and given. The illusory quest for immortality through the practice of regenerative medicine using stem cells is a gross violation of that virtue.

  5. Tandem recombineering by SLIC cloning and Cre-LoxP fusion to generate multigene expression constructs for protein complex research.

    PubMed

    Haffke, Matthias; Viola, Cristina; Nie, Yan; Berger, Imre

    2013-01-01

    A robust protocol to generate recombinant DNA containing multigene expression cassettes by using sequence and ligation independent cloning (SLIC) followed by multiplasmid Cre-LoxP recombination in tandem for multiprotein complex research is described. The protocol includes polymerase chain reaction (PCR) amplification of the desired genes, seamless insertion into the target vector via SLIC, and Cre-LoxP recombination of specific donor and acceptor plasmid molecules, optionally in a robotic setup. This procedure, called tandem recombineering, has been implemented for multiprotein expression in E. coli and mammalian cells, and also for insect cells using a recombinant baculovirus.

  6. Elongation and gene expression in bovine cloned embryos transferred to temporary recipients.

    PubMed

    Rodríguez-Alvarez, Lleretny; Cox, José; Navarrete, Felipe; Valdés, Cristián; Zamorano, Teresa; Einspanier, Ralf; Castro, Fidel Ovidio

    2009-11-01

    SummaryElongated embryos provide a unique source of information about trophoblastic differentiation, gene expression and maternal-embryonic interactions; however they are difficult and costly to obtain, especially elongated cloned embryos. One alternative is their production in heterologous temporary recipients such as sheep and goats. We aimed to produce elongated bovine cloned embryos using heterologous transfer to temporary recipients. Day-7 cloned cattle blastocysts were transferred to the uteri of ewes and goats and recovered as elongated structures at day 17. We evaluated elongation, length, presence of embryonic disc and expression of several important genes for embryonic development. We also produced homologous (cloned cattle embryos transferred into cattle uteri). Cloned bovine blastocysts were able to proceed with preimplantation development through elongation with high efficiency despite the species to which they were transferred. In qualitative and quantitative RT-PCR experiments we found differences in the pattern of gene expression among embryos recovered from different species. Sox2, Nanog and FGF-4 were markedly deregulated. No previous reports about the expression pattern of the studied genes had been published for elongated bovine cloned embryos produced in intermediate recipients, furthermore, the pattern of expression of Nanog, Oct4, Eomes, Cdx2, IFN-tau, Dicer, FGF-4 and Sox2 shown here are novel for elongated cloned bovine embryos created by hand-made cloning. Our data confirmed that sheep and goats can be used as temporary recipients. This model could serve as a basis for further research on gene expression and cellular changes during bovine peri-implantation development.

  7. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

    PubMed

    Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

    2016-01-01

    Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

  8. 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.

  9. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

    PubMed Central

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

  10. [Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

    PubMed

    An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

    2012-12-01

    We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

  11. 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.

  12. 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.

  13. Replication of IMR-32-adapted JC virus clones in human embryonic kidney cells.

    PubMed

    Nukuzuma, Souichi; Sugiura, Shigeki; Nakamichi, Kazuo; Kameoka, Masanori; Nukuzuma, Chiyoko; Tasaki, Takafumi; Takegami, Tsutomu

    2015-04-01

    It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT-PCR assay revealed that large T antigen expression in cells transfected with IMR-32-adapted JCVs was significantly greater than in those transfected with Mad-1 or CY. DNA replication assay and viral load verified that the IMR-32-adapted JCVs were replication-competent in 293 cells, but not Mad-1 or CY JCVs. These results suggest that a 293 culture system with IMR-32-adapted JCVs may be a useful tool for assessing replication of JCV in vitro.

  14. [Cloning of mMR-1 gene and expression in Pichia pastoris systems].

    PubMed

    Li, Tian-Bo; Hu, Yang; Wang, Yi-Guang; Xia, Huan-Zhang

    2005-01-01

    hMR-1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our laboratory. The former studies revealed that hMR-1 is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain, alpha-enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR-1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR-1 gene homologous sequence on mouse genome chromosome 1. The mouse MR-1 gene (mMR-1) was cloned by PCR following the first round RT-PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR-1 gene and amino acids sequence showed 90.4% and 90.1% identity with hMR-1, respectively. The prediction of hydrophobic transmembrane structure of mMR-1 suggested it is also a transmembrane protein. The mMR-1 Pichia pastoris expression vector pPIC9-mMR-1 was constructed by fusion of the flanking mMR-1 ORF in the pPIC9 plasmid. After linearization of pPIC9-mMR-1 with Sal I, the 8.5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut+ pPIC9-mMR-1 transformants were selected on minimal methanol medium. Integration of mMR-1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR-1 protein was expressed by induction under the concentration of 0.5 % methanol. The specific induced protein of 25 kD molecular mass in SDS-PAGE was confirmed to be the mMR-1 protein by Western blot rsing hMR-1 polyclonal antibody. The expression level of this recombinant mMR-1 protein was about 50 mg/L. The successful expression of mMR-1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR-1 in animal model.

  15. Assessment and characterization of Ca2+-ATPase expression in selected isolates and clones of Plasmodium falciparum.

    PubMed

    Bolaji, O M; Happi, T C; Bababunmi, E A

    2012-06-07

    Ca2+-ATPase expression in 15 selected isolates from malaria patients at the University College Hospital (UCH) Ibadan and two cloned strains (W2-chloroquine resistant, D6-chloroquine sensitive) of P.falciparum was assessed using spectrophotometric assay method. The kinetics of activity of Ca2+- ATPase in three isolates (NCP 14, NCP5, NCP1) and two clones (W2, D6) also assessed. 12% SDS-PAGE analysis of total proteins in one isolate (NCP14) and two clones (W2, D6) was also investigated. All the selected isolates and the two cloned strains exhibited measurable Ca2+-ATPase activity. The Ca2+-ATPase activity in cloned strain D6 (6.50 + 0.74mmolPi/min/mg protein) was higher than in cloned strain W2 (3.93 + 0.61mmolPi/min/mg protein. The Ca2+-ATPase activity in isolates from malaria patients varied widely (1.95 + 0.74 - 21.56 +1.43mmolPi/min/mg protein). The kinetic constants obtained for the two cloned strains showed that clone W2 had a higher Vmax (Vmax = 363mmolPi/min/mg protein) than clone D6 (Vmax = 74mmolPi/min/mg protein). All the isolates and the two cloned strains showed similar affinity for ATP (Km ~ 10mM). Scan of SDS-PAGE gel of total proteins in the isolate and cloned strains showed the presence of oligopeptide bands of molecular weights range of 148-176 kDa; 116-123 kDa respectively. These suggest the presence of predicted polypeptide of Ca2+-ATPase nature of molecular weight estimate of 139 kDa. The study agrees with previous findings that Ca2+-ATPase is functionally expressed in P.falciparum, The study also indicates that Ca2+-ATPase functional expression may vary with isolate or clone but the ATP binding mechanism to the enzyme is similar in all isolates and clones of P. falciparum. The study further suggests a possible association between acquisition of chloroquine resistance and Ca2+-ATPase functional expression in P. falciparum.

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

    PubMed

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

    1992-07-01

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

  17. Cloning and expression of a rat brain GABA transporter

    SciTech Connect

    Guastella, J.; Czyzyk, L.; Davidson, N.; Lester, H.A. ); Nelson, N.; Nelson, H.; Miedel, M.C. ); Keynan, S.; Kanner, B.I. )

    1990-09-14

    A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter {gamma}-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated ({sup 3}H)GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium- and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

  18. Generation of Isogenic D4Z4 Contracted and Noncontracted Immortal Muscle Cell Clones from a Mosaic Patient

    PubMed Central

    Krom, Yvonne D.; Dumonceaux, Julie; Mamchaoui, Kamel; den Hamer, Bianca; Mariot, Virginie; Negroni, Elisa; Geng, Linda N.; Martin, Nicolas; Tawil, Rabi; Tapscott, Stephen J.; van Engelen, Baziel G.M.; Mouly, Vincent; Butler-Browne, Gillian S.; van der Maarel, Silvère M.

    2013-01-01

    In most cases facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat in the 4q subtelomere. This contraction is associated with local chromatin decondensation and derepression of the DUX4 retrogene. Its complex genetic and epigenetic cause and high clinical variability in disease severity complicate investigations on the pathogenic mechanism underlying FSHD. A validated cellular model bypassing the considerable heterogeneity would facilitate mechanistic and therapeutic studies of FSHD. Taking advantage of the high incidence of somatic mosaicism for D4Z4 repeat contraction in de novo FSHD, we have established a clonal myogenic cell model from a mosaic patient. Individual clones are genetically identical except for the size of the D4Z4 repeat array, being either normal or FSHD sized. These clones retain their myogenic characteristics, and D4Z4 contracted clones differ from the noncontracted clones by the bursts of expression of DUX4 in sporadic nuclei, showing that this burst-like phenomenon is a locus-intrinsic feature. Consequently, downstream effects of DUX4 expression can be observed in D4Z4 contracted clones, like differential expression of DUX4 target genes. We also show their participation to in vivo regeneration with immunodeficient mice, further expanding the potential of these clones for mechanistic and therapeutic studies. These cell lines will facilitate pairwise comparisons to identify FSHD-specific differences and are expected to create new opportunities for high-throughput drug screens. PMID:22871573

  19. Molecular cloning and expression of the IL-10 gene from guinea pigs.

    PubMed

    Dirisala, Vijaya R; Jeevan, Amminikutty; Bix, Gregory; Yoshimura, Teizo; McMurray, David N

    2012-04-25

    The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project

  20. Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium.

    PubMed

    Biase, Fernando H; Rabel, Chanaka; Guillomot, Michel; Hue, Isabelle; Andropolis, Kalista; Olmstead, Colleen A; Oliveira, Rosane; Wallace, Richard; Le Bourhis, Daniel; Richard, Christophe; Campion, Evelyne; Chaulot-Talmon, Aurélie; Giraud-Delville, Corinne; Taghouti, Géraldine; Jammes, Hélène; Renard, Jean-Paul; Sandra, Olivier; Lewin, Harris A

    2016-12-20

    A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

  1. Graft-versus-host resistance induced by class II major histocompatibility complex-specific T cell clones

    PubMed Central

    1991-01-01

    Possible mechanisms of graft-vs.-host (GVH) resistance have been studied using a panel of seven class II major histocompatibility complex-specific T cell clones for elicitation and challenge. One clone recognized I-Ak,d,f, and expressed V beta 8.3 together with J beta 1.5. The remaining six clones were I-Ek specific and expressed V beta 15 rearranged to J beta 1.1 or J beta 1.3. The I-Ek-specific clones were also homologous to each other and different from the I-A-reactive one in the D and N regions. Four of the seven clones exhibited I-Ek- specific cytolytic activity. Each clone, when injected in sublethal numbers into appropriate recipients, could induce resistance to a subsequent lethal dose of any other clone in the panel. The resistance did not require sharing of either T cell receptor beta chains or antigen specificity, or MHC molecules by the eliciting and challenging clone. Cytolytic and noncytolytic clones were equally efficient in inducing GVH resistance. A prerequisite of resistance induction was the activation of eliciting clone subsequent to recognition of class II molecules in the host. Clones preactivated with high concentrations of recombinant interleukin 2, in vitro, could induce GVH resistance also in syngeneic hosts, suggesting that resistance induction was associated with the activated state of clone, rather than antigen recognition per se. In all instances of resistance, the challenging clones failed to induce vascular leakage, which was the cause of death in susceptible recipients (Lehmann, P. V., G. Schumm, D. Moon, U. Hurtenbach, F. Falcioni, S. Muller, and Z. A. Nagy. 1990. J. Exp. Med. 171:1485). Lipopolysaccharide (LPS) induced resistance to vascular leakage did not provide crossresistance to GVH and vice versa, suggesting that interleukin 1 alpha and tumor necrosis factor alpha implicated in LPS resistance are not involved in GVH resistance. Although the mechanism remains unclear, the most likely explanation for GVH resistance in this

  2. Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit.

    PubMed

    Kaomek, Mana; Mizuno, Kouichi; Fujimura, Tatsuhito; Sriyotha, Poonsook; Cairns, James R Ketudat

    2003-04-01

    Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by PCR amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening. Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids. Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli produced high chitinase activity in the cell lysate. The recombinant thioredoxin fusion protein was purified and cleaved to yield a 32-kDa chitinase. The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity. It also inhibited growth of 13 of the 14 fungal strains tested.

  3. Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

    PubMed Central

    MATSUYAMA, Satoshi; NAKANO, Yuko; NAKAMURA, Mieko; YAMAMOTO, Ryohei; SHIMADA, Terumasa; OHASHI, Fumihito; KUBO, Kihei

    2014-01-01

    Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors. PMID:25312047

  4. Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Liu, Yang; Ge, Hui; Qiu, Xuemei

    2010-11-01

    The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene ( flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.

  5. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  6. Cloning and expression of N-glycosylation-related glucosidase from Glaciozyma antarctica

    NASA Astrophysics Data System (ADS)

    Yajit, Noor Liana Mat; Kamaruddin, Shazilah; Hashim, Noor Haza Fazlin; Bakar, Farah Diba Abu; Murad, Abd. Munir Abd.; Mahadi, Nor Muhammad; Mackeen, Mukram Mohamed

    2016-11-01

    The need for functional oligosaccharides in various field is ever growing. The enzymatic approach for synthesis of oligosaccharides is advantageous over traditional chemical synthesis because of the regio- and stereo- selectivity that can be achieved without the need for protection chemistry. In this study, the α-glucosidase I protein sequence from Saccharomyces cerevisiae (UniProt database) was compared using Basic Local Alignment Search Tool (BLAST) with Glaciozyma antarctica genome database. Results showed 33% identity and an E-value of 1 × 10-125 for α-glucosidase I. The gene was amplified, cloned into the pPICZα C vector and used to transform Pichia pastoris X-33 cells. Soluble expression of α-Glucosidase I (˜91 kDa) was achieved at 28 °C with 1.0 % of methanol.

  7. Molecular cloning and expression analysis of porcine ghrelin o-acyltransferase.

    PubMed

    Lin, Tonghui; Meng, Qingyong; Sui, Dandan; Peng, Dezhi; Li, Yang; Liu, Xiaofang; Xie, Longfei; Li, Ning

    2011-10-01

    The peptide hormone ghrelin is secreted in the stomach, with unique N-octanoylation at serine 3, which is a requirement for its functionality. These functions include growth hormone release, appetite stimulation, gastrointestinal motility, glucose regulation, and cell proliferation. The enzyme responsible for ghrelin acylation was recently identified as ghrelin O-acyltransferase (GOAT). In this study, porcine GOAT was cloned and characterized. A full-length cDNA of GOAT of 2013 bp was obtained, which included a 70-bp 5' UTR, a 635-bp 3' UTR, and a 1308-bp open reading frame encoding a protein of 415 amino acids. The GOAT and ghrelin mRNAs are co-expressed in stomach, pancreas, and duodenum at high levels. GOAT was also detected in liver, lung, brain, testis, spleen, kidney, heart, muscle, lipid, and ovary. Our results provide an important basis for further research on GOAT function and the relationship between ghrelin and GOAT.

  8. Molecular cloning and developmental expression of foxP2 in zebrafish.

    PubMed

    Bonkowsky, Joshua L; Chien, Chi-Bin

    2005-11-01

    Forkhead domain transcription factors are a large gene family with multiple roles in development. FOXP2, a recently identified member of this family, has been shown to be critical for normal development of language in humans, but little is known of its broader function during nervous system development. We report here the cloning of foxP2, the zebrafish ortholog of FOXP2. Zebrafish FoxP2 is highly conserved in its zinc-finger and forkhead domains, but lacks the large glutamine repeat characteristic of its orthologs. In examining the spatial and temporal distribution of foxP2 during development, we find that it is specifically expressed in many domains of the nervous system, including the telencephalon, diencephalon, cerebellum, hindbrain, tectum, retinal ganglion cells, and spinal cord. Thus, in addition to specific roles in language development, foxP2 likely has a more general conserved role in nervous system development.

  9. Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Mycoplasma Genitalium Protein MG289

    SciTech Connect

    Sippel, K.; Boehlein, S; Sakai, Y; Quirit, J; Agbandje-McKenna, M; Rosser, C; McKenna, R

    2009-01-01

    Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 {angstrom} resolution. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 {angstrom}. The diffraction data after processing had an overall R{sub merge} of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

  10. Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

    PubMed Central

    Garrow, T A; Admon, A; Shane, B

    1992-01-01

    A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria. PMID:1409616

  11. Cloning and expression analysis of three novel CC chemokine genes from Japanese flounder (Paralichthys olivaceus).

    PubMed

    Zou, Gang-gang; Nozaki, Reiko; Kondo, Hidehiro; Hirono, Ikuo

    2014-10-01

    Chemokines are small cytokines secreted by various cell types. They not only function in cell activation, differentiation and trafficking, but they also have influences on many biological processes. In this study, three novel CC chemokine genes Paol-SCYA105, 106 and 107 in Japanese flounder (Paralichthys olivaceus) were cloned and characterized. Paol-SCYA105 was mainly detected in gill, kidney and spleen, Paol-SCYA106 was detected in all tissues examined and Paol-SCYA107 was mainly detected in the spleen and kidney. Paol-SCYA105 and Paol-SCYA106 gene expressions peaked in kidney at day 3 after viral hemorrhagic septicemia virus infection and decreased at day 6, but Paol-SCYA106 still remained at a high level at day 6. Paol-SCYA107 gene expression was significantly up-regulated in kidney at day 6 after viral hemorrhagic septicemia virus infection. In response to infection by Gram-negative Edwardsiella tarda and Gram-positive Streptococcus iniae in kidney, only Paol-SCYA106 gene expression significantly increased. Together, these results indicate that these three novel CC chemokines are involved in the immune response against pathogen infections.

  12. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase

    SciTech Connect

    Holst, L.S.; Laurell, H.; Holm, C.

    1996-08-01

    By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSL{sub tes}. Due to an addition of amino acids at the NH{sub 2}-termini, rat and human HSL{sub tes} consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSL{sub adi}). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSL{sub adi}. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSL{sub adi} sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M{sub r} {approximately}120,000) that exhibited catalytic activity similar to that of HSL{sub adi}. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells. 34 refs., 5 figs.

  13. Molecular cloning, tissue expression, and subcellular localization of porcine peptidoglycan recognition proteins 3 and 4.

    PubMed

    Ueda, Wataru; Tohno, Masanori; Shimazu, Tomoyuki; Fujie, Hitomi; Aso, Hisashi; Kawai, Yasushi; Numasaki, Muneo; Saito, Tadao; Kitazawa, Haruki

    2011-09-15

    Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.

  14. Molecular cloning and prokaryotic expression of vp5 gene of grass carp reovirus strain GCRV096.

    PubMed

    Jian, Ji-chang; Wang, Ya; Yan, Xiu-ying; Ding, Yu; Wu, Zao-he; Lu, Yi-shan

    2013-12-01

    VP5 is an outer capsid protein of grass carp reovirus (GCRV). It is predicted to involve in helping GCRV enter the host cells. In this study, the full-length vp5 gene (accession number in GenBank: JN206664.1) was cloned from GCRV strain GCRV096, which was isolated from diseased grass carp (Ctenopharyngodon idella) in southern China by RT-PCR technique using the primers designed from the known vp5 gene sequences of other strains of GCRV published in GenBank. The ORF sequence of vp5 is composed of 1,947 nucleotides encoding a 648-residues protein with a calculated molecular mass of 68.6 kDa and an estimated isoelectric point of 6.1. Sequence analysis results showed that VP5 might serve as a penetration protein and play an important role in GCRV penetration into the host cells. A full length of vp5 gene was subcloned into the prokaryotic expression vector pET-28a (+) and the recombinant plasmid (pET/GCRV-VP5) was then transduced into Escherichia coli BL21 (DE3) cells to express VP5 in vitro. SDS-PAGE and western blotting analysis indicated that the protein expressed successfully. Results also showed that the fusion protein expressed in the form of inclusion body, and it expressed in the highest level when induced with 0.2-mM IPTG at 28 °C for 4 h. These results are important for the future study on the molecular structure, function, and immunogenicity of GCRV capsid protein.

  15. Molecular cloning and functional expression of a novel Helicobacter pylori alpha-1,4 fucosyltransferase.

    PubMed

    Rabbani, Said; Miksa, Viktoria; Wipf, Beat; Ernst, Beat

    2005-11-01

    Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is associated with gastric cancer and lymphoma. This microorganism synthesizes fucosylated oligosaccharides, predominantly the Galb-1,4GlcNAc (Type II) blood group antigens Lewis X and Y, whereas a small population also expresses the Galb-1,3GlcNAc (Type I) blood group antigens Lewis A and B. These carbohydrate structures are known to mimic host cell antigens and permit the bacteria to escape from the host immune response. Here, we report the cloning and characterization of a novel H. pylori alpha-1,4 fucosyltransferase (FucT). In contrast to the family members characterized to date, this enzyme shows exclusively Type I acceptor substrate specificity. The enzyme consisting of 432 amino acids (MW 50,502 Da) was cloned using a polymerase chain reaction (PCR)-based approach. It exhibits a high degree of identity (75-87%) and similar structural features, for example, in the heptamer repeat pattern, with other H. pylori FucTs. The kinetic characterization revealed a very efficient transferase (k(cat)/Km = 229 mM(-1) s(-1)) for the Type I acceptor substrate (Gal)-1,3 GlcNAc-Lem (1). Additionally, the enzyme possesses a broad tolerance toward nonnatural Type I acceptor substrate analogs and therefore represents a valuable tool for the chemoenzymatic synthesis of Lewis A, sialyl Lewis A as well as mimetics thereof.

  16. Cloning, sequencing, and expression of interferon-γ from elk in North America

    USGS Publications Warehouse

    Sweeney, Steven J.; Emerson, Carlene; Eriks, Inge S.

    2001-01-01

    Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-γ) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-γ from Cervidae. To begin to address this problem, the IFN-γ gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-γ (rElkIFN-γ) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-γ epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.

  17. Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon

    PubMed Central

    Barmeyer, Christian; Rahner, Christoph; Yang, Youshan; Sigworth, Frederick J.; Binder, Henry J.

    2010-01-01

    KCNN4 channels that provide the driving force for cAMP- and Ca2+-induced anion secretion are present in both apical and basolateral membranes of the mammalian colon. However, only a single KCNN4 has been cloned. This study was initiated to identify whether both apical and basolateral KCNN4 channels are encoded by the same or different isoforms. Reverse transcriptase-PCR (RT-PCR), real-time quantitative-PCR (RT-QPCR), and immunofluorescence studies were used to clone and identify tissue-specific expression of KCNN4 isoforms. Three distinct KCNN4 cDNAs that are designated as KCNN4a, KCNN4b, and KCNN4c encoding 425, 424, and 395 amino acid proteins, respectively, were isolated from the rat colon. KCNN4a differs from KCNN4b at both the nucleotide and the amino acid level with distinct 628 bp at the 3′-untranslated region and an additional glutamine at position 415, respectively. KCNN4c differs from KCNN4b by lacking the second exon that encodes a 29 amino acid motif. KCNN4a and KCNN4b/c are identified as smooth muscle- and epithelial cell-specific transcripts, respectively. KCNN4b and KCNN4c transcripts likely encode basolateral (40 kDa) and apical (37 kDa) membrane proteins in the distal colon, respectively. KCNN4c, which lacks the S2 transmembrane segment, requires coexpression of a large conductance K+ channel β-subunit for plasma membrane expression. The KCNN4 channel blocker TRAM-34 inhibits KCNN4b- and KCNN4c-mediated 86Rb (K+ surrogate) efflux with an apparent inhibitory constant of 0.6 ± 0.1 and 7.8 ± 0.4 μM, respectively. We conclude that apical and basolateral KCNN4 K+ channels that regulate K+ and anion secretion are encoded by distinct isoforms in colonic epithelial cells. PMID:20445171

  18. Cloning and In Situ Expression Studies of the Hydrogenobaculum Arsenite Oxidase Genes▿

    PubMed Central

    Clingenpeel, Scott R.; D'Imperio, Seth; Oduro, Harry; Druschel, Greg K.; McDermott, Timothy R.

    2009-01-01

    Novel arsenite [As(III)] oxidase structural genes (aoxAB) were cloned from Hydrogenobaculum bacteria isolated from an acidic geothermal spring. Reverse transcriptase PCR demonstrated expression throughout the outflow channel, and the aoxB cDNA clones exhibited distribution patterns relative to the physicochemical gradients in the spring. Microelectrode analyses provided evidence of quantitative As(III) transformation within the microbial mat. PMID:19304831

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

    PubMed

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

    1993-04-15

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

  20. [SPREADING OF NCTC CLONE 929 CELLS AFTER RESEEDING].

    PubMed

    Petrov, Yu P; Negulyaev, Yu A; Tsupkina, N V

    2015-01-01

    The period (1 h after reseeding) of behaviour of mouse NCTC clone 929 cells to the conditions of artificial cultivation was studied. The time-lapse imaging followed the processing of the cells with ImageJ program was applied. To characterize the parametres cell status we used the cell area (projection of the cell on substrate) and Rp/Ra ratio introduced earlier as a spreading coefficient (Kuz'minykh, Petrov, 2004). After attaching a substratum, cells have a form of sphere (the phase "sphere") as the daughter cells after a mitosis. We revealed however that after this phase the reseeded cells do not start usual spreading and migration along substratum. They pass a phase of equally spreading in all directions and shaping their area as a circle (phase "circle"). This phase is absent of the daughter cells spreading after mitosis. We assume that the phase "circle" is a result of adaptation of the cells to reseedings at artificial cultivation. It is necessary for formation of a substrate composed of own extracellular matrix components (ECM) of the cells. Own ECM facilitates transition of the cells to their usual spreading and migration along substratum.

  1. Cloning, expression and molecular analysis of Iranian Brucella melitensis Omp25 gene for designing a subunit vaccine

    PubMed Central

    Yousefi, Soheil; Tahmoorespur, Mojtaba; Sekhavati, Mohammad Hadi

    2016-01-01

    Brucellosis is a well-known domestic animal infectious disease, which is caused by Brucella bacterium. The outer membrane protein 25 kDa (Omp25) gene plays an important role in simulating of TNF-α, IFN-α, macrophage, and cytokines cells. In the current study molecular cloning and expression analysis of Omp25 gene for designing a subunit vaccine against Brucella was investigated. Amplifying the full length of candidate gene was performed using specific primers. Sub-cloning of this gene conducted using pTZ57R/T vector in TOP10F strain of Escherichia coli(E.coli) as the host. Also, pET32(a)+ vector used for expression in BL21 (DE3) strain of E.coli. Omp25 gene with 642 bp size was amplified and cloned successfully. The expression results were confirmed by sequencing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses which showed 42 kDa protein band correctly. Also, phylogenic analysis showed this gene has a near genetic relation with other Brucella strains. According to our results we can propose this gene as a candidate useful for stimulation of cell-mediated and humoral immunity system in future study. PMID:27920824

  2. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DsrEFH from Allochromatium vinosum

    SciTech Connect

    Dahl, Christiane; Schulte, Andrea; Shin, Dong Hae

    2007-10-01

    DsrEFH from Allochromatium vinosum has been cloned, expressed, purified, and crystallized. A preliminary X-ray study of DsrEFH has been performed with a good quality crystal. In purple sulfur bacteria, the proteins encoded by dsr genes play an essential role in the oxidation of intracellular sulfur, which is an obligate intermediate during the oxidation of sulfide and thiosulfate. One such gene product, DsrEFH from Allochromatium vinosum, has been cloned, expressed, purified and crystallized. Synchrotron data were collected to 2.5 Å from a crystal of selenomethionine-substituted DsrEFH. The crystal belongs to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 56.6, b = 183.1, c = 107.8 Å, β = 99.6°. A full structure determination is under way in order to provide insight into the structure–function relationships of this protein.

  3. [Cloning and expression of VLRB of Lampetra japonica and generation of the corresponding monoclonal antibodies].

    PubMed

    Wu, Fen-Fang; Ma, Ning; Chen, Li-Yong; Su, Peng; Li, Qing-Wei

    2012-04-01

    The agnathans (lampreys and hagfishes) are representatives of the jawless vertebrates. The receptor molecules of adaptive immune system in lampreys are different from the antigen receptors in mammal vertebrates. The unique receptor molecules of lampreys are known as variable lymphocyte receptors (VLR). There are three types of VLRs in lampreys, VLRA, VLRB, and VLRC. Multimeric antigen-specific VLRB antibodies are secreted by VLRB+ lymphocytes and constitute the major components of the humoral arm of the lamprey adaptive immune system. Oligomeric VLRB antibodies are composed of four or five disulfide-linked dimeric subunits, which are similar to IgM antibodies in structure and function. In this study, the conservative c-terminal of Lampetra japonica VLRB was cloned and expressed in BL21 E. coli. The recombinant VLRB protein was purified by Ni2+ affinity chromatography column. After Balb/c mice immunity, cell fusion, the positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA). Finally, the hybridoma cells that produced specific anti-VLRB monoclonal antibodies were obtained. In order to get a large number of antibodies against VLRB, the hybridoma cells were injected into the abdominal cavity of Balb/c mice and the antibodies were purified by protein G sepharose. The results of ELISA indicated that the valence of anti-VLRB antibodies was 1:40000. Western blotting assay showed that the antibodies were able to detect both recombinant VLRB and secreted VLRB in lamprey sera. Flow cytometry analysis also revealed the existence of VLRB on the surface of lymphocytes. In summary, the anti-VLRB monoclonal antibodies provided a major tool for studying lamprey adaptive immune system.

  4. Cloning higher plants from aseptically cultured tissues and cells

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  5. Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones.

    PubMed

    Wolf, Kyle J; Emerson, Ryan O; Pingel, Jeanette; Buller, R Mark; DiPaolo, Richard J

    2016-01-01

    Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4+ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4+Foxp3- and CD4+Foxp3+ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4+ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.

  6. Express Primer Tool for high-throughput gene cloning and expression

    SciTech Connect

    2002-12-01

    A tool to assist in the design of primers for DNA amplification. The Express Primer web-based tool generates primer sequences specifically for the generation of expression clones for both lab 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 simultaneous amplification of multiple target genes. The Express Primer Tool enables the user to specify various experimental parameters (e.g. optimal Tm, Tm range, maximum Tm difference) for single or multiple candidate sequence(s) in FASTA format input as a flat text (ASCII) file. The application generates condidate primers, selects optimal primer pairs, and writes the forward and reverse primers pairs to an Excel file that is suitable for electronic submission to a synthesis facility. The program parameters emphasize high-throughput but allow for target atrition at various stages of the project.

  7. Immortalization of human myogenic progenitor cell clone retaining multipotentiality

    SciTech Connect

    Hashimoto, Naohiro . E-mail: nao@nils.go.jp; Kiyono, Tohru; Wada, Michiko R.; Shimizu, Shirabe; Yasumoto, Shigeru; Inagawa, Masayo

    2006-10-06

    Human myogenic cells have limited ability to proliferate in culture. Although forced expression of telomerase can immortalize some cell types, telomerase alone delays senescence of human primary cultured myogenic cells, but fails to immortalize them. In contrast, constitutive expression of both telomerase and the E7 gene from human papillomavirus type 16 immortalizes primary human myogenic cells. We have established an immortalized primary human myogenic cell line preserving multipotentiality by ectopic expression of telomerase and E7. The immortalized human myogenic cells exhibit the phenotypic characteristics of their primary parent, including an ability to undergo myogenic, osteogenic, and adipogenic terminal differentiation under appropriate culture conditions. The immortalized cells will be useful for both basic and applied studies aimed at human muscle disorders. Furthermore, immortalization by transduction of telomerase and E7 represents a useful method by which to expand human myogenic cells in vitro without compromising their ability to differentiate.

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

    SciTech Connect

    Zarlenga, D.; Gamble, H.R.

    1987-05-01

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

  9. Molecular cloning and expression analysis of Foxp3 from Nile tilapia.

    PubMed

    Wei, Jing; Yu, Lintian; Sun, Lina; Zhang, Xiaoping; Li, Minghui; Qi, Wenchuang; Zhou, Linyan; Wang, Deshou

    2013-09-01

    Foxp3 is a crucial transcription factor for the development and function of CD4(+)CD25(+) regulatory T cells (Tr) which play a key role in preventing autoimmune diseases and maintaining maternal-fetal tolerance in mammals. However, the knowledge of Foxp3 and its regulation in teleosts is limited. In the present study, the Foxp3 cDNA was cloned from Nile tilapia and characterized. The full length cDNA of Nile tilapia (nt)Foxp3 contained a 5'UTR of 104 bp, a 3'UTR of 239 bp and an open reading frame of 1134 bp. The putative ntFoxp3 contained a C2H2 zinc finger domain, a winged-helix/fork head domain and a leucine zipper-like domain with a consensus of V-x(6)-L-x(6)-L-x(6)-L, which are typical motifs of a Foxp3. The highest mRNA expression of ntFoxp3 was observed in the spleen, with moderate expression in the head kidney, intestine, kidney, liver and brain. Stimulation of peripheral blood mononuclear cells with PHA and LPS led to a significant increase of ntFoxp3 mRNA expression at 6 and 24h, respectively. In contrast, stimulation with PMA had no effect during the sampling period. After injecting 1×10(7) cells of live Aeromonas hydrophila into adult female Nile tilapias, the mRNA expression of ntFoxp3 significantly increased in the gill and intestine at 6h, while unchanged in the spleen. In all the tissues examined, a clearly up-regulation of ntFoxp3 expression was detected at 24h after the second challenge. These results suggest that ntFoxp3 might be involved into lymphocyte activation and immune responses as reported in mammals. Meanwhile, the correlation between the expression profile of ntFoxp3 and serum estrogen (17-beta-estradiol, E2) concentration was investigated by real-time PCR and enzyme immunoassay. The results showed that E2 could regulate the expression of ntFoxp3 in the intestine and kidney but not in the spleen. This work will help future investigations into Tr cells and extend our understanding of interactions between sex hormones and immune related

  10. Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes.

    PubMed

    Kranz, D M; Tonegawa, S; Eisen, H N

    1984-12-01

    The molecular basis for the dependence of antigen recognition by T cells on products of the major histocompatibility complex (MHC) is unknown, and the antigenic structures that are actually bound by T-cell receptors are ill-defined. In this study, we asked whether a monoclonal antibody (mAb) that reacts with the T-cell receptor of a clone of murine cytotoxic T lymphocytes (CTL) and not with the receptors of other CTL clones can substitute for that clone's natural ligand in specific cytolytic reactions. To answer the question, a mAb (1B2) to the receptor of a CTL clone (2C) was attached covalently to 51Cr-labeled cells that were not otherwise susceptible to lysis by clone 2C, and the cells thus modified were then tested as targets for clone 2C and other CTL clones of similar specificity. All labeled cells modified in this way, including a murine cell line that expresses no cell-surface MHC class I molecules and a human cell line, were lysed by clone 2C but not by other CTL clones. If, however, instead of attaching the mAb to the receptor of clone 2C, the cells were modified by attaching to them mAbs to other surface antigens on CTL [lymphocyte function-associated antigen (LFA-1), Thy-1.2], they were not lysed. In cytolytic titrations, the cells that had been converted by attachment of mAb 1B2 into specific targets for clone 2C were just as susceptible to lysis by that clone as the clone's natural H-2d targets (e.g., P815 cells). However, some accessory surface molecules (LFA-1, Lyt-2) that are required for clone 2C to lyse its natural H-2d targets seemed not to be required for this clone to lyse the mAb-converted target cells. By demonstrating that a variety of different cell types can be thus converted into target cells for CTL, the approach described in this study may provide opportunities to analyze further the mechanisms by which CTL destroy target cells.

  11. Imprinted Genes and Satellite Loci Are Differentially Methylated in Bovine Somatic Cell Nuclear Transfer Clones

    PubMed Central

    Shen, Chih-Jie; Lin, Chiao-Chieh; Shen, Perng-Chih; Cheng, Winston T.K.; Chen, Hsiao-Ling; Chang, Tsung-Chou; Liu, Shyh-Shyan

    2013-01-01

    Abstract In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygotes. These reprogramming systems play crucial roles in regulating genome functions during critical stages of embryonic development, and they confer the stability of gene expression during mammalian development. The frequent unexpected loss of progeny from somatic cell nuclear transfer (SCNT) is an ongoing problem. In this study, we used six cloned bovines (named NT-1 to NT-6), which were created by ear fibroblast nuclear transfer and displayed short life spans with multiple organ defects, as an experimental model. We focus here on three imprinted genes (IGF2, H19, and XIST) and four satellite loci (Satellite I, Satellite II, Art2, and VNTR) to investigate their methylation changes. The results revealed that aberrant methylation frequently occurred in the analyzed imprinted genes, but not in the satellite loci, of the cloned bovines. After the bovine fibroblast cells were treated with the 5-aza-2(′)-deoxycytidine (5-Aza-dc) demethylation agent, the methylation percentages of the XIST and H19 putative differentially methylated region (DMR) were significantly decreased (XIST, p<0.01; H19, p<0.05) followed by an increase in their mRNA expression levels (p<0.01). Furthermore, we found that five short-lived cloned bovines (NT-1 to NT-5) exhibited more severe aberrant methylation changes in the three imprinted genes examined than the little longer-lived clone (NT-6) compared with wild-type (WT) cows. Our data suggest that the reprogramming of the methylation-controlled regions between the imprinted genes and satellite loci are differences and may be involved with additional mechanisms that need further elucidation. PMID:23961768

  12. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

    PubMed

    Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn

    2014-06-15

    Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

  13. Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4.

    PubMed

    Suetake, Hiroaki; Araki, Kyosuke; Suzuki, Yuzuru

    2004-08-01

    We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15-20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.

  14. Cloning, expression, purification, and biological activity of five feline type I interferons.

    PubMed

    Wonderling, Ramani; Powell, Tim; Baldwin, Susan; Morales, Tony; Snyder, Scott; Keiser, Kathy; Hunter, Shirley; Best, Elaine; McDermott, Martin J; Milhausen, Michael

    2002-10-08

    Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals. In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity. Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses. We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity. FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes. Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60%. Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site. Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated. The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro. In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris. The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.

  15. Genomic cloning, structure, expression pattern, and chromosomal location of the human SIX3 gene.

    PubMed

    Granadino, B; Gallardo, M E; López-Ríos, J; Sanz, R; Ramos, C; Ayuso, C; Bovolenta, P; Rodríguez de Córdoba, S

    1999-01-01

    The Drosophila gene sine oculis (so) is a nuclear homeoprotein that is required for eye development. Homologous genes to so, denoted SIX genes, have been found in vertebrates. Among the SIX genes, SIX3 is considered to be the functional homologue of so. To provide insight into the potential implications of SIX3 in human ocular malformations, we have cloned and characterized the human SIX3 gene. In human eye, SIX3 produces a 3-kb transcript that codes for a 332-amino-acid polypeptide that is virtually identical to its mouse and chick homologues. Expression of SIX3 was detected in human embryos as early as 5-7 weeks of gestation and found to be maintained in the eye throughout the entire period of fetal development. At 20 weeks of gestation, expression of SIX3 in the human retina was detected in the ganglion cells and in cells of the inner nuclear layer. The human SIX3 gene spans 4.4 kb of genomic DNA and is split in two exons separated by a 1659-bp intron. SIX3 was mapped to human chromosome 2p16-p21, between the genetic markers D2S119 and D2S288. Interestingly, the map position of human SIX3 overlaps the locations of two dominant disorders with ocular phenotypes that have been assigned to this chromosomal region, holoprosencephaly type 2 and Malattia Leventinese.

  16. Molecular cloning of natriuretic peptide receptor A from bullfrog (Rana catesbeiana) brain and its functional expression.

    PubMed

    Sekiguchi, T; Miyamoto, K; Mizutani, T; Yamada, K; Yazawa, T; Yoshino, M; Minegishi, T; Takei, Y; Kangawa, K; Minamino, N; Saito, Y; Kojima, M

    2001-08-08

    A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.

  17. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms.

    PubMed

    Rouleau, Agnès; Héron, Anne; Cochois, Véronique; Pillot, Catherine; Schwartz, Jean-Charles; Arrang, Jean-Michel

    2004-09-01

    The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

  18. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

    PubMed Central

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-01-01

    Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

  19. The regulation of science and the Charter of Rights: would a ban on non-reproductive human cloning unjustifiably violate freedom of expression?

    PubMed

    Billingsley, Barbara; Caulfield, Timothy

    2004-01-01

    Non-Reproductive Human Cloning (NRHC) allows researchers to develop and clone cells, including non-reproductive cells, and to research the etiology and transmission of disease. The ability to clone specific stem cells may also allow researchers to clone cells with genetic defects and analyze those cells with more precisions. Despite those potential benefits, Parliament has banned such cloning due to a myriad of social and ethical concerns. In May 2002, the Canadian Government introduced Bill C-13 on assisted human reproductive technologies. Bill C-13 deals with both the scientific and the clinical use of human reproductive materials, and it prohibits a number of other activities, including NRHC. Although the Supreme Court of Canada has never ruled on whether scientific experiments area form of expression, academic support exists for this notion. The authors go through the legal analysis that would be required to find that scientific experiments are expression, focusing in part on whether NRHC could be considered violent and thus fall outside the protection of section 2(b). The latter question is complicated by the ongoing policy debate over whether an "embryonic cell" is property of human life. The authors then consider whether a ban on NRHC could be justified under section 1 of the Charter. They conclude that both the breadth of the legislative purpose and the proportionality of the measure are problematic. Proportionality is a specific concern because the ban could be viewed as an outright denial of scientific freedom of expression. Although consistent with current jurisprudence on freedom of expression, this paper runs against the flow of government policy in the areas of regulation and prohibition of non-reproductive human cloning. As there has been no Charter litigation to date on whether scientific research is a form of expression, the authors introduce a new way of looking at the legality of the regulation of new reproductive technologies.

  20. Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells

    SciTech Connect

    Pendse, G.J.; Bailey, J.E. . Dept. of Chemical Engineering)

    1994-12-01

    Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA cells show a reduced specific growth rate in the VHb-expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture.

  1. Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein

    PubMed Central

    Azarnezhad, Asaad; Sharifi, Zohreh; Seyedabadi, Rahmatollah; Hosseini, Arshad; Johari, Behrooz; Sobhani Fard, Mahsa

    2016-01-01

    Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli (E. coli) BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 μg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA. Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests. PMID:27920885

  2. Cloning, functional expression, and characterization of the human prostaglandin E2 receptor EP2 subtype.

    PubMed

    Bastien, L; Sawyer, N; Grygorczyk, R; Metters, K M; Adam, M

    1994-04-22

    A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 > iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.

  3. Cloning and expression of the first gene for biodegrading microcystin LR by Sphingopyxis sp. USTB-05.

    PubMed

    Yan, Hai; Wang, Huasheng; Wang, Junfeng; Yin, Chunhua; Ma, Song; Liu, Xiaolu; Yin, Xueyao

    2012-01-01

    Harmful cyanobacterial blooms are a growing environmental problem worldwide in natural waters, the biodegradation is found to be the most efficient method for removing microcystins (MCs) produced by harmful cyanobacteria. Based on the isolation of a promising bacterial strain of Sphingopyxis sp. USTB-05 for biodegrading MCs, we for the first time cloned and expressed a gene USTB-05-A (HM245411) that is responsible for the first step in the biodegradation of microcystin LR (MC-LR) in E. coli DH5alpha, with a cloning vector of pGEM-T easy and an expression vector of pGEX-4T-1, respectively. The cell-free extracts (CE) of recombinant E. coli DH5alpha containing USTB-05-A had high activity for biodegrading MC-LR. The initial MC-LR concentration of 40 mg/L was completely biodegraded within 1 hr in the presence of CE with a protein concentration of 0.35 mg/mL. Based on an analysis of the liquid chromatogram-mass spectrum (LC-MS), the enzyme encoded by gene USTB-OS-A was found to be active in cleaving the target peptide bond between 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda) and arginine of MC-LR, and converting cyclic MC-LR to linear MC-LR as a first product that is much less toxic than parent MC-LR, which offered direct evidence for the first step on the pathway of MC-LR biodegradation by Sphingopyxis sp. USTB-05.

  4. Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.

    PubMed

    Yokoi, Ken-ji; Shinohara, Masayuki; Kawahigashi, Nobutaka; Nakagawa, Kazuaki; Kawasaki, Ken-Ichi; Nakamura, Shogo; Taketo, Akira; Kodaira, Ken-Ichi

    2005-04-01

    Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.

  5. An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene.

    PubMed

    Jang, Goo; Bhuiyan, M M U; Jeon, Hyun Yong; Ko, Kyeong Hee; Park, Hee Jung; Kim, Min Kyu; Kim, Joung Ju; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk

    2006-06-01

    In an attempt to produce transgenic cloned cows secreting alpha 1-antitrypsin (alpha1-AT) protein into milk, bovine cumulus cells were transfected with a plasmid containing an alpha1-AT gene and green fluorescent protein (GFP) reporter gene using Fugene 6 as a lipid carrier. The GFP-expressing cells were selected and transferred into enucleated bovine oocytes. Couplets were fused, chemically activated and cultured. Developmental competence was monitored and the number of inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts were counted after differential staining. The percentage of blastocysts was lower (P < 0.05) in transgenic cloned embryos compared to non-transgenic cloned embryos (23% versus 35%). No difference in the numbers of ICM and TE cells between the two groups of embryos was observed. One or two GFP-expressing blastocysts were transferred into the uterus of each recipient cow. Out of 49 recipient cows, three pregnancies were detected by non-return estrus and rectal palpation. However, the pregnancies failed to maintain to term; two fetuses were aborted at Day 60 and 150, respectively, and one fetus at Day 240. The genomic DNA from the aborted fetus was amplified by polymerase chain reaction (PCR) to investigate integration of the transgene in the fetus. The expected PCR product was sequenced and was identical to the sequence of alpha1-AT transgene. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower than embryos derived from non-transfected donor cells. Although we failed to obtain a viable transgenic cloned calf, integration of alpha1-AT gene into the fetus presents the possibility of producing transgenic cloned cows by somatic cell nuclear transfer.

  6. Cloning and expression of propionyl acylase gene of Streptomyces mycarofaciens mutant.

    PubMed

    Li, Y A; Sun, Y P; Shi, L Y; Li, X P; Li, H L

    1991-01-01

    The midecamycin producer, S. mycarofaciens mutant, which has propionyl acylase activity, can convert spiramycin into propionyl spiramycin. Plasmid pIJ702 was used as a vector for the cloning of propionyl acylase gene. After shot-gun cloning, the DNA fragments of the mutant were cloned in S. lividans TK54. The results of TLC and HPLC showed that No.9 transformant can convert spiramycin into propionyl spiramycin. It demonstrated that the propionyl acylase gene was cloned and expressed in S. lividans TK54. The insert fragment of No.9 recombinant plasmid is about 4.16 kb. Southern hybridization showed that the fragment originated from S. mycarofaciens mutant. The restriction endonuclease map of No.9 recombinant plasmid was constructed.

  7. CD79a is heterogeneously expressed in neoplastic and normal myeloid precursors and megakaryocytes in an antibody clone-dependent manner.

    PubMed

    Bhargava, Parul; Kallakury, Bhaskar V S; Ross, Jeffrey S; Azumi, Norio; Bagg, Adam

    2007-08-01

    CD79a, a component of the B-cell antigen receptor complex, can also be expressed in certain non-B-cell malignancies. The reported frequency of CD79a expression in acute myeloid leukemias (AML) ranges from 0% to 90%. We evaluated 39 bone marrow biopsy specimens (29 AML and 10 normal cases) using 5 different commercially available anti-CD79a monoclonal antibody (MoAb) clones. Of 7 acute promyelocytic leukemia (APL) cases, 6 (86%) stained for CD79a with clones HM47/A9 (Novocastra, Newcastle Upon Tyne, England) and HM57 (DAKO, Carpinteria, CA) but were negative with clones 11E3 (Novocastra), and JCB117 (DAKO). Half of 6 acute megakaryoblastic leukemia (AMKL) cases and normal megakaryocytes in 14 (67%) of 21 cases were immunoreactive using clone 11D10 (Novocastra). Approximately one third of non-APL/non-AMKL AML and myeloid precursors in normal marrow specimens stained with clones HM57 and 11D10. This heterogeneity of CD79a expression in AML, megakaryocytes, and myeloid precursors is MoAb clone-dependent, likely owing to different epitope detection, and may be of diagnostic usefulness.

  8. Protective natural autoantibodies to apoptotic cells: evidence of convergent selection of recurrent innate-like clones.

    PubMed

    Silverman, Gregg J

    2015-12-01

    During murine immune development, recurrent B cell clones arise in a predictable fashion. Among these B cells, an archetypical clonotypic set that recognizes phosphorylcholine (PC) antigens and produces anti-PC IgM, first implicated for roles in microbial protection, was later found to become expanded in hyperlipidemic mice and in response to an increased in vivo burden of apoptotic cells. These IgM natural antibodies can enhance clearance of damaged cells and induce intracellular blockade of inflammatory signaling cascades. In clinical populations, raised levels of anti-PC IgM correlate with protection from atherosclerosis and may also downmodulate the severity of autoimmune disease. Human anti-PC-producing clones without hypermutation have been isolated that can similarly discriminate apoptotic from healthy cells. An independent report on unrelated adults has described anti-PC-producing B cells with IgM genes that have conserved CDR3 motifs, similar to stereotypic clonal sets of B cell chronic lymphocytic leukemia. Taken together, emerging evidence suggests that, despite the capacity to form an effectively limitless range of Ig receptors, the human immune system may often recurrently generate lymphocytes expressing structurally convergent B cell receptors with protective and homeostatic roles.

  9. Development of K562 cell clones expressing β-globin mRNA carrying the β039 thalassaemia mutation for the screening of correctors of stop-codon mutations

    PubMed Central

    Salvatori, Francesca; Cantale, Vera; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30 % of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039-thalassaemia. In this context, we started the development of a cellular model of the β039-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing

  10. Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.

    PubMed Central

    Davis, A H; Blanton, R; Rottman, F; Maurer, R; Mahmoud, A

    1986-01-01

    Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms. Images PMID:3461448

  11. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    PubMed

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  12. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  13. To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.

    PubMed

    Willmann, Charlotte A; Hemeda, Hatim; Pieper, Lisa A; Lenz, Michael; Qin, Jie; Joussen, Sylvia; Sontag, Stephanie; Wanek, Paul; Denecke, Bernd; Schüler, Herdit M; Zenke, Martin; Wagner, Wolfgang

    2013-01-01

    Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.

  14. Using Transcriptomics to Identify Differential Gene Expression in Response to Salinity among Australian Phragmites australis Clones

    PubMed Central

    Holmes, Gareth D.; Hall, Nathan E.; Gendall, Anthony R.; Boon, Paul I.; James, Elizabeth A.

    2016-01-01

    Common Reed (Phragmites australis) is a frequent component of inland and coastal wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically diverse range of salt-tolerant P. australis plants. This has prompted an interest in examining the variation in salinity tolerance among clones and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L-1 TDS) and three from low salinity sites (<6 g L-1) were grown in containers irrigated with either fresh (<0.1 g L-1) or saline water (16 g L-1). An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the higher relative expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of certain clones to salinity

  15. Using Transcriptomics to Identify Differential Gene Expression in Response to Salinity among Australian Phragmites australis Clones.

    PubMed

    Holmes, Gareth D; Hall, Nathan E; Gendall, Anthony R; Boon, Paul I; James, Elizabeth A

    2016-01-01

    Common Reed (Phragmites australis) is a frequent component of inland and coastal wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically diverse range of salt-tolerant P. australis plants. This has prompted an interest in examining the variation in salinity tolerance among clones and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L(-1) TDS) and three from low salinity sites (<6 g L(-1)) were grown in containers irrigated with either fresh (<0.1 g L(-1)) or saline water (16 g L(-1)). An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the higher relative expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of certain clones to

  16. Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis.

    PubMed

    Izacc, S M; Gomez, F J; Jesuino, R S; Fonseca, C A; Felipe, M S; Deepe, G S; Soares, C M

    2001-10-01

    A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).

  17. Cloning, expression and antiviral activity of IFNγ from the Australian fruit bat, Pteropus alecto.

    PubMed

    Janardhana, Vijaya; Tachedjian, Mary; Crameri, Gary; Cowled, Chris; Wang, Lin-Fa; Baker, Michelle L

    2012-03-01

    Bats are natural reservoir hosts to a variety of viruses, many of which cause morbidity and mortality in other mammals. Currently there is a paucity of information regarding the nature of the immune response to viral infections in bats, partly due to a lack of appropriate bat specific reagents. IFNγ plays a key role in controlling viral replication and coordinating a response for long term control of viral infection. Here we describe the cloning and expression of IFNγ from the Australian flying fox, Pteropus alecto and the generation of mouse monoclonal and chicken egg yolk antibodies specific to bat IFNγ. Our results demonstrate that P. alecto IFNγ is conserved with IFNγ from other species and is induced in bat splenocytes following stimulation with T cell mitogens. P. alecto IFNγ has antiviral activity on Semliki forest virus in cell lines from P. alecto and the microbat, Tadarida brasiliensis. Additionally recombinant bat IFNγ was able to mitigate Hendra virus infection in P. alecto cells. These results provide the first evidence for an antiviral role for bat IFNγin vitro in addition to the application of important immunological reagents for further studies of bat antiviral immunity.

  18. Frequent occurrence of highly expanded but unrelated B-cell clones in patients with multiple myeloma.

    PubMed

    Kriangkum, Jitra; Motz, Sarah N; Debes Marun, Carina S; Lafarge, Sandrine T; Gibson, Spencer B; Venner, Christopher P; Johnston, James B; Belch, Andrew R; Pilarski, Linda M

    2013-01-01

    Clonal diversity in multiple myeloma (MM) includes both MM-related and MM-unrelated clonal expansions which are subject to dominance exerted by the MM clone. Here we show evidence for the existence of minor but highly expanded unrelated B-cell clones in patients with MM defined by their complementary determining region 3 (CDR3) peak. We further characterize these clones over the disease and subsequent treatment. Second clones were identified by their specific IgH-VDJ sequences that are distinct from those of dominant MM clones. Clonal frequencies were determined through semi-quantitative PCR, quantitative PCR and single-cell polymerase chain reaction of the clone-specific sequence. In 13/74 MM patients, more than one dominant CDR3 peak was identified with 12 patients (16%) being truly biclonal. Second clones had different frequencies, were found in different locations and were found in different cell types from the dominant MM clone. Where analysis was possible, they were shown to have chromosomal characteristic distinct from those of the MM clone. The frequency of the second clone also changed over the course of the disease and often persisted despite treatment. Molecularly-defined second clones are infrequent in monoclonal gammopathy of undetermined significance (MGUS, 1/43 individuals or 2%), suggesting that they may arise at relatively late stages of myelomagenesis. In further support of our findings, biclonal gammopathy and concomitant MM and CLL (chronic lymphocytic leukemia) were confirmed to originate from two unrelated clones. Our data supports the idea that the clone giving rise to symptomatic myeloma exerts clonal dominance to prevent expansion of other clones. MM and second clones may arise from an underlying niche permissive of clonal expansion. The clinical significance of these highly expanded but unrelated clones remains to be confirmed. Overall, our findings add new dimensions to evaluating related and unrelated clonal expansions in MM and the

  19. Transfer of protective immunity in murine histoplasmosis by a CD4+ T-cell clone.

    PubMed

    Allendoerfer, R; Magee, D M; Deepe, G S; Graybill, J R

    1993-02-01

    We have reported that a murine Histoplasma capsulatum-reactive CD4+ T-cell line and clones thereof did not adoptively transfer protection against H. capsulatum infection in normal or cyclophosphamide-treated C57BL/6 mice. One explanation for the results was that the T cells failed to traffic to lymphoid organs in these animals. In this study, we have sought to determine whether one of these clones, 2.3H3, could mediate protection in nude (C57BL/10) or irradiated (5 Gy) heterozygous nude (nu/+) C57BL/6 mice. Mice were inoculated intravenously with 10(7) resting 2.3H3 cells or with an equal number of cells of the ovalbumin-reactive clone 1S6; 2 h later, the mice were challenged intranasally with 5 x 10(6) yeast cells. By day 5 of infection, lungs, livers, and spleens of nude and irradiated nu/+ mice given 2.3H3 contained significantly fewer (P < 0.05) CFU than the same organs from mice inoculated with 1S6. This effect was specific for H. capsulatum, since 2.3H3 did not reduce the number of Coccidioides immitis CFU in lungs, livers, and spleens of irradiated nu/+ mice. By day 10, the amounts of H. capsulatum CFU in lungs, livers, or spleens of nude and irradiated nu/+ mice inoculated with 2.3H3 were smaller than those in 1S6-inoculated mice, but these differences did not reach statistical significance (P > 0.05). The mortality rate of mice inoculated with 2.3H3 and that of mice inoculated with 1S6 were similar. Histopathological examination of tissues from 2.3H3- and 1S6-inoculated mice demonstrated the presence of granulomatous inflammation in organs from both groups. Tissues from 2.3H3-treated mice contained fewer yeasts per high-power field than tissues from 1S6-treated mice. Thus, irradiated or nude mice are permissive for the expression of protective immunity by a CD4+ T-cell clone. Although the protective capacity of T cells in these animals is transient, these animals will be useful for differentiating protective from nonprotective T-cell clones.

  20. A steroid-inducible promoter for the controlled overexpression of cloned genes in eukaryotic cells.

    PubMed Central

    Mader, S; White, J H

    1993-01-01

    Previous studies have shown that members of the steroid receptor family of transcriptional regulators can function synergistically when bound to multiple arrays of specific DNA binding sites known as hormone response elements, usually located upstream of target genes. We have constructed a mammalian expression vector containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (termed GRE5) placed upstream of the adenovirus 2 major late promoter "TATA" region. In transiently transfected HeLa cells in the presence of dexamethasone, the GRE5 promoter was at least 50-fold more efficient than the mouse mammary tumor virus long terminal repeat in expressing bacterial chloramphenicol acetyltransferase activity. When the GRE5 vector was introduced stably into the HeLa cell genome, chloramphenicol acetyltransferase activity was induced from 10- to >50-fold by dexamethasone in six of eight responsive clones. The levels of both basal and induced expression varied from one clone to the next, probably due to an effect of chromosomal location on promoter activity. When propagated stably in HeLa cells in an Epstein-Barr virus episomal vector, the GRE5 promoter was > 50-fold inducible and its activity was strictly dependent on the presence of dexamethasone. We also show that the GRE5 promoter stably propagated in HeLa cells is inducible by progesterone in the presence of a transiently transfected progesterone receptor expression vector. The GRE5 promoter should be widely applicable for the strictly controlled high-level expression of target genes in eukaryotic cells that contain either the glucocorticoid or progesterone receptors. Images Fig. 1 Fig. 2 Fig. 3 PMID:8390672

  1. The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli.

    PubMed

    Yokoi, Ken-Ji; Kawahigashi, Nobutaka; Uchida, Maiko; Sugahara, Kazuki; Shinohara, Masayuki; Kawasaki, Ken-Ichi; Nakamura, Shogo; Taketo, Akira; Kodaira, Ken-Ichi

    2005-05-23

    From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes (holWMY and lytWMY) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and pI were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli, lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N-acetylmuramoyl-L-alanine amidases encoded by the staphylococcal phages phi11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for D-alanyl-glycyl endopeptidase), amidase (L-muramoyl-L-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys-His-Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and pI of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, phi11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (phiWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY-lysWMY genes in the phage genome.

  2. [Detection of hybrid DQ molecules by the use of T cell clone and 2D-gel analyses].

    PubMed

    Hawkin, S

    1986-11-01

    The HLA-D region incorporates three subregions, DR, DQ and DP, encoding for three sets of Ia molecules. Whereas DR antigens consist of a constant alpha chain and an extremely polymorphic beta chain, both of alpha and beta chain of DQ antigens show moderate polymorphism. This indicated us the existence of hybrid HLA-DQ molecules in HLA-D heterozygous cells, resulting from the association of an alpha chain and a beta chain encoded by genes located on the two separate haplotypes. In this report, hybrid DQ antigens were demonstrated by using cytotoxic T cell-clone. A cytotoxic T cell clone, which was generated by mixed lymphocyte reaction against a lymphoblastoid B cell line, EBV-Fuk (HLA-DR1/4, DQw1/Wa), recognized only heterogenous lymphoblastoid B cell lines (HLA-DR1/4, DQw1/Wa). Cytotoxic T cell clone, however, didn't react with B cell lines which are homozygous for HLA-DR1, DQw1 or DR4/DQWa. This suggests the T cell clone recognized the hybrid DQ molecules expressed only on heterozygous cell lines. Further confirmation was obtained by inhibition test using monoclonal antibody and biochemically by 2-D gel analyses. Biological significance of hybrid DQ antigens were discussed.

  3. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    PubMed

    Ma, LiBing; Liu, XiYu; Wang, FengMei; He, XiaoYing; Chen, Shan; Li, WenDa

    2015-01-01

    It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT). Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1) cultured to 70-80% confluency (control group), (2) cultured to full confluency, (3) starved in low serum medium for 4 d, or (4) cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in G0/G1 phase

  4. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1999-02-09

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  5. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1997-12-02

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  6. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

    1999-02-09

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

  7. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

    1997-12-02

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

  8. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1990-01-01

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  9. Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases.

    PubMed

    Massart, C; Caroff, G; Maugendre, D; Genetet, N; Gibassier, J

    1999-01-01

    For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.

  10. Molecular cloning, characterization and expression analysis of Tim-3 and Galectin-9 in the woodchuck model.

    PubMed

    Liu, Yanan; Wang, Junzhong; Wang, Lu; Wang, Baoju; Yang, Shangqing; Wang, Qin; Luo, Jinzhuo; Feng, Xuemei; Yang, Xuecheng; Lu, Yinping; Roggendorf, Michael; Lu, Mengji; Yang, Dongliang; Liu, Jia

    2017-03-01

    In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model.

  11. Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli.

    PubMed

    Anbu Rajan, Lawrance; Joseph, Toms C; Thampuran, Nirmala; James, Roswin; Ashok Kumar, Kesavan; Viswanathan, Chinnusamy; Bansal, Kailash C

    2008-08-01

    The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.

  12. Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant, Polyrhachis vicina.

    PubMed

    Lü, Shu-Min; Zhao, Zhuo; Li, Ke; Zhang, Ya-Lin; Xi, Geng-Si

    2011-09-01

    Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full-length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599-amino acid protein that exhibits a high degree of homology with other mAchRs. Real-time quantitative RT-PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains.

  13. Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli.

    PubMed

    Damak, Naourez; Abdeljalil, Salma; Koubaa, Aida; Trigui, Sameh; Ayadi, Malika; Trigui-Lahiani, Hèla; Kallel, Emna; Turki, Nadia; Djemal, Lamia; Belghith, Hafeth; Taieb, Noomen Hadj; Gargouri, Ali

    2013-11-01

    The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel β-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.

  14. Molecular cloning and expression of nanos in the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae).

    PubMed

    Ogaugwu, Christian E; Wimmer, Ernst A

    2013-01-01

    The gene nanos (nos) is a maternal-effect gene that plays an important role in posterior patterning and germ cell development in early stage embryos. nos is known from several diverse insect species, but has so far not been described for any Tephritid fruit fly. Here, we report the molecular cloning and expression pattern of the nos orthologous gene, Ccnos, in the Mediterranean fruit fly Ceratitis capitata, which is a destructive pest of high agricultural importance. CcNOS contains 398 amino acids and has a C-terminal region with two conserved CCHC zinc-binding motifs known to be essential for NOS function. Transcripts of Ccnos were confirmed by in situ hybridization to be maternally-derived and localized to the posterior pole of early stage embryos. Regulatory regions of nos have been employed in genetic engineering in some dipterans such as Drosophila and mosquitoes. Given the similarity in spatial and temporal expression between Ccnos and nos orthologs from other dipterans, its regulatory regions will be valuable to generate additional genetic tools that can be applied for engineering purposes to improve the fight against this devastating pest.

  15. Molecular cloning, expression, and characterization of a Sophora alopecuroides lectin from Escherichia coli.

    PubMed

    Li, Yang; Li, Tingting; Li, Jinyao; Liu, Dongliang; Yang, Jie; Yang, Jianhua; Zhang, Fuchun; Sun, Surong

    2014-09-01

    Sophora alopecuroides lectin (SAL) has been isolated from the seeds and confirmed to have antifungal and antitumor activities, and presently the preparation of the natural lectin was cumbersome, time-consuming, and the yield was relatively low for further analysis. In this study, the signal peptide of lectin, the modification sites, and the secondary structure were analyzed, and the three-dimensional structures of SAL were modeled. The gene of SAL was amplified by the reverse transcription polymerase chain reaction, and cloned into the pET-30a vector and expressed in Escherichia coli BL21(DE3) by the induction of isopropyl-beta-d-thiogalactopyranoside. Totally, 400 mg of recombinant SAL (rSAL) was purified from 1 l of bacterial culture through Ni-NTA agarose column and the purity reached 95%. The recombinant protein was further confirmed by western blot using rSAL-specific antibody. The biological activity analysis results showed that rSAL exclusively bound to d-galactose and had universal hemagglutinating activities to human A, B, O, and AB, and rabbit and mouse erythrocytes. rSAL also inhibited the growth of fungi, the proliferation of cancer cells, and the HIV-I reverse transcriptase activity. In conclusion, this study indicates that rSAL can be produced in large quantities in the prokaryotic expression system and the recombinant protein still retains the various biological activities, which will make the large-scale production of SAL recombinant protein at dramatically reduced cost possible.

  16. Interferon-gamma of the giant panda (Ailuropoda melanoleuca): complementary DNA cloning, expression, and phylogenetic analysis.

    PubMed

    Tao, Yaqiong; Zeng, Bo; Xu, Liu; Yue, Bisong; Yang, Dong; Zou, Fangdong

    2010-01-01

    Interferon-gamma (IFN-gamma) is the only member of type II IFN and is vital in the regulation of immune and inflammatory responses. Herein we report the cloning, expression, and sequence analysis of IFN-gamma from the giant panda (Ailuropoda melanoleuca). The open reading frame of this gene is 501 base pair in length and encodes a polypeptide consisting of 166 amino acids. All conserved N-linked glycosylation sites and cysteine residues among carnivores were found in the predicted amino acid sequence of the giant panda. Recombinant giant panda IFN-gamma with a V5 epitope and polyhistidine tag was expressed in HEK293 host cells and confirmed by Western blotting. Phylogenetic analysis of mammalian IFN-gamma-coding sequences indicated that the giant panda IFN-gamma was closest to that of carnivores, then to ungulates and dolphin, and shared a distant relationship with mouse and human. These results represent a first step into the study of IFN-gamma in giant panda.

  17. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  18. Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase.

    PubMed

    Kalhori, Nahid; Nulit, R; Go, Rusea

    2013-10-01

    Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.

  19. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos

    PubMed Central

    Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D. Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  20. Construction of a directional T vector for cloning PCR products and expression in Escherichia coli.

    PubMed

    Liang, Xiu-Yi; Liang, Zhi-Cheng; Zhang, Zhi; Zhou, Jiao-Jiao; Liu, Shi-Yu; Tian, Sheng-Li

    2015-05-01

    In order to clone PCR products and express them effectively in Escherichia coli, a directional cloning system was constructed by generating a T vector based on pQE-30Xa. The vector was prepared by inserting an XcmI cassette containing an endonuclease XcmI site, a kanamycin selective marker, a multiple-cloning-site (MCS) region and an opposite endonuclease XcmI site into the vector pQE-30Xa. The T vector pQE-T with single overhanging dT residues at both 3' ends was obtained by digesting with the restriction enzyme XcmI. For directional cloning, a BamHI site was introduced to the ends of the PCR products. A BamHI site was also located on the multiple cloning site of pQE-T. The PCR products were ligated with pQE-T. The directionally inserted recombinants were distinguished by using BamHI to digest the recombinants because there are two BamHI sites located on the both sides of PCR fragment. In order to identify the T-vector functions, the 14-3-3-ZsGreen and hRBP genes were amplified and a BamHI site was added to the ends of the genes to confirm this vector by ligation with pQE-T. Results showed that the 14-3-3-ZsGreen and hRBP were cloned to the vector pQE-T directly and corresponding proteins were successfully produced. It was here demonstrated that this directional vector is capable of gene cloning and is used to manipulate gene expression very easily. The methodology proposed here involves easy incorporation of the construct into other vectors in various hosts.

  1. High-level protein expression following single and dual gene cloning of infectious bronchitis virus N and S genes using baculovirus systems.

    PubMed

    Abdel-Moneim, Ahmed S; Giesow, Katrin; Keil, Günther M

    2014-03-01

    Baculovirus is an efficient system for the gene expression that can be used for gene transfer to both insect and different vertebrate hosts. The nucleocapsid gene (N) of the infectious bronchitis virus was cloned in a baculovirus expression system for insect cell expression. Dual expression vectors containing IBV N and spike (S) proteins of the avian infectious bronchitis virus were engineered under the control of human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in both vertebrate and insect cells. Transduction of the N gene in the insect Sf9 cells revealed a high level of protein expression. The expressed protein, used in ELISA, effectively detected chicken anti-IBV antibodies with high specificity. Transduction of mammalian and avian cells with BacMam viruses revealed that dual expression cassettes yielded high levels of protein from both transcription units.

  2. Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-ß-1,4-glucanase we named TcEG1 (...

  3. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  4. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    EPA Science Inventory

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
    S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE

    Stephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

  5. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    EPA Science Inventory

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)

    Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

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

    SciTech Connect

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

    1989-11-01

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

  7. Cloning assay thresholds on cells exposed to ultrafast laser pulses

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Riemann, Iris; Fischer, Peter; Becker, Thomas P.; Oehring, Hartmut; Halbhuber, Karl-Juergen

    1999-06-01

    The influence of the peak power, laser wavelength and the pulse duration of near infrared (NIR) ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of-cavity pulse- stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two-photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two- photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond layers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

  8. Cloning assay thresholds on cells exposed to ultrafast laser pulses

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Riemann, Iris; Fischer, Peter; Becker, Thomas P.; Oehring, Hartmut; Halbhuber, Karl-Juergen

    1999-06-01

    The influence of the peak power, laser wavelength and the pulse duration of near infrared ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular, we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond(s) pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of- cavity pulse-stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two- photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two-photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond lasers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

  9. Nitric oxide synthase in the nervous system and ink gland of the cuttlefish Sepia officinalis: molecular cloning and expression.

    PubMed

    Scheinker, Vladimir; Fiore, Gabriella; Di Cristo, Carlo; Di Cosmo, Anna; d'Ischia, Marco; Enikolopov, Grigori; Palumbo, Anna

    2005-12-16

    Nitric oxide (NO) signaling is involved in numerous physiological processes in mollusks, e.g., learning and memory, feeding behavior, neural development, and defence response. We report the first molecular cloning of NOS mRNA from a cephalopod, the cuttlefish Sepia officinalis (SoNOS). SoNOS was cloned using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with RACE procedure. Two splicing variants of SoNOS, differing by 18 nucleotides, were found in the nervous system and the ink gland of Sepia. In situ hybridization shows that SoNOS is expressed in the immature and mature cells of the ink gland and in the regions of the nervous system that are related to the ink defence system.

  10. Brain-specific expression of MAP2 detected using a cloned cDNA probe

    PubMed Central

    1986-01-01

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low

  11. Cloning and expression analysis of a hsp70 gene from Saussurea medusa.

    PubMed

    Cheng, Liqin; Jin, Zhiping; Fu, Chunxiang; Zhao, Dexiu

    2006-04-01

    Members of the 70 kD heat-shock gene family are highly conserved across a wide range of organisms. In an effort to learn more about the evolution and possible functions of extreme environment plant Saussurea medusa Maxim hsp70, we isolated a cDNA clone encoding a putative cytosolic member (Smhsp70) of this family of proteins from a cDNA library of S. medusa cell cultures. The cDNA clone was 2224 bp in length and contained a 1941 bp open reading frame (ORF) encoding a polypeptide of 647 amino acid residues with a predicted molecular mass of 70,794 Da. The predicted protein was found to contain a C-terminal amino acid motif of "PKIEEVD" indicating that Smhsp70 was related to cytosolic members of the hsp70 family in higher plant. The secondary and three-dimensional structures of Smhsp70 were analyzed by molecular modeling. The genomic structure of Smhsp70 included one intron of 1134 bp in length. The deduced Smhsp70 protein has 93.7 and 93.2% similarity with the hsp70 of tobacco and tomato, 73.2% with the hsp70 of human, and 43.7% with DnaK of Escherichia. coli, respectively. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that the cytosolic Smhsp70 protein was constitutively expressed and markedly increased after relatively short periods of heat shock (37 degrees C) as well as by low temperature (4 degree C) treatments.

  12. Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.

    PubMed Central

    Diu, A; Gougeon, M L; Moreau, J L; Reinherz, E L; Thèze, J

    1987-01-01

    The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle. PMID:2962196

  13. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  14. Cloning and expression of quorum sensing N-acyl-homoserine synthase (LuxI) gene detected in Acinetobacter baumannii

    PubMed Central

    Modarresi, Farzan; Azizi, Omid; Shakibaie, Mohammad Reza; Motamedifar, Mohammad; Mansouri, Shahla

    2016-01-01

    Background and Objectives: In present study we aimed to clone the luxI gene encoding N-acyl-homoserine synthase detected in clinical isolates of Acinetobacter baumannii and study its expression in Escherichia coli transformants. Materials and Methods: Four A. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation. The presence of luxI gene was detected using PCR technique. Purified PCR product DNA was initially cloned into pTG19 and transformed to E. coli DH5α. The gene was then recovered from agarose gel and ligated by T4 DNA ligase into pET28a expression vector using NdeI and XhoI enzymes. pET28a + luxI was transformed into E. coli BL21 (DE3). The luxI putative gene was further detected in the transformants by colony PCR. Expression of the luxI gene in the recombinant E. coli BL21 cells was studied by quantitative real time PCR (qRT-PCR) and the presence of N-acylhomoserine lactone (AHL) was checked by colorimetric assay and Fourier Transform Infra-Red (FT-IR) spectroscopy. Results: We successfully cloned AHL gene from A. baumannii strain 23 to pET28a expression vector. There was four fold increases in expression of luxI in the transformants (P ≤ 0.05). It was found that, strain 23 and the transformants showed highest amount of AHL activity (OD = 1.524). The FT-IR analysis indicated stretching C=O bond of the lactone ring and primary amides (N=H) at 1764.69 cm−1 and 1659.23 cm−1 respectively. Conclusion: From above results we concluded that, luxI in A. baumannii is indeed responsible for AHL production and not regulation and pET28a vector allows efficient AHL expression in E. coli BL21 transformants. PMID:27307980

  15. Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system.

    PubMed

    Breier, G; Breviario, F; Caveda, L; Berthier, R; Schnürch, H; Gotsch, U; Vestweber, D; Risau, W; Dejana, E

    1996-01-15

    An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells. Hematopoietic cells in the center of blood islands were not labeled. At later embryonic stages, VE-cadherin transcripts were detected in vascular structures of all organs examined, eg, in the ventricle of the heart, the inner cell lining of the atrium and the dorsal aorta, in intersomitic vessels, and in the capillaries of the developing brain. A comparison with flk-1 expression during brain angiogenesis revealed that brain capillaries expressed relatively low amounts of VE-cadherin. In the adult brain, the level of VE-cadherin transcript was further reduced. By

  16. The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host.

    PubMed

    Weiler, Frank; Rehfeldt, Klaus; Bautz, Frank; Schmitt, Manfred J

    2002-11-01

    Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

  17. Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line.

    PubMed

    Goldberg, D A; Posakony, J W; Maniatis, T

    1983-08-01

    We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.

  18. Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

    PubMed Central

    Gaeng, Susanne; Scherer, Siegfried; Neve, Horst; Loessner, Martin J.

    2000-01-01

    Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa l-alanoyl-d-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-l-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SPslpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SPslpA–ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3′-end coding sequence of SPslpA–ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511Δ(S262–K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SPSlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it

  19. Gene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis.

    PubMed

    Gaeng, S; Scherer, S; Neve, H; Loessner, M J

    2000-07-01

    Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the (SP)slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of (SP)slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3'-end coding sequence of (SP)slpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511 Delta(S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent (SP)SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it

  20. Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

    PubMed

    Asano, K; Okamoto, K

    1991-12-01

    To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4+, Lyt-2.2- phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-gamma transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.

  1. Cloning and functional expression of rKCNQ2 K(+) channel from rat brain.

    PubMed

    Jow, F; Wang, K

    2000-09-15

    By homologue cloning, we have isolated a cDNA encoding a voltage-gated K(+) channel, rKCNQ2, from a rat brain cDNA library using RACE. The open reading frame of the translated protein comprises 852 amino acids with 6 transmembrane segments and a pore motif between S5 and S6. rKCNQ2 shares 96% amino acid identity with human KCNQ2 in which mutations cause a form of epilepsy known as benign familial neonatal convulsions (BFNC). Northern blotting with a rKCNQ2-specific probe revealed a robust single band of 8.6-kb transcript expressed in brain not in other tissues. Functional expression of rKCNQ2 in an HEK 293 cell line by whole-cell current recording and in Xenopus oocytes by two-electrode voltage clamp showed outward K(+) selective currents that displayed delayed rectifier-type kinetics. The G-V curve, fitted with a Boltzmann function, showed voltage dependence of activation with a threshold of activation approximately -60 mV. The rKCNQ2 currents were sensitive to TEA block with a Ki of 0.1 mM. In addition, rKCNQ2 currents were down-regulated upon exposure of cells to either a broad-spectrum tyrosine kinase inhibitor genistein or a Src-like tyrosine kinase inhibitor herbimycin A. Our findings add a rodent member to the KCNQ channel subfamily, providing new information of the channel modulation, and will facilitate generation of rodent models of epilepsy.

  2. Cloning, Expression, and Cost Effective Purification of Authentic Human Epidermal Growth Factor With High Activity

    PubMed Central

    Pouranvari, Sara; Ebrahimi, Firouz; Javadi, Gholamreza; Maddah, Bozorgmehr

    2016-01-01

    Background: Epidermal growth factor (EGF) plays a fundamental role in the healing of wounds relating to skin damage, the cornea, and the gastrointestinal tract. Objectives: The aim of this study is the cloning, expression, and purification of recombinant human EGF (rhEGF), and an assessment of its activity. Materials and Methods: In the present experimental study, a synthetic pET28a (+) -hEGF construct was prepared. In order to ligate hEGF into pET24a (+), the PCR technique was performed, using special primers that possess restriction enzyme sites, which are also located in appropriate sites in pET24a (+). After transferring this construct into E. coli cells, protein expression was performed under standard conditions. Protein solubilization was done by urea. hEGF purification and refolding were carried out using gradient dialysis against the urea. We used RP-HPLC to compare between rhEGF and commercial rhEGF as a control. Finally, an MTT assay was performed to assess the viability of the NIH 3T3 cells treated with various concentrations of rhEGF. Results: Dialysis after urea solubilization caused precipitation of unwanted proteins, resulting in achievement of purified EGF with > 90% purity, without the need for expensive and time-consuming process. The MTT assay results showed that our rhEGF activate significantly higher proliferation of NIH 3T3 cells in comparison to the control (P-values were < 0.0001), in total concentrations and times evaluated Conclusions: Via our purification protocol, a sufficient amount of bioactive recombinant human epidermal growth factor was obtained in just a few affordable steps, with superlative purity. PMID:27247796

  3. Malignant progressive tumor cell clone exhibits significant up-regulation of cofilin-2 and 27-kDa modified form of cofilin-1 compared to regressive clone.

    PubMed

    Kuramitsu, Yasuhiro; Wang, Yufeng; Okada, Futoshi; Baron, Byron; Tokuda, Kazuhiro; Kitagawa, Takao; Akada, Junko; Nakamura, Kazuyuki

    2013-09-01

    QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p<0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p<0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

  4. Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis.

    PubMed

    Bonfim, Sheyla M R C; Cruz, Aline H S; Jesuino, Rosália S A; Ulhoa, Cirano J; Molinari-Madlum, Eugênia E W I; Soares, Célia M A; Pereira, Maristela

    2006-03-01

    A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.

  5. Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation.

    PubMed Central

    Mondello, F J

    1989-01-01

    Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400. Images PMID:2493454

  6. Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

    PubMed

    Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji

    2016-12-21

    l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

  7. Molecular cloning and functional expression in heterologous mammalian cells of the cDNAs encoding lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) from man and mouse

    SciTech Connect

    Sakai, I.; Hou, E.W.; Sharief, F.S.; Hiraoka, B.Y.; Takano, T.; Li, S.S.L.

    1987-05-01

    The full-length cDNAs encoding human LHD-A (muscle), human and mouse LDH-B (heart), and mouse LDH-C (testis) isozymes have been isolated and their nucleotide sequences determined. The cDNAs for human LDH-A and mouse LDH-C isozymes were inserted into mammalian expression vectors and were shown to direct the synthesis of functional LDH isozymes in Chinese hamster ovary cells. The exogeneous LDH-A and LDH-C subunits were found to form hybrid LDH tetrameric isozymes with endogeneous LDH-A subunit of CHO cells. The genomic DNA containing LDH cDNA insert and the levels of mRNA expression are being characterized.

  8. Molecular cloning and expression of a larval immunogenic protein from the cattle tick Boophilus annulatus.

    PubMed

    Shahein, Yasser Ezzat

    2008-02-15

    A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936bp cloned fragment was sequenced and showed an open reading frame of 516bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.

  9. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  10. Amphioxus allantoicase: molecular cloning, expression and enzymatic activity.

    PubMed

    Wang, Yongjun; Zhang, Shicui; Liu, Zhenhui; Li, Hongyan; Wang, Lei

    2005-06-01

    Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.

  11. T-Cell Receptor (TCR) Clonotype-Specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T-Cell Clones Is Not Mediated by TCR Alone.

    PubMed

    Flerin, Nina C; Chen, Huabiao; Glover, Tynisha D; Lamothe, Pedro A; Zheng, Jian Hua; Fang, Justin W; Ndhlovu, Zaza M; Newell, Evan W; Davis, Mark M; Walker, Bruce D; Goldstein, Harris

    2017-03-15

    Functional analysis of T-cell responses in HIV-infected individuals has indicated that virus-specific CD8(+) T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progressors. To define the role of individual T-cell receptor (TCR) clonotypes in differential antiviral CD8(+) T-cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8(+) T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8(+) T-cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained nonlytic cytokine and chemokine secretion than ineffective clones. To evaluate the TCR clonotype contribution to CD8(+) T-cell function, we cloned the TCR α and β chain genes from one effective and two ineffective CD8(+) T-cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8(+) T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited a level of activation by cognate peptide and inhibition of in vitro HIV-1 infection, respectively, that were comparable to those of the effective clonotype. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8(+) T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8(+) T cells in elite controllers to inhibit HIV infection.IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8(+) T cells from elite controllers than from HIV-1 progressors supports the crucial role of effective HIV-specific CD8(+) T cells in controlling HIV-1

  12. Molecular cloning, expression, purification, and functional characterization of dammarenediol synthase from Panax ginseng.

    PubMed

    Hu, Wei; Liu, Ning; Tian, Yuhua; Zhang, Lianxue

    2013-01-01

    The objective of this study is to clone and charecterize the expression of dammarenediol synthase gene and then to determine the relationship between the expression of dammarenediol synthase gene that is involved in the ginsenoside biosynthetic pathway and the ginsenoside content. A cDNA phage library was constructed from a five-year-old ginseng root. The cDNA library was screened for the dammarenediol synthase gene by using its specific primers. It was further cloned and expressed in pET-30a vector. The recombinant plasmid pET-30a-DS was expressed in Rosetta E. coli. The recombinant DS protein was purified by affinity chromatography. The production of dammarenediol was detected by liquid chromatography-mass spectrometry (LC-MS). Results showed that dammarenediol synthase gene was cloned from the cDNA library and was expressed in Rosetta E. coli and the SDS-PAGE analysis showed the presence of purified DS protein. LS-MS showed the activity of DS protein, as the protein content increases the dammarenediol increases. Our results indicate that the recombinant dammarenediol synthase protein could increase the production of dammarenediol and the expression of DS played a vital role in the biosynthesis of ginsenosides in P. ginseng.

  13. Production of Computationally Designed Small Soluble- and Membrane-Proteins: Cloning, Expression, and Purification.

    PubMed

    Tripathy, Barsa; Acharya, Rudresh

    2017-01-01

    This book chapter focuses on expression and purification of computationally designed small soluble proteins and membrane proteins that are ordinarily difficult to express in good amounts for experiments. Over-expression of such proteins can be achieved by using the solubility tag such as maltose binding protein (MBP), Thioredoxin (Trx), and Gultathione-S-transferase (GST) fused to the protein of interest. Here, we describe and provide the protocols for cloning, expression and purification of such proteins using the solubility tag.

  14. Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells.

    PubMed

    Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng

    2016-02-01

    Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.

  15. Generation of Novel Thyroid Cancer Stem-Like Cell Clones: Effects of Resveratrol and Valproic Acid.

    PubMed

    Hardin, Heather; Yu, Xiao-Min; Harrison, April D; Larrain, Carolina; Zhang, Ranran; Chen, Jidong; Chen, Herbert; Lloyd, Ricardo V

    2016-06-01

    Anaplastic thyroid cancer is an aggressive and highly lethal cancer for which conventional therapies have proved ineffective. Cancer stem-like cells (CSCs) represent a small fraction of cells in the cancer that are resistant to chemotherapy and radiation therapy and are responsible for tumor reoccurrence and metastasis. We characterized CSCs in thyroid carcinomas and generated clones of CSC lines. Our study showed that anaplastic thyroid cancers had significantly more CSCs than well-differentiated thyroid cancers. We also showed that Aldefluor-positive cells revealed significantly higher expression of stem cell markers, self-renewal properties, thyrosphere formation, and enhanced tumorigenicity. In vivo passaging of Aldefluor-positive cells resulted in the growth of larger, more aggressive tumors. We isolated and generated two clonal spheroid CSC lines derived from anaplastic thyroid cancer that were even more enriched with stem cell markers and more tumorigenic than the freshly isolated Aldefluor-positive cells. Resveratrol and valproic acid treatment of one of the CSC lines resulted in a significant decrease in stem cell markers, Aldefluor expression, proliferation, and invasiveness, with an increase in apoptosis and thyroid differentiation markers, suggesting that these cell lines may be useful for discovering new adjuvant therapies for aggressive thyroid cancers. For the first time, we have two thyroid CSC lines that will be useful tools for the study of thyroid CSC targeted therapies.

  16. Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology.

    PubMed

    Cuna, W R; Cuna, C R

    1995-01-01

    Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC) and cloned. These T cell clones (TCC) were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%). On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%), bradycardia with megacolon (75%) and bradycardia (75%). Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

  17. Bovine Sex Determining Region Y: Cloning, Optimized Expression, and Purification.

    PubMed

    Soleymani, Bijan; Hafezian, Sayed Hassan; Mianji, Ghodratollah Rahimi; Mansouri, Kamran; Chaharaein, Broomand; Tajehmiri, Ahmad; Sharifi Tabar, Mehdi; Mostafaie, Ali

    2017-01-02

    Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.

  18. Molecular cloning, characterization and expression of goose Toll-like receptor 5.

    PubMed

    Fang, Qiang; Pan, Zhiming; Geng, Shizhong; Kang, Xilong; Huang, Jinlin; Sun, Xiaolin; Li, Qiuchun; Cai, Yinqiang; Jiao, Xinan

    2012-10-01

    Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain; moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-κB-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3 d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3 d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-γ in PBMCs

  19. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 CYP4A15.

    PubMed

    Ngo, Suong Ngoc Thi; McKinnon, Ross Allan; Stupans, Ieva

    2006-07-05

    In the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala (Phascolarctos cinereus), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not ingest eucalyptus leaves as food [Ngo, S., Kong, S., Kirlich, A., Mckinnon, R.A., Stupans, I., 2000. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comp. Biochem. Physiol., C 127, 327-334]. A 1544 bp koala liver CYP4A cDNA, designated CYP4A15, was cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CYP4A15 cDNA encodes a protein of 500 amino acids and shares 69% nucleotide and 65% amino acid sequence identity to human CYP4A11. Transfection of the koala CYP4A15 cDNA into Cos-7 cells resulted in the expression of a protein with lauric acid hydroxylase activity. The koala CYP4A15 cDNA-expressed enzyme catalysed lauric acid hydroxylation at the rates of 0.45+/-0.18 nmol/min/mg protein and 4.79+/-1.91 nmol/min/nmol CYP (mean+/-SD, n=3), which were comparable to that of rat CYP4A subfamilies. Total CYP content for koala CYP4A15-expressed protein in Cos-7 cells was 0.094+/-0.001 nmol/mg protein (mean+/-SD, n=3) with negligible CYP content in untransfected Cos-7 cells lysate. Immunoblot analysis, using a sheep anti-rat CYP4A polyclonal antibody, detected multiple CYP4A immunoreactive bands in the liver from all species studied. The koala bands were found to be fainter and less confined but appeared much broader as compared to rat, human and wallaby. Northern blot analysis, utilising the koala CYP4A15 cDNA 417 bp probe, detected a mRNA species of approximately 2.6 kb in the koala liver and a mRNA species of approximately 2.4 kb in other species studied. Relative to the intensity of the beta

  20. Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

    PubMed Central

    Ng, J D; Ko, T P; McPherson, A

    1993-01-01

    Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures. PMID:8310055

  1. Identification, cloning, and expression of Pseudomonas aeruginosa phosphorylcholine phosphatase gene.

    PubMed

    Massimelli, María J; Beassoni, Paola R; Forrellad, Marina A; Barra, José L; Garrido, Mónica N; Domenech, Carlos E; Lisa, Angela T

    2005-05-01

    Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.

  2. Images of cloning and stem cell research in editorial cartoons in the United States.

    PubMed

    Giarelli, Ellen

    2006-01-01

    Through semiotic analysis of manifest and latent meanings in editorial cartoons, the author uncovers how cloning and stem cell research are represented in a popular mass medium. She identified 86 editorial cartoons published in the United States between 2001 and 2004 that referred to cloning and 20 that referred to stem cell research. Cartoonists portrayed people individually 224 times and 4 times in groups of more than 10. Men were portrayed in 64% of cartoons. Stem cell research was depicted as having a potential positive value, and cloning was depicted negatively. Some major messages are that cloning will lead to the mass production of evil, cloning creates monsters, and politics will influence who or what will be cloned. Analyzing popular images can allow access to public understanding about genetic technology and evaluation of public beliefs, preconceptions, and expectations as the public is educated on the use and value of services.

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

    PubMed

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

    2012-12-01

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

  4. Cloning of a marine cyanobacterial promoter for foreign gene expression using a promoter probe vector

    SciTech Connect

    Sode, Koji; Hatano, Naoaki; Tatara, Masahiro

    1996-06-01

    A marine cyanobacterial promoter was cloned to allow efficient foreign gene expression. This was carried out using chloramphenicol acetyl transferase (CAT) as a marker protein. For rapid and simple measurement of CAT activity, a method based on a fluorescently labeled substrate was improved by utilizing HPLC equipped with a flow-through fluorescent spectrophotometer. This method was used in conjunction with a newly constructed promoter probe vector. Cyanobacterial transformants, harboring plasmid containing a cloned 2-kbp marine cyanobacterial genomic fragment, showed a 10-fold higher CAT activity, compared with that achieved using the kanamycin-resistant gene promoter. From the sequence analysis of the cloned fragment, a putative promoter region was found. 20 refs., 7 figs., 2 tabs.

  5. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

    PubMed

    Ringer, Kerry L; Davis, Edward M; Croteau, Rodney

    2005-03-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene

  6. Cloning, expression and properties of porcine trachea UDP-galnac: polypeptide N-acetylgalactosaminyl transferase.

    PubMed

    Sangadala, Sreedhara; Swain, Ja Baris; McNear, Adrian; Mendicino, Joseph

    2004-11-01

    A UDP-GalNAc:polypeptide N-acetyl-galactosaminyl transferase which catalyses the transfer of GalNAc from UDP-GalNAc to serine and threonine residues in mucin polypeptide chains was purified to homogeneity from swine trachea epithelium (Mendicino J, Sangadala S: Mol Cell Biochem 185: 135-145, 1998). Peptides obtained by proteolysis of the purified enzyme were isolated, sequenced and used to prepare degenerate oligonucleotide primers. Amplified segments of a gene encoding GalNAc transferase were synthesised using the primers and a swine trachea epithelial cDNA library. Selected cDNA fragments were then used to screen the cDNA library, and a clone containing an open reading frame encoding 559 amino acids was isolated. The predicted amino acid sequence contains type II transmembrane region, three potential N-glycosylation sites as well as all of the isolated peptide sequences. The nucleotide sequence and predicted primary protein structure of the transferase were very similar to those of type T-1 GalNAc transferases. The isolated clone was transiently expressed in COS 7 cells and the recombinant enzyme, which contained an N-terminal hexa-histidine tag, was purified to homogeneity and its enzymatic properties were examined. The Vmax of the recombinant enzyme, 2.08 micromol/(min mg), was nearly the same as the native enzyme, 2.12 micromol/(min mg), when assayed with partially deglycosylated mucins as glycosyl acceptors. Both enzymes showed much higher activities when assayed with peptides prepared by limited acid hydrolysis of incompletely deglycosylated Cowper's gland, swine, and human respiratory mucins and tryptic peptides isolated from deglycosylated mucin polypeptide chains. However, as noted earlier (Mendicino J, Sangadala S: Mol Cell Biochem 185: 135-145, 1998), these enzymes showed very little activity with completely deglycosylated mucin polypeptide chains. When completely deglycosylated polypeptide chains were partially glycosylated by incubation with microsome

  7. Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development.

    PubMed

    Golding, Michael C; Williamson, Gayle L; Stroud, Todd K; Westhusin, Mark E; Long, Charles R

    2011-05-01

    In studies of somatic cell nuclear transfer (SCNT), the ability of factors within the oocyte to epigenetically reprogram transferred nuclei is essential for embryonic development of the clone to proceed. However, irregular patterns of X-chromosome inactivation, abnormal expression of imprinted genes, and genomic DNA hypermethylation are frequently observed in reconstructed embryos, suggesting abnormalities in this process. To better understand the epigenetic events underlying SCNT reprogramming, we sought to determine if the abnormal DNA methylation levels observed in cloned embryos result from a failure of the oocyte to properly reprogram transcription versus differential biochemical regulation of the DNA methyltransferase family of enzymes (DNMTs) between embryonic and somatic nuclei. To address this question, we conducted real-time quantitation of Dnmt transcripts in bovine preimplantation embryos generated though in vitro fertilization (IVF), parthenogenic activation, and SCNT. By the 8-cell stage, transcripts encoding Dnmt1 become significantly down-regulated in cloned embryos, likely in response to the state of genomic hypermethylation, while the de novo methyltransferases maintain an expression pattern indistinguishable from their IVF and parthenote counterparts. Depletion of embryonic/maternal Dnmt1 transcripts within IVF embryos using short-interfering RNAs, while able to lower genomic DNA methylation levels, resulted in developmental arrest at the 8/16-cell stage. In contrast, SCNT embryos derived from a stable, Dnmt1-depleted donor cell line develop to blastocyst stage, but failed to carry to term. Our results indicate an essential role for Dnmt1 during bovine preimplantation development, and suggest proper transcriptional reprogramming of this gene family in SCNT embryos.

  8. Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform.

    PubMed

    Zhao, Li; Zhao, Kate Q; Hurst, Robin; Slater, Michael R; Acton, Thomas B; Swapna, G V T; Shastry, Ritu; Kornhaber, Gregory J; Montelione, Gaetano T

    2010-09-01

    Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT((R)) SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354-359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two (15)N-, (13)C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.

  9. Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

    PubMed

    Aceves-Ramos, A; de la Torre, P; Hinojosa, L; Ponce, A; García-Villegas, R; Laclette, J P; Bobes, R J; Romano, M C

    2014-07-01

    The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

  10. [Molecular cloning and expression of Nattokinase gene in Bacillus subtilis].

    PubMed

    Liu, B Y; Song, H Y

    2002-05-01

    In order to characterize biochemically the nattokinase,the nucleotide sequence of the nattokinase gene was amplified from the chromosomal DNA of B.subtilis (natto) by PCR. The expression plasmid pBL NK was constructed and was used to transform Bacillus subtilis containing a chromosomal deletion in its subtilisin gene. The supernatant of the culture was collected after 15 h culture. The target proteins were identified by SDS-PAGE. Nattokinase was purified by a method including ultrafiltration, Sephacryl S-100 gel filtration and S-Sepharose ion-exchange chromatography, and 100 mg of purified nattokinase was obtained from one liter of culture. The purity of the protein and the specific activity were 95% and 12 000 u/mg (compared to tPA), respectively.

  11. Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient

    PubMed Central

    BANGA, J P; MOORE, J K; DUHINDAN, N; MADEC, A M; VAN ENDERT, P M; ORGIAZZI, J; ENDL, J

    2004-01-01

    We used a GAD65-specific human B–T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B–T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation. PMID:14678267

  12. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

    NASA Astrophysics Data System (ADS)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

    1983-11-01

    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  13. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    PubMed

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

  14. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

  15. Molecular cloning and expression of key gene encoding hypothetical DNA polymerase from B. mori parvo-like virus.

    PubMed

    Zhang, Junhong; Li, Guohui; Chen, Huiqing; Li, Xiaogang; Lv, Meng; Chen, Keping; Yao, Qin

    2010-10-01

    BmPLV-Z is the abbreviation for Bombyx mori parvo-like virus (China isolate). This is a novel virus with two single-stranded linear DNA molecules, viz., VD1 (6543 bp) and VD2 (6022 bp), which are encapsidated respectively into separate virions. Analysis of the deduced amino acid sequence of VD1-ORF4 indicated the existence of a putative DNA-polymerase with exonuclease activity, possibly involved in the replication of BmPLV-Z. In the present study, a recombinant baculovirus was constructed to express the full length of the protein encoded by the VD1-ORF4 gene (3318 bp). In addition, a 2163-bp fragment amplified from the very same gene was cloned into prokaryotic expression vector pET-30a and expressed in E.coli Rosetta 2 (DE3) pLysS. The expressed fusion protein was employed to immunize New Zealand white rabbits for the production of an antiserum, afterwards used for examining the expression of the protein encoded by VD1-ORF4 gene in Sf-9 cells infected with recombinant baculovirus. Western blot analysis of extracts from thus cells infected revealed a specific band of about 120 kDa, thereby indicating that the full length protein encoded by the VD1-ORF4 gene had been successfully and stably expressed in Sf-9 cells.

  16. Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli.

    PubMed

    Yari, Kheirollah; Afzali, Saboor; Mozafari, Hadi; Mansouri, Kamran; Mostafaie, Ali

    2013-02-01

    Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.

  17. Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles.

    PubMed

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; López-Ruano, Guillermo; Muntión, Sandra; Preciado, Silvia; Hernández-Ruano, Montserrat; Rosado, Belén; de las Heras, Natalia; Chillón, M Carmen; Hernández-Hernández, Ángel; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, Consuelo

    2015-08-01

    The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML.

  18. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

    PubMed Central

    Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship be