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Sample records for recombinant adenovirus-mediated gene

  1. Modulation of adenovirus-mediated gene transfer by nitric oxide.

    PubMed

    Haddad, I Y; Sorscher, E J; Garver, R I; Hong, J; Tzeng, E; Matalon, S

    1997-05-01

    We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that

  2. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting. PMID:14970588

  3. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  4. Effects of bronchopulmonary inflammation induced by pseudomonas aeruginosa on adenovirus-mediated gene transfer to airway epithelial cells in mice.

    PubMed

    van Heeckeren, A; Ferkol, T; Tosi, M

    1998-03-01

    Cystic fibrosis (CF) patients have endobronchial inflammation caused by infection with mucoid Pseudomonas aeruginosa. Since adenovirus vectors are being studied for gene therapy for CF, we sought to determine whether bronchopulmonary inflammation would influence adenovirus-mediated gene transfer. We hypothesized that bronchopulmonary inflammation in mice inoculated with mucoid P. aeruginosa would be associated with a decrease in the efficacy of adenovirus-mediated gene transfer. Agarose beads embedded with mucoid P. aeruginosa (6 x 10(4) c.f.u. per mouse) were inoculated transtracheally into C57BL/6 mice. Control mice received sterile agarose beads. Ten days after inoculation with agarose beads, recombinant adenovirus containing the beta-galactosidase reporter gene (Ad2/beta Gal-2) was administered intranasally (1.1 x 10(9) IU per mouse), and mice were killed 3 days later. The extent of inflammation, determined by neutrophil numbers in bronchoalveolar lavage fluid and by areal lung inflammation, was significantly greater in mice inoculated with P. aeruginosa-laden agarose beads and Ad2/beta Gal-2 compared with controls. Mice that had received Pseudomonas-laden agarose beads and Ad2/beta Gal-2 had significantly fewer (P < 0.015) airway epithelial cells transduced (4.1 +/- 0.9%) compared with mice that received sterile agarose beads and Ad2/beta Gal-2 (9.4 +/- 1.4%). These results indicate that the efficacy of adenovirus-mediated gene transfer is reduced in Pseudomonas-induced bronchopulmonary inflammation.

  5. Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene

    PubMed Central

    Zhao, Chen; Wu, Ningning; Deng, Fang; Zhang, Hongmei; Wang, Ning; Zhang, Wenwen; Chen, Xian; Wen, Sheng; Zhang, Junhui; Yin, Liangjun; Liao, Zhan; Zhang, Zhonglin; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Wu, Di; Ye, Jixing; Deng, Youlin; Zhou, Guolin; Luu, Hue H.; Haydon, Rex C.; Si, Weike; He, Tong-Chuan

    2014-01-01

    Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells. PMID:24658746

  6. Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression

    SciTech Connect

    Yu Hui; Wu Jihong; Li Huiming; Wang Zhanli; Chen Xiafang; Tian Yuhua; Yi Miaoying; Ji Xunda; Ma Jialie; Huang Qian

    2007-10-05

    The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10{sup 8} PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization.

  7. Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells.

    PubMed

    Kajitani, Koji; Honda, Ken-Ichi; Terada, Hiroyuki; Yasui, Tomoyo; Sumi, Toshiyuki; Koyama, Masayasu; Ishiko, Osamu

    2015-01-01

    The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor β, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower β-tubulin levels and comparatively higher p53/β-tubulin or CAR /β-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.

  8. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

    PubMed

    Régulier, E; Paul, S; Marigliano, M; Kintz, J; Poitevin, Y; Ledoux, C; Roecklin, D; Cauet, G; Calenda, V; Homann, H E

    2001-01-01

    Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors. PMID:11219493

  9. Ex vivo adenovirus-mediated gene transfer and immunomodulatory protein production in human cornea.

    PubMed

    Oral, H B; Larkin, D F; Fehervari, Z; Byrnes, A P; Rankin, A M; Haskard, D O; Wood, M J; Dallman, M J; George, A J

    1997-07-01

    One attractive strategy to prevent or control allograft rejection is to genetically modify the donor tissue before transplantation. In this study, we have examined the feasibility of gene transfer to human corneal endothelium, using a number of recombinant adenovirus constructs. Ex vivo infection of human corneas with adenoviral vectors containing lacZ, under transcriptional control of either cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters, provided high-level gene expression, which was largely restricted to endothelium. Expression of the reporter gene persisted at relatively high levels for up to 7 days, followed by a decline to indetectable levels by 28 days. RT-PCR analysis of lacZ transcription showed a similar picture with a short period (3-7 days) of RNA transcription after infection. In contrast, adenoviral DNA persisted for at least 56 days. Subsequently, we examined the expression of a potential therapeutic gene, CTLA-4 Ig fusion protein. Following infection of human corneas with adenoviral vectors encoding CTLA-4 Ig protein, high levels of the fusion protein were detected in corneal culture supernatants for up to 28 days. This protein was functionally active, as determined by binding to B7.1 (CD80)-expressing transfectants. This study suggests that genetic alteration of donor cornea before transplantation is a feasible approach for preventing or controlling allograft rejection. Similar gene-based strategies might also be feasible to prevent rejection of other transplanted tissues or organs. PMID:9282165

  10. Synergistic tumor suppression by adenovirus-mediated ING4/PTEN double gene therapy for gastric cancer.

    PubMed

    Zhang, H; Zhou, X; Xu, C; Yang, J; Xiang, J; Tao, M; Xie, Y

    2016-01-01

    Both inhibitor of growth 4 (ING4) and phosphatase and tensin homolog (PTEN) have been shown to be strong candidate tumor suppressors. However, the combined efficacy of ING4 and PTEN for human gastric cancer remains to be determined. In this report, we constructed a multiple promoter expression cassette-based recombinant adenovirus coexpressing ING4 and PTEN (AdVING4/PTEN), assessed the combined effects of AdVING4/PTEN on gastric cancer using wild-type p53 AGS and SNU-1 human gastric cancer cell lines, and elucidated its underlying mechanisms. We found that AdVING4/PTEN-induced synergistic growth inhibition and apoptosis in vitro AGS or SNU-1 tumor cells and in vivo AGS xenografted tumors subcutaneously inoculated in athymic BALB/c nude mice. Mechanistically, AdVING4/PTEN exhibited an enhanced effect on upregulation of p53, Ac-p53 (K382), P21, Bax, PUMA, Noxa, cleaved Caspase-9, cleaved Caspase-3 and cleaved PARP as well as downregulation of Bcl-2 in vitro and in vivo. In addition, AdVING4/PTEN synergistically downregulated tumor vessel CD34 expression and reduced microvessel density, and additively inhibited vascular endothelial growth factor (VEGF) expression in vivo. The synergistic tumor suppression elicited by AdVING4/PTEN was closely associated with the synergistic induction of apoptosis possibly via enhancement of endogenous p53 responses through cooperatively facilitating p53's stability and acetylation, and the synergistic inhibition of tumor angiogenesis probably via overlapping reduction of VEGF through cooperatively downregulating hypoxia inducible factor-1α's level and transcription activity. Thus, our results indicate that cancer gene therapy combining ING4 and PTEN may constitute a novel and effective therapeutic modality for human gastric cancer and other cancers.

  11. Effects of Adenovirus-Mediated Delivery of the Human Hepatocyte Growth Factor Gene in Experimental Radiation-Induced Heart Disease

    SciTech Connect

    Hu Shunying; Chen Yundai; Li Libing; Chen Jinlong; Wu Bin; Zhou, Xiao; Zhi Guang; Li Qingfang; Wang Rongliang; Duan Haifeng; Guo Zikuan; Yang Yuefeng; Xiao Fengjun; Wang Hua; Wang Lisheng

    2009-12-01

    Purpose: Irradiation to the heart may lead to late cardiovascular complications. The purpose of this study was to investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor gene could reduce post-irradiation damage of the rat heart and improve heart function. Methods and Materials: Twenty rats received single-dose irradiation of 20 Gy gamma ray locally to the heart and were randomized into two groups. Two weeks after irradiation, these two groups of rats received Ad-HGF or mock adenovirus vector intramyocardial injection, respectively. Another 10 rats served as sham-irradiated controls. At post-irradiation Day 120, myocardial perfusion was tested by myocardial contrast echocardiography with contrast agent injected intravenously. At post-irradiation Day 180, cardiac function was assessed using the Langendorff technique with an isolated working heart model, after which heart samples were collected for histological evaluation. Results: Myocardial blood flow was significantly improved in HGF-treated animals as measured by myocardial contrast echocardiography at post-irradiation Day 120 . At post-irradiation Day 180, cardiac function was significantly improved in the HGF group compared with mock vector group, as measured by left ventricular peak systolic pressure (58.80 +- 9.01 vs. 41.94 +- 6.65 mm Hg, p < 0.05), the maximum dP/dt (5634 +- 1303 vs. 1667 +- 304 mm Hg/s, p < 0.01), and the minimum dP/dt (3477 +- 1084 vs. 1566 +- 499 mm Hg/s, p < 0.05). Picrosirius red staining analysis also revealed a significant reduction of fibrosis in the HGF group. Conclusion: Based on the study findings, hepatocyte growth factor gene transfer can attenuate radiation-induced cardiac injury and can preserve cardiac function.

  12. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  13. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  14. Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, cetuximab

    PubMed Central

    Xing, Man; Wang, Xiang; Chi, Yudan; Zhou, Dongming

    2016-01-01

    Cetuximab is a chimeric monoclonal antibody, approved to treat patients with metastatic colorectal cancer (mCRC), head and neck squamous cell carcinoma (HNSCC), non-small-cell lung cancer (NSCLC) for years. It functions by blocking the epidermal growth factor receptor (EGFR) from receiving signals or interacting with other proteins. Although the demand for cetuximab for the treatment of cancer patients in clinics is increasing, the complicated techniques involved and its high cost limit its wide applications. Here, a new, cheaper form of cetuximab was generated for cancer gene therapy. This was achieved by cloning the full-length cetuximab antibody into two serotypes of adenoviral vectors, termed as AdC68-CTB and Hu5-CTB. In vivo studies showed that a single dose of AdC68-CTB or Hu5-CTB induced sustained cetuximab expression and dramatically suppressed tumor growth in NCI-H508– or DiFi-inoculated nude mice. In conclusion, gene therapy using adenovirus expressing full-length cetuximab could be a novel alternative method for the effective treatment of colorectal cancer. PMID:27058423

  15. Silk-Elastinlike Hydrogel Improves the Safety of Adenovirus-Mediated Gene-Directed Enzyme-Prodrug Therapy

    PubMed Central

    Gustafson, Joshua A.; Price, Robert A.; Greish, Khaled; Cappello, Joseph; Ghandehari, Hamidreza

    2010-01-01

    Recombinant Silk-Elastinlike Protein polymers (SELPs) are well-known for their highly tunable properties on both the molecular and macroscopic hydrogel level. One specific structure of these polymers, SELP-815K, has been investigated as an injectable controlled delivery system for the treatment of head and neck cancer via a gene-directed enzyme prodrug therapy (GDEPT) approach. Due to its pore size and gelation properties in vivo, SELP restricts the distribution and controls the release of therapeutic viruses for up to one month. It has been shown that SELP-mediated delivery significantly improves therapeutic outcome of the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system in xenograft models of human head and neck cancer. However little is known about potential benefits of this approach with regard to toxicity in the presence of a fully intact immune system. The studies presented here were designed to assess the change in toxicity of the SELP mediated viral delivery compared to free viral injection in a non-tumor bearing immune competent mouse model. Toxicity was assessed at 1, 2, 4, and 12 weeks via body weight monitoring, complete blood count (CBC), and blood chemistry. It was found that in the acute and subacute phases (weeks 1-4) there is significant toxicity in groups combining the virus and the prodrug, and matrix-mediated gene delivery with SELP demonstrates a reduction in toxicity from the 2 week time point through the 4 week time point. At the end of the subchronic phase (12 weeks), signs of toxicity had subsided in both groups. Based on these results, recombinant SELPs offer a significant reduction in toxicity of virus-mediated GDEPT treatment compared to free virus injection in the acute and subacute phases. PMID:20586469

  16. Adenovirus-mediated ING4 Gene Transfer in Osteosarcoma Suppresses Tumor Growth via Induction of Apoptosis and Inhibition of Tumor Angiogenesis.

    PubMed

    Xu, Ming; Xie, Yufeng; Sheng, Weihua; Miao, Jingcheng; Yang, Jicheng

    2015-08-01

    The inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors. ING4 as a novel member of ING family has potential tumor-suppressive effects via multiple pathways. However, the therapeutic effect of adenovirus-mediated ING4 (Ad-ING4) gene transfer in human osteosarcoma is still unknown. In this study, we explored the in vitro and in vivo antitumor activity of Ad-ING4 in human osteosarcoma and its potential mechanism using a MG-63 human osteosarcoma cell line. We demonstrated that Ad-ING4 induced significant growth inhibition and apoptosis, upregulated the expression of P21, P27 and Bax, downregulated the Bcl-2 expression and activated Caspase-3 in MG-63 human osteosarcoma cells. Moreover, intratumoral injections of Ad-ING4 in athymic nude mice bearing MG-63 human osteosarcoma tumors significantly suppressed osteosarcoma xenografted tumor growth, increased the expression of P21, P27 and Bax, reduced the Bcl-2 and CD34 expression and microvessel density (MVD) in tumors. This retarded MG-63 osteosarcoma growth in vitro and in vivo in an athymic nude mouse model elicited by Ad-ING4 was closely associated with the increase in the expression of cell cycle-related molecules P21 and P27, decrease in the ratio of anti- to pro-apoptotic molecules Bcl-2/Bax followed by the activation of Caspase-3 leading to apoptosis via intrinsic apoptotic pathways, and the inhibition of tumor angiogenesis. Thus, our results indicate that Ad-ING4 is a potential candidate for human osteosarcoma gene therapy.

  17. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

    SciTech Connect

    Freytag, Svend O.; Stricker, Hans; Lu, Mei; Elshaikh, Mohamed; Aref, Ibrahim; Pradhan, Deepak; Levin, Kenneth; Kim, Jae Ho; Peabody, James; Siddiqui, Farzan; Barton, Kenneth; Pegg, Jan; Zhang, Yingshu; Cheng, Jingfang; Oja-Tebbe, Nancy; Bourgeois, Renee; Gupta, Nilesh; Lane, Zhaoli; Rodriguez, Ron; DeWeese, Theodore; and others

    2014-06-01

    Purpose: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. Methods and Materials: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. Results: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. Conclusions: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

  18. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.

    PubMed Central

    Newman, K D; Dunn, P F; Owens, J W; Schulick, A H; Virmani, R; Sukhova, G; Libby, P; Dichek, D A

    1995-01-01

    Adenovirus vectors are capable of high efficiency in vivo arterial gene transfer, and are currently in use as therapeutic agents in animal models of vascular disease. However, despite substantial data on the ability of viruses to cause vascular inflammation and proliferation, and the presence in current adenovirus vectors of viral open reading frames that are translated in vivo, no study has examined the effect of adenovirus vectors alone on the arterial phenotype. In a rabbit model of gene transfer into a normal artery, we examined potential vascular cell activation, inflammation, and neointimal proliferation resulting from exposure to replication-defective adenovirus. Exposure of normal arteries to adenovirus vectors resulted in: (a) pronounced infiltration of T cells throughout the artery wall; (b) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (c) neointimal hyperplasia. These findings were present both 10 and 30 d after gene transfer, with no evidence of a decline in severity over time. Adenovirus vectors have pleiotropic effects on the arterial wall and cause significant pathology. Interpretation of experimental protocols that use adenovirus vectors to address either biological or therapeutic issues should take these observations into account. These observations should also prompt the design of more inert gene transfer vectors. Images PMID:8675667

  19. Adenovirus-mediated gene therapy specific for small cell lung cancer cells using a Myc-Max binding motif.

    PubMed

    Nishino, K; Osaki, T; Kumagai, T; Kijima, T; Tachibana, I; Goto, H; Arai, T; Kimura, H; Funakoshi, T; Takeda, Y; Tanio, Y; Hayashi, S

    2001-03-15

    Recent clinical trials of gene therapy for patients with thoracic cancers have shown that these treatments were well tolerated with minimal side effects and that we need to further enhance specificity as well as efficiency of gene transfer to target cancer cells. We previously reported that myc-overexpressing SCLC cell lines became selectively sensitive to ganciclovir (GCV) by transducing the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the Myc-Max response elements (a core nucleotide sequence, CACGTG) and that this construct (MycTK) could be utilized to develop a novel treatment against chemo-radio-resistant SCLC. We report here in vivo antitumor effects and safety of a replication-deficient adenoviral vector containing the Myc-Max binding motif (AdMycTK) on SCLC cells. In vitro infection with AdMycTK selectively rendered myc-overexpressing SCLC cell lines 63- to 307-fold more sensitive to GCV. In vivo injections with AdMycTK followed by GCV administration markedly suppressed the growth of myc-overexpressing tumors established in the subcutis or in the peritoneal cavity of athymic mice. On the other hand, infection with AdMycTK did not significantly affect either in vitro GCV sensitivity of the cells expressing very low levels of the myc genes or the growth of their subcutaneous tumors. Moreover, we observed no apparent side effects of this treatment including body weight loss or biochemical abnormalities in contrast to the treatment with AdCATK that conferred strong but nonspecific expression of the HSV-TK gene. These results suggested that AdMycTK/GCV therapy is effective on SCLC patients whose tumors overexpress myc family oncogenes.

  20. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

    PubMed Central

    Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

    1993-01-01

    To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

  1. Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport.

    PubMed

    Gnudi, L; Frevert, E U; Houseknecht, K L; Erhardt, P; Kahn, B B

    1997-01-01

    Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in

  2. Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

    SciTech Connect

    Mu, Haixi; Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu; Li, Lili; Ren, Guosheng; Xu, Yongzhu; Zhou, Xiangyang; Xiang, Tingxiu

    2015-03-15

    Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.

  3. [Adenovirus-mediated delivery of nm23-H1 gene inhibits growth of colorectal carcinoma cell line Lovo].

    PubMed

    Wang, Qi; He, Xueling; Liu, Yan; Yin, Hailin

    2010-12-01

    This experimental study sought to find out the inhibitory effects of Ad-GFP-nm23-H1 on proliferation and metastasis of human colorectal carcinoma cell line Lovo, and, further, to gain an insight into some theoretical and methodical basis for instituting nm23-H1 gene therapy of cancers. MTT assay and Transwell chamber were used to detect the rates of proliferation and invasion as well as the adhesion of Lovo cells in vitro. The results demonstrated that the proliferation inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 84.9% +/- 1.51%, 48.5% +/- 7.23% and 22.5% +/- 5.47%, that the adherence inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 70.3% +/- 2.40%, 60.1% +/- 5.68% and 18.5% +/- 3.61%, and that the invasiveness inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 83.2% +/- 5.71%, 52.2% +/- 6.94% and 28.1% +/- 8.21%. These data suggested that Ad-GFP-nm23-H1 exerted significant inhibitory effects on the proliferation and metastasis of human colorectal carcinoma cell line Lovo in a dose-dependent way.

  4. Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo.

    PubMed

    Saito, Y; Swanson, X; Mhashilkar, A M; Oida, Y; Schrock, R; Branch, C D; Chada, S; Zumstein, L; Ramesh, R

    2003-11-01

    The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers. PMID:14528320

  5. Oncolytic adenovirus-mediated short hairpin RNA targeting MYCN gene induces apoptosis by upregulating RKIP in neuroblastoma.

    PubMed

    Li, Yuan; Zhang, Hongwei; Zhu, Xiaoyu; Feng, Dongchuan; Zhang, Deyong; Zhuo, Baobiao; Zheng, Junnian

    2015-08-01

    The amplification of MYCN is a typical characteristic of aggressive neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases. We had previously examined the applicability of the replication-competent oncolytic adenovirus, ZD55-shMYCN, to deliver a short hairpin RNA targeting MYCN gene for p53-null and MYCN-amplified neuroblastoma cell line LA1-55N. Our data have shown that ZD55-shMYCN has an additive tumor growth inhibitory response through shRNA-mediated MYCN knockdown and ZD55-mediated cancer cell lysis. In this regard, ZD55-shMYCN can downregulate MYCN and perform anticancer effects, thereby acquiring significance in the administration of MYCN-amplified and p53-null neuroblastomas. Hence, we further investigated the anticancer properties of ZD55-shMYCN in neuroblastomas. Our data showed that ZD55-shMYCN induced G2/M arrest via decreasing the levels of cyclin D1 and cyclin B1 irrespective of p53 status. ZD55-shMYCN effectively induced apoptosis in neuroblastomas through activation of caspase-3 and enhancing PARP cleavage. Furthermore, ZD55-shMYCN could downregulate phosphoinositide 3-kinase and pAkt and upregulate RKIP levels. Similarly, pro-apoptosis was revealed by the histopathologic examination of paraffin-embedded section of resected tumors of mice xenograft. In vitro and in vivo studies, we elucidate the apoptosis properties and mechanisms of action of ZD55-shMYCN, which provide a promising approach for further clinical development.

  6. Adenovirus-mediated bone morphogenetic protein-2 gene transfection of bone marrow mesenchymal stem cells combined with nano-hydroxyapatite to construct bone graft material in vitro.

    PubMed

    Li, W C; Wang, D P; Li, L J; Zhu, W M; Zeng, Y J

    2013-04-01

    To study the adhesion, proliferation and expression of bone marrow mesenchymal stem cells (BMSCs) on nano-hydroxyapatite (Nano-HA) bone graft material after transfection of adenovirus-mediated human bone morphogenetic protein-2 expression vector (Ad-BMP-2). BMSCs were transfected using Ad-BMP-2. Immunohistochemistry and Western blot were used to detect BMP-2 expression in transfected cells. After transfection, BMP-2 protein was highly expressed in BMSCs; MTT test assay showed that the Nano-HA bone graft material could not inhibit in vitro proliferation of BMSCs. Ad-BMP-2-transfected BMSCs are well biocompatible with Nano-HA bone graft material, the transfected cells in material can secrete BMP-2 stably for a long time.

  7. Adenovirus-mediated p53 and ING4 gene co-transfer elicits synergistic antitumor effects through enhancement of p53 acetylation in breast cancer.

    PubMed

    Wu, Jie; Zhu, Yanbo; Xu, Chun; Xu, Hong; Zhou, Xiumin; Yang, Jicheng; Xie, Yufeng; Tao, Min

    2016-01-01

    Multigene-based combination therapy may be an effective practice in cancer gene therapy. Substantial studies have demonstrated that tumor suppressor p53 acetylation is indispensable for p53 activation. Inhibitor of growth 4 (ING4), as a novel tumor suppressor, is capable of remarkably enhancing p53 acetylation and its transcriptional activity. Hence, we assumed that combined treatment of p53 and ING4 double tumor suppressors would exhibit enhanced antitumor effects. The combined therapeutic efficacy of p53 and ING4 for human cancers has not been previously reported. We thus generated multiple promoter expression cassette-based recombinant adenovirus-co-expressing ING4 and p53 double tumor suppressor genes (AdVING4/p53), evaluated the combined effects of AdVING4/p53 on breast cancer using the MDA-MB-231 (mutant p53) human breast cancer cell line, and also elucidated its underlying molecular mechanisms. We demonstrated that AdVING4/p53-mediated p53 and ING4 co-expression induced synergistic growth inhibition and apoptosis as well as enhanced effects on upregulation of acetylated p53, P21, Bax, PUMA, Noxa, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, and downregulation of Bcl-2, CD31 and microvessel density (MVD) in MDA-MB-231 breast cancer in vitro and/or in vivo subcutaneous (s.c.) xenografted tumors. The synergistic antitumor activity elicited by AdVING4/p53 was closely associated with the enhanced activation of the intrinsic apoptotic pathway and synergistic inhibition of tumor angiogenesis, very possibly via ING4-mediated enhancement of p53 acetylation and activity. Thus, our results indicate that cancer gene therapy combining two or more tumor suppressors such as p53 and ING4 may constitute a novel and effective therapeutic modality for human breast cancer and other cancers.

  8. In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.

    PubMed

    Erbs, P; Regulier, E; Kintz, J; Leroy, P; Poitevin, Y; Exinger, F; Jund, R; Mehtali, M

    2000-07-15

    Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies. PMID:10919655

  9. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    NASA Astrophysics Data System (ADS)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  10. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    PubMed Central

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-01-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma. PMID:27625116

  11. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy.

    PubMed

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-01-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma. PMID:27625116

  12. Adenovirus-mediated expression of p53 or p21 in a papillary serous endometrial carcinoma cell line (SPEC-2) results in both growth inhibition and apoptotic cell death: potential application of gene therapy to endometrial cancer.

    PubMed

    Ramondetta, L; Mills, G B; Burke, T W; Wolf, J K

    2000-01-01

    Papillary serous endometrial carcinoma is an aggressive tumor characterized by late-stage presentation, i.p. spread, and poor prognosis. It is histologically similar to serous papillary carcinoma of the ovary. Preclinical studies have shown that adenovirus-mediated expression of p53 in ovarian cancer cell lines causes growth inhibition and apoptosis in vitro and in vivo. Such studies provide the rationale for Phase I Adp53 gene therapy clinical trials in ovarian cancer. In the present study, we compared the efficacy of adenoviral vectors containing p53 (Adp53) or p21 (Adp21) in a papillary serous endometrial tumor cell line (SPEC-2) that contains mutated p53. Growth assays revealed that both Adp53 and Adp21 were efficacious in decreasing cell proliferation as assessed by anchorage-dependent and anchorage-independent growth assays. However, as compared with Adp53, the effects of Adp21 tended to be more transient and less marked. Strikingly, Adp21, but not Adp53, induced a G1 arrest in SPEC-2 endometrial adenocarcinoma cells. In contrast, as assessed by induction of hypodiploid peaks, free DNA ends detected by a terminal deoxynucleotidyl transferase-based assay, and annexin V positivity, p53 was more effective than p21 in inducing cell death by apoptosis. Compatible with the more efficient induction of apoptosis, Adp53, but not Adp21, induced a marked increase in expression of the preapoptotic molecule BAX without a concomitant change in expression of the antiapoptotic mediator Bcl-2. The differential effects of Adp53 and Adp21 on cell cycle progression and apoptosis may be related to the reversibility of p21-induced cell cycle arrest and the irreversibility of p53-induced apoptosis. Thus, at least in the papillary serous endometrial carcinoma cell line SPEC-2, Adp53 may be more effective than Adp21 as a gene therapeutic. Nevertheless, these preclinical studies suggest that papillary serous endometrial carcinoma is a potential target for p53- or p21-mediated gene

  13. Increase in muscarinic stimulation-induced Ca(2+) response by adenovirus-mediated Stim1-mKO1 gene transfer to rat submandibular acinar cells in vivo.

    PubMed

    Morita, Takao; Nezu, Akihiro; Tojyo, Yosuke; Tanimura, Akihiko

    2013-10-01

    Adenoviruses have been used for gene transfer to salivary gland cells in vivo. Their use to study the function of salivary acinar cells was limited by a severe inflammatory response and by the destruction of fluid-secreting acinar cells. In the present study, low doses of adenovirus were administered to express Stim1-mKO1 by retrograde ductal injection to submandibular glands. The approach succeeded in increasing muscarinic stimulation-induced Ca(2+) responses in acinar cells without inflammation or decreased salivary secretions. This increased Ca(2+) response was notable upon weak muscarinic stimulation and was attributed to increased Ca(2+) release from internal stores and increased Ca(2+) entry. The basal Ca(2+) level was higher in Stim1-mKO1-expressing cells than in mKO1-expressing and non-expressing cells. Exposure of permeabilized submandibular acinar cells, where Ca(2+) concentration was fixed at 50 nM, to inositol 1,4,5-trisphosphate (IP3) produced similar effects on the release of Ca(2+) from stores in Stim1-mKO1-expressing and non-expressing cells. The low toxicity and relative specificity to acinar cells of the mild gene transfer method described herein are particularly useful for studying the molecular functions of salivary acinar cells in vivo, and may be applied to increase salivary secretions in experimental animals and human in future.

  14. Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

    PubMed

    Nalbantoglu, J; Pari, G; Karpati, G; Holland, P C

    1999-04-10

    Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

  15. Retrograde Ductal Administration of the Adenovirus-mediated NDRG2 Gene Leads to Improved Sialaden Hypofunction in Estrogen-deficient Rats

    PubMed Central

    Li, Yan; Liu, Changhao; Hou, Wugang; Li, Yang; Ma, Ji; Lin, Kaifeng; Situ, Zhenqiang; Xiong, Lize; Li, Shaoqing; Yao, Libo

    2014-01-01

    One of the most common oral manifestations of menopause is xerostomia. Oral dryness can profoundly affect quality of life and interfere with basic daily functions, such as chewing, deglutition, and speaking. Although the feeling of oral dryness can be ameliorated after estrogen supplementation, the side effects of estrogen greatly restrict its application. We previously found that N-myc downstream-regulated gene 2 (NDRG2) is involved in estrogen-mediated ion and fluid transport in a cell-based model. In the present study, we used an ovariectomized rat model to mimic xerostomia in menopausal women and constructed two adenovirus vectors bearing NDRG2 to validate their therapeutic potential. Ovariectomized rats exhibited severe sialaden hypofunction, including decreased saliva secretion and ion reabsorption as well as increased water intake. Immunohistochemistry revealed that the expression of NDRG2 and Na+ reabsorption-related Na+/K+-ATPase and epithelial sodium channels (EnaC) decreased in ovariectomized rat salivary glands. We further showed that the localized delivery of NDRG2 improved the dysfunction of Na+ and Cl− reabsorption. In addition, the saliva flow rate and water drinking recovered to normal. This study elucidates the mechanism of estrogen deficiency-mediated xerostomia or sialaden hypofunction and provides a promising strategy for therapeutic intervention. PMID:24343104

  16. Adenovirus-mediated ING4/PTEN double tumor suppressor gene co-transfer modified by RGD enhances antitumor activity in human nasopharyngeal carcinoma cells.

    PubMed

    Wang, Yihong; Yang, Jicheng; Sheng, Weihua; Xie, Yufeng; Liu, Jisheng

    2015-03-01

    Inhibitor of growth-4 (ING4) is a member of the inhibitor of growth (ING) family and acts as a tumor suppressor protein. PTEN is a phosphatase and shows potent and extensive antitumor activity. In this study, we constructed an RGD-modified bicistronic ING4/PTEN adenovirus (Ad.RGD-ING4-PTEN) and comprehensively investigated its effects following modification of the CNE human nasopharyngeal carcinoma cell line both in vitro and in vivo. We demonstrated that Ad.RGD-ING4-PTEN enhanced growth inhibition and apoptosis. Furthermore, expression of P21, Bax and cleaved caspase-3 was upregulated, while that of Bcl-2 and survivin was downregulated in CNE cells and CNE xenografted tumors. Moreover, Ad.RGD-ING4-PTEN treatment additively downregulated CD34, VEGF and microvessel density in subcutaneously (s.c.) xenografted CNE cell tumors. The enhanced antitumor activity generated by Ad.RGD-ING4-PTEN was closely associated with activation of the intrinsic and extrinsic apoptotic pathways and additive inhibition of tumor angiogenesis both in vitro and in vivo. On the basis of this evidence, it is believed that cancer gene therapy combining two tumor suppressors such as ING4 and PTEN can be used to establish an effective and novel therapeutic strategy for nasopharyngeal carcinoma and other cancers.

  17. Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells*

    PubMed Central

    Luo, Xu-wei; Liu, Kang; Chen, Zhu; Zhao, Ming; Han, Xiao-wei; Bai, Yi-guang; Feng, Gang

    2016-01-01

    Objective: To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 (GDF-5) gene, investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells, and explore a candidate gene therapy method for intervertebral disc degeneration (IDD). Methods: Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan (sGAG), deoxyribonucleic acid (DNA) and hydroxyproline (Hyp), synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results: The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium (BM) or Ad-green fluorescent protein (GFP) groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions: The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP cells. PMID:26739524

  18. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-12-29

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

  19. Adenovirus-mediated overexpression of soluble ST2 provides a protective effect on lipopolysaccharide-induced acute lung injury in mice

    PubMed Central

    Yin, H; Li, X Y; Yuan, B H; Zhang, B B; Hu, S L; Gu, H B; Jin, X B; Zhu, J Y

    2011-01-01

    Acute lung injury is characterized by a diffuse inflammatory parenchymal process, implicated in the context of significant morbidity and mortality. Previously, we have reported that soluble ST2 (sST2), a member of the Toll-interleukin (IL)-1 receptor (TIR) superfamily, represses proinflammatory cytokine production of macrophage exposed to lipopolysaccharide (LPS). In this study, we examined the possibility of modulating LPS-induced murine inflammatory pulmonary damage by recombinant adenovirus-mediated sST2-Fc (Ad-sST2-Fc) gene transfer. Single intranasal administration of Ad-sST2-Fc led to a profound decrease in LPS-induced bronchoalveolar lavage leucocyte exudation and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Histological examination revealed alveolitis with inflammatory cell infiltration and alveolar haemorrhage in the alveolar airspace was less severe in Ad-sST2-Fc-treated mice when compared with control groups. In addition, high levels of sST2-Fc in vivo reduced the transcription of tumour necrosis factor-α, IL-6 and Toll-like receptor-4 gene remarkably, and suppressed the nuclear translocation of nuclear factor-κB in lung tissues in response to LPS challenge. Taken together, these results suggested that administration of Ad-sST2-Fc gene transfer may have therapeutic potential for the immunomodulatory treatment of LPS-mediated inflammatory lung injury. PMID:21352201

  20. Adenovirus-mediated wild-type p53 gene transfer in combination with bronchial arterial infusion for treatment of advanced non-small-cell lung cancer, one year follow-up

    PubMed Central

    Guan, Yong-song; Liu, Yuan; Zou, Qing; He, Qing; La, Zi; Yang, Lin; Hu, Ying

    2009-01-01

    Objective: In the present study, we have examined the safety and efficacy of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) injection in patients with advanced non-small-cell lung cancer (NSCLC) in the combination with the therapy of bronchial arterial infusion (BAI). Methods: A total of 58 patients with advanced NSCLC were enrolled in a non-randomized, two-armed clinical trial. Of which, 19 received a combination treatment of BAI and rAd-p53 (the combo group), while the remaining 39 were treated with only BAI (the control group). Patients were followed up for 12 months, with safety and local response evaluated by the National Cancer Institute’s Common Toxicity Criteria and response evaluation criteria in solid tumor (RECIST), respectively. Time to progression (TTP) and survival rates were also analyzed by Kaplan-Meier method. Results: In the combo group, 19 patients received a total of 49 injections of rAd-p53 and 46 times of BAI, respectively, while 39 patients in the control group received a total of 113 times of BAI. The combination treatment was found to have less adverse events such as anorexia, nausea and emesis, pain, and leucopenia (P<0.05) but more arthralgia, fever, influenza-like symptom, and myalgia (P<0.05), compared with the control group. The overall response rates (complete response (CR)+partial response (PR)) were 47.3% and 38.4% for the combo group and the control group, respectively (P>0.05). Patients in the combo group had a longer TTP than those in the control group (a median 7.75 vs 5.5 months, P=0.018). However, the combination treatment did not lead to better survival, with survival rates at 3, 6, and 12 months in the combo group being 94.74%, 89.47%, and 52.63%, respectively, compared with 92.31%, 69.23%, and 38.83% in the control group (P=0.224). Conclusion: Our results show that the combination of rAd-p53 and BAI was well tolerated in patients with NSCLC and may have improved the quality of life and delayed

  1. Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

    PubMed Central

    Ghivizzani, Steven C.; Lechman, Eric R.; Kang, Richard; Tio, Caroline; Kolls, Jay; Evans, Christopher H.; Robbins, Paul D.

    1998-01-01

    Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides. PMID:9539786

  2. Downregulation of PI3Kcb utilizing adenovirus-mediated transfer of siRNA attenuates bone cancer pain.

    PubMed

    Huang, Huan-Jun; Zhang, Mei

    2014-01-01

    Phosphatidylinositol 3-kinase (PI3K) signaling plays a pivotal role in intracellular signal transduction pathways involved in chronic pain states. PI3K is implicated in pathomechanisms of enhanced synaptic strength, such as wind-up and central sensitization in the spinal dorsal horn. The PI3Kcb gene encoding the class 1A PI3K catalytic subunit p110beta is one of the most important molecular of the P13K signaling pathway. Here, we used small interfering RNA (siRNA) targeted to PI3Kcb by adenovirus-mediated transfer, to determine whether inhibition of PI3Kcb was a potential therapeutic target for bone cancer pain (BCP). In this study, treatment of BCP model in rats with PI3Kcb-specific siRNA resulted in inhibited pain-related behavior. Depletion of PI3Kcb decreased the protein levels of spinal PI3Kcb and phospho-Akt (P-Akt)-downstream targets of PI3K. Knockdown of PI3Kcb by siRNA also induced decreased expression of GFAP and OX42, suggesting that the upregulation of spinal PI3Kcb may increase glia excitability, at least in part by regulating glia message. Our findings suggest that siRNA-mediated gene silencing of PI3Kcb may be a useful therapeutic strategy for BCP. PMID:25550861

  3. Downregulation of PI3Kcb utilizing adenovirus-mediated transfer of siRNA attenuates bone cancer pain

    PubMed Central

    Huang, Huan-Jun; Zhang, Mei

    2014-01-01

    Phosphatidylinositol 3-kinase (PI3K) signaling plays a pivotal role in intracellular signal transduction pathways involved in chronic pain states. PI3K is implicated in pathomechanisms of enhanced synaptic strength, such as wind-up and central sensitization in the spinal dorsal horn. The PI3Kcb gene encoding the class 1A PI3K catalytic subunit p110beta is one of the most important molecular of the P13K signaling pathway. Here, we used small interfering RNA (siRNA) targeted to PI3Kcb by adenovirus-mediated transfer, to determine whether inhibition of PI3Kcb was a potential therapeutic target for bone cancer pain (BCP). In this study, treatment of BCP model in rats with PI3Kcb-specific siRNA resulted in inhibited pain-related behavior. Depletion of PI3Kcb decreased the protein levels of spinal PI3Kcb and phospho-Akt (P-Akt)-downstream targets of PI3K. Knockdown of PI3Kcb by siRNA also induced decreased expression of GFAP and OX42, suggesting that the upregulation of spinal PI3Kcb may increase glia excitability, at least in part by regulating glia message. Our findings suggest that siRNA-mediated gene silencing of PI3Kcb may be a useful therapeutic strategy for BCP. PMID:25550861

  4. Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

    SciTech Connect

    Li, Xiaohua; Fan, Rui; Zou, Xue; Gao, Lin; Jin, Haifeng; Du, Rui; Xia, Lin; Fan, Daiming . E-mail: fandaim@yahoo.com.cn

    2007-06-29

    Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma.

  5. HSV Recombinant Vectors for Gene Therapy

    PubMed Central

    Manservigi, Roberto; Argnani, Rafaela; Marconi, Peggy

    2010-01-01

    The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges. PMID:20835362

  6. Major psychological factors affecting acceptance of gene-recombination technology.

    PubMed

    Tanaka, Yutaka

    2004-12-01

    The purpose of this study was to verify the validity of a causal model that was made to predict the acceptance of gene-recombination technology. A structural equation model was used as a causal model. First of all, based on preceding studies, the factors of perceived risk, perceived benefit, and trust were set up as important psychological factors determining acceptance of gene-recombination technology in the structural equation model. An additional factor, "sense of bioethics," which I consider to be important for acceptance of biotechnology, was added to the model. Based on previous studies, trust was set up to have an indirect influence on the acceptance of gene-recombination technology through perceived risk and perceived benefit in the model. Participants were 231 undergraduate students in Japan who answered a questionnaire with a 5-point bipolar scale. The results indicated that the proposed model fits the data well, and showed that acceptance of gene-recombination technology is explained largely by four factors, that is, perceived risk, perceived benefit, trust, and sense of bioethics, whether the technology is applied to plants, animals, or human beings. However, the relative importance of the four factors was found to vary depending on whether the gene-recombination technology was applied to plants, animals, or human beings. Specifically, the factor of sense of bioethics is the most important factor in acceptance of plant gene-recombination technology and animal gene-recombination technology, and the factors of trust and perceived risk are the most important factors in acceptance of human being gene-recombination technology.

  7. Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy.

    PubMed

    Tosch, C; Geist, M; Ledoux, C; Ziller-Remi, C; Paul, S; Erbs, P; Corvaia, N; Von Hoegen, P; Balloul, J-M; Haegel, H

    2009-04-01

    The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumor-induced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-gamma. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad-P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy. PMID:18949016

  8. Genetic recombination is targeted towards gene promoter regions in dogs.

    PubMed

    Auton, Adam; Rui Li, Ying; Kidd, Jeffrey; Oliveira, Kyle; Nadel, Julie; Holloway, J Kim; Hayward, Jessica J; Cohen, Paula E; Greally, John M; Wang, Jun; Bustamante, Carlos D; Boyko, Adam R

    2013-01-01

    The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.

  9. Genetic Recombination Is Targeted towards Gene Promoter Regions in Dogs

    PubMed Central

    Auton, Adam; Rui Li, Ying; Kidd, Jeffrey; Oliveira, Kyle; Nadel, Julie; Holloway, J. Kim; Hayward, Jessica J.; Cohen, Paula E.; Greally, John M.; Wang, Jun; Bustamante, Carlos D.; Boyko, Adam R.

    2013-01-01

    The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred. PMID:24348265

  10. Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

    PubMed

    Choi, Kyuha; Reinhard, Carsten; Serra, Heïdi; Ziolkowski, Piotr A; Underwood, Charles J; Zhao, Xiaohui; Hardcastle, Thomas J; Yelina, Nataliya E; Griffin, Catherine; Jackson, Matthew; Mézard, Christine; McVean, Gil; Copenhaver, Gregory P; Henderson, Ian R

    2016-07-01

    Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

  11. Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes

    PubMed Central

    Serra, Heïdi; Ziolkowski, Piotr A.; Yelina, Nataliya E.; Jackson, Matthew; Mézard, Christine; McVean, Gil; Henderson, Ian R.

    2016-01-01

    Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity. PMID:27415776

  12. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  13. Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model.

    PubMed

    Tsung, Andrew J; Kargiotis, Odysseas; Chetty, Chandramu; Lakka, Sajani S; Gujrati, Meena; Spomar, Daniel G; Dinh, Dzung H; Rao, Jasti S

    2008-03-01

    Matrix metalloproteinases (MMPs) comprise a class of secreted zinc-dependent endopeptidases implicated in the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix (ECM) and basement membrane. Matrix metalloproteinase-2 (MMP-2) has been detected in high levels and correlates with invasiveness in human melanoma. We have studied the effect of adenovirus-mediated transfer of small interfering RNA (siRNA) against MMP-2 in the human melanoma cell line A2058. The delivery of these double-stranded RNA molecules represents an efficient technology in silencing disease-causing genes with known sequences at the post-transcriptional level. siRNA against MMP-2 mRNA (Ad-MMP-2) was found to decrease MMP-2 protein expression and activity in melanoma cells as demonstrated by western blotting and gelatin zymography. Furthermore, infection of cells with Ad-MMP-2 inhibited cellular migration and invasion as indicated by spheroid and matrigel assays. We also observed dose-dependent suppression of vascular network formation in an angiogenesis assay. Finally, we developed a nude mouse spinal metastatic model to investigate the local effects of tumor metastasis. Intravenous tail vein injection with Ad-MMP-2 on days 5, 9 and 11 after tumor implantation resulted in complete retention of neurological function as compared to control and scrambled vector (Ad-SV)-treated groups that showed complete paraplegia by day 14+/-2 days. Hematoxylin and eosin staining revealed decreased tumor size in the Ad-MMP-2-treated animals. This novel experimental model revealed that adenoviral-mediated transfer of RNA interference against MMP-2 results in the retention of neurological function and significantly inhibited tumor growth.

  14. [Knock out of bovine beta casein gene by homologous recombination].

    PubMed

    Xue, Ke; Li, Feng; Luo, Guang-Bin; Huang, Wei-Wei; Chen, Xue-Jin

    2007-05-01

    It has been reported that homologous recombination with Red system has been successfully used for knock-out. We try to work on the construction of the expression vector of Mammary Gland with Red system. This study takes CSN2 as a vector for gene target, which contains the complete bovine beta casein gene. Different homologous arms were designed and the CDS region of the beta casein gene was successfully knocked out. The efficiency was also explored for knocking out different DNA fragment. Based on the study, it is very convenient for making a deep research of the foreign gene expression under the regulation of CSN2 flanking region.

  15. Recombination and Gene Flux Caused by Gene Conversion and Crossing over in Inversion Heterokaryotypes

    PubMed Central

    Navarro, A.; Betran, E.; Barbadilla, A.; Ruiz, A.

    1997-01-01

    A theoretical analysis of the effects of inversions on recombination and gene flux between arrangements caused by gene conversion and crossing over was carried out. Two different mathematical models of recombination were used: the Poisson model (without interference) and the Counting model (with interference). The main results are as follows. (1) Recombination and gene flux are highly site-dependent both inside and outside the inverted regions. (2) Crossing over overwhelms gene conversion as a cause of gene flux in large inversions, while conversion becomes relatively significant in short inversions and in regions around the breakpoints. (3) Under the Counting model the recombination rate between two markers depends strongly on the position of the markers along the inverted segment. Two equally spaced markers in the central part of the inverted segment have less recombination than if they are in a more extreme position. (4) Inversions affect recombination rates in the uninverted regions of the chromosome. Recombination increases in the distal segment and decreases in the proximal segment. These results provide an explanation for a number of observations reported in the literature. Because inversions are ubiquitous in the evolutionary history of many Drosophila species, the effects of inversions on recombination are expected to influence DNA variation patterns. PMID:9178017

  16. Recombineering: using drug cassettes to knock out genes in vivo.

    PubMed

    Sawitzke, James A; Thomason, Lynn C; Bubunenko, Mikhail; Li, Xintian; Costantino, Nina; Court, Donald L

    2013-01-01

    A 'gene knockout' or 'knockout' is a mutation that inactivates a gene function. These mutations are very useful for classical genetic studies as well as for modern techniques including functional genomics. In the past, knockouts of bacterial genes were often made by transposon mutagenesis. In this case, laborious screens are required to find a knockout in the gene of interest. Knockouts of other organisms have traditionally been made by first using in vitro genetic engineering to modify genes contained on plasmids or bacterial artificial chromosomes (BACs) and later moving these modified constructs to the organism of interest by cell culture techniques. Other methods utilizing a combination of genetic engineering and in vivo homologous recombination were inefficient at best. Recombineering provides a new way to generate knockout mutations directly on the bacterial chromosome or to modify any plasmid or BAC in vivo as a prelude to making knockouts in other organisms. The constructs are designed to the base pair and are not dependent on suitable restriction sites. A drug cassette can be placed anywhere within a gene or the open reading frame of the gene can be replaced with the drug cassette. Either way, the desired construct is selected for.

  17. Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety

    NASA Astrophysics Data System (ADS)

    Moss, Bernard

    1996-10-01

    Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

  18. Recombination events suggest potential sites for the Huntington's disease gene.

    PubMed

    MacDonald, M E; Haines, J L; Zimmer, M; Cheng, S V; Youngman, S; Whaley, W L; Wexler, N; Bucan, M; Allitto, B A; Smith, B

    1989-08-01

    The Huntington's disease gene (HD) maps distal to the D4S10 marker in the terminal 4p16.3 subband of chromosome 4. Directed cloning has provided several DNA segments that have been grouped into three clusters on a physical map of approximately 5 X 10(6) bp in 4p16.3. We have typed RFLPs in both reference and HD pedigrees to produce a fine-structure genetic map that establishes the relative order of the clusters and further narrows the target area containing the HD gene. Despite the large number of meiotic events examined, the HD gene cannot be positioned relative to the most distal cluster. One recombination event with HD suggests that the terminal-most markers flank the disease gene; two others favor a telomeric location for the defect. Efforts to isolate the HD gene must be divided between these two distinct intervals until additional genetic data resolve the apparent contradiction in localization.

  19. Adenovirus-mediated siRNA targeting CXCR2 attenuates titanium particle-induced osteolysis by suppressing osteoclast formation.

    PubMed

    Wang, Chen; Liu, Yang; Wang, Yang; Li, Hao; Zhang, Ran-Xi; He, Mi-Si; Chen, Liang; Wu, Ning-Ning; Liao, Yong; Deng, Zhong-Liang

    2016-01-01

    BACKGROUND Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. MATERIAL AND METHODS Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. RESULTS Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. CONCLUSIONS CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening. PMID:26939934

  20. Adenovirus-Mediated siRNA Targeting CXCR2 Attenuates Titanium Particle-Induced Osteolysis by Suppressing Osteoclast Formation

    PubMed Central

    Wang, Chen; Liu, Yang; Wang, Yang; Li, Hao; Zhang, Ran-Xi; He, Mi-Si; Chen, Liang; Wu, Ning-Ning; Liao, Yong; Deng, Zhong-Liang

    2016-01-01

    Background Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. Material/Methods Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. Results Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. Conclusions CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening. PMID:26939934

  1. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  2. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  3. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  4. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  5. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  6. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  7. Therapeutic potential of adenovirus-mediated delivery of β-defensin 2 for experimental otitis media.

    PubMed

    Woo, Jeong-Im; Kil, Sung-Hee; Brough, Douglas E; Lee, Yoo Jin; Lim, David J; Moon, Sung K

    2015-02-01

    Otitis media (OM), one of the most prevalent diseases in young children, is clinically important owing to its high incidence in children and its potential impact on language development and motor coordination. OM is the most common reason for the prescription of antibiotics (accounting for 25% of prescriptions) due to its extremely high incidence. A recent increase in antibiotic resistance among OM pathogens is emerging as a major public health concern globally, which led us to consider non-antibiotic approaches for the management of OM. In this study, we evaluated gene transfer of an antimicrobial peptide, human β-defensin 2 (DEFB4), using an adenoviral vector (Ad5 with deletions of E1/E3/E4) as a potential therapeutic approach. We demonstrated that the transduction of human β-defensin 2 induces the production of human β-defensin 2 and suppresses non-typeable Haemophilus influenzae (NTHi) adhesion to human middle ear epithelial cells. Moreover, intratympanic inoculation of Ad-DEFB4 was found to attenuate NTHi-induced middle ear effusions without eliciting a significant immune response. Most importantly, intratympanic inoculation of Ad-DEFB4 appeared to significantly augment clearance of NTHi from middle ear cavity. Collectively, our results suggest that intratympanic gene delivery of antimicrobial molecules may serve as an alternative/adjuvant approach for the management of OM.

  8. Characterization of recombinant bacteriophages containing mosquito ribosomal RNA genes

    SciTech Connect

    Park, Y.J.

    1988-01-01

    A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been isolated by screening two different genomic DNA libraries - Charon 30 and EMBL 3 using {sup 32}P-labeled 18S and 28S rRNA as probes. These nine recombinant bacteriophages were characterized by restriction mapping, Southern blotting, and S1 nuclease analysis. The 18S rRNA coding region contains an evolutionarily conserved EcoRI site near the 3{prime}-end, and measures 1800 bp. The 28S rRNA genes were divided into {alpha} and {beta} coding regions measuring 1750 bp and 2000 bp, respectively. The gap between these two regions measures about 340 bp. No insertion sequences were found in the rRNA coding regions. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region. The non-transcribed spacer region of cloned A. albopictus rDNA contained a common series of seven PvuI sites within a 1250 bp region upstream of the 18S rRNA coding region, and a proportion of this region also showed heterogeneity both in the length and in the restriction sites.

  9. Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

    SciTech Connect

    Joshi, Ayesha; Ellenson, Lora Hedrick

    2011-07-01

    Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten{sup +/-} mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten{sup +/-} mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ER{alpha} as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

  10. Identification of recombination between Muscovy duck parvovirus and goose parvovirus structural protein genes.

    PubMed

    Shen, Hongxing; Zhang, Wen; Wang, Hua; Zhou, Yang; Shao, Shihe

    2015-10-01

    Waterfowl parvoviruses are divided into Muscovy duck parvoviruses (MDPVs) and goose parvoviruses (GPVs). Phylogenetic analysis based on structural gene nucleotide sequences showed that the strains of three GPVs (DY, PT and D strains) and two MDPVs (GX5 and SAAH-SHNH) are closely related and formed one cluster. Recombination analysis showed that recombination between GPV-GDFsh and MDPV-89384/FRANCE strains led to five recombinant strains: GPV-DY, GPV-PT, GPV-D, MDPV-GX5 and MDPV-SAAH-SHNH. The recombinant event was confirmed using the Simplot program and phylogenetic analysis. This is the first comprehensive investigation of recombination between MDPV and GPV structural genes.

  11. Molecular Mechanisms of Recombination Restriction in the Envelope Gene of the Human Immunodeficiency Virus

    PubMed Central

    Simon-Loriere, Etienne; Galetto, Roman; Hamoudi, Meriem; Archer, John; Lefeuvre, Pierre; Martin, Darren P.; Robertson, David L.; Negroni, Matteo

    2009-01-01

    The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology. PMID:19424420

  12. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    PubMed Central

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  13. Identification of recombination in the NS1 and VPs genes of parvovirus B19.

    PubMed

    Shen, Hongxing; Zhang, Wen; Wang, Hua; Shao, Shihe

    2016-08-01

    Human parvovirus B19 (B19V), a member of the genus Erythrovirus of the family Parvoviridae, is a pathogenic virus distributed worldwide in the human population. In this study, we performed phylogenetic and recombination analysis of B19V based on the available nonstructural gene (NS1) and capsid proteins (VPs) genes in GenBank. Results indicated that recombination occurred between genotypes 3 and 1, leading to the recombinant cluster genotype 2. Other three inter-genotype recombination events were also discovered. Moreover, our results showed that among the four recombinant events in the present study, all of the major parents belonged to genotype 1, the minor parents were from genotypes 3 or 2, and all of the recombinants belonged to genotype 2. These recombinant events were confirmed by SimPlot Program and phylogenetic analysis. J. Med. Virol. 88:1457-1461, 2016. © 2016 Wiley Periodicals, Inc.

  14. Divergence of human [alpha]-chain constant region gene sequences: A novel recombinant [alpha]2 gene

    SciTech Connect

    Chintalacharuvu, K. R.; Morrison, S.L. ); Raines, M. )

    1994-06-01

    IgA is the major Ig synthesized in humans and provides the first line of defense at the mucosal surfaces. The constant region of IgA heavy chain is encoded by the [alpha] gene on chromosome 14. Previous studies have indicated the presence of two [alpha] genes, [alpha]1 and [alpha]2 existing in two allotypic forms, [alpha]2 m(1) and [alpha]2 m(2). Here the authors report the cloning and complete nucleotide sequence determination of a novel human [alpha] gene. Nucleotide sequence comparison with the published [alpha] sequences suggests that the gene arose as a consequence of recombination or gene conversion between the two [alpha]2 alleles. The authors have expressed the gene as a chimeric protein in myeloma cells indicating that it encodes a functional protein. The novel IgA resembles IgA2 m(2) in that disulfide bonds link H and L chains. This novel recombinant gene provides insights into the mechanisms of generation of different constant regions and suggests that within human populations, multiple alleles of [alpha] may be present providing IgAs of different structures.

  15. Genetic Analysis of Fusion Recombinants in Bacillus Subtilis: Function of the Rece Gene

    PubMed Central

    Ftouhi, N.; Guillen, N.

    1990-01-01

    Bacillus subtilis protoplast fusion allows the study of the genetic recombination of an entire procaryotic genome. Protoplasts from bacterial strains marked genetically by chromosomal mutations were fused using polyethylene glycol and the regenerated cells analyzed. Recombinants represent 19.3% of heterozygotic cells; they are haploids. Individual characterization of clones show a unique particular phenotype in each colony suggesting that recombination takes place immediately after fusion, probably before the first cellular division. Recombination occurs in the whole chromosome; in one-third of the cases both reciprocal recombinants could be shown in the colony. The genetic interval that includes the chromosome replication origin shows the highest recombination level. Our results suggest that the RecE protein accounts for most of the fused protoplast recombination; however, some ``replication origin-specific'' recombination events were independent of the recE gene product. PMID:2123461

  16. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    PubMed

    Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  17. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

    PubMed Central

    Spring-Pearson, Senanu M.; Stone, Joshua K.; Doyle, Adina; Allender, Christopher J.; Okinaka, Richard T.; Mayo, Mark; Broomall, Stacey M.; Hill, Jessica M.; Karavis, Mark A.; Hubbard, Kyle S.; Insalaco, Joseph M.; McNew, Lauren A.; Rosenzweig, C. Nicole; Gibbons, Henry S.; Currie, Bart J.; Wagner, David M.; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order. PMID:26484663

  18. Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF

    PubMed Central

    Im, S-A; Kim, J-S; Gomez-Manzano, C; Fueyo, J; Liu, T-J; Cho, M-S; Seong, C-M; Lee, S N; Hong, Y-K; Yung, W K A

    2001-01-01

    Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-αVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-αVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-αVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF 165 cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-αVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336478

  19. Oncolytic adenovirus-mediated transfer of the antisense chk2 selectively inhibits tumor growth in vitro and in vivo.

    PubMed

    Chen, G; Zhou, J; Gao, Q; Huang, X; Li, K; Zhuang, L; Huang, M; Xu, G; Wang, S; Lu, Y; Ma, D

    2006-10-01

    Screening and identifying molecules target to checkpoint pathways has fostered the development of checkpoint-based anticancer strategies. Among these targets, inhibition of chk2 may induce cell death for tumors whose growth depends on enhanced chk2 activity. However, improvement of the potency and specificity of such therapeutics remains a major challenge. To resolve this problem, we constructed M3, a novel recombinant adenovirus with a 27-bp deletion in E1A CR2 region by which to realize tumor-specific replication, and an 829-bp of antisense chk2 fragment inserted into the E3 coding region. In this design, M3 exploited the native adenovirus E3 promoters to express antisense chk2 cDNA in a viral replication-dependent fashion, and preferentially silenced the chk2 gene in tumor cells. In vitro and in vivo assays confirmed that downregulated chk2 expression induced by M3 infection was tumor-specific and virus replication-dependent. Furthermore, systemic administration of M3 combined with a low dose of cisplatin cured 75% (9/12) of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. Our results indicated that the upcoming development in this field would improve the antitumor efficacy and maximize the synergistic effect of oncolytic viruses administered with traditional chemotherapy or radiotherapy. PMID:16741520

  20. The essential helicase gene RAD3 suppresses short-sequence recombination in Saccharomyces cerevisiae.

    PubMed Central

    Bailis, A M; Maines, S; Negritto, M T

    1995-01-01

    We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae. Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells. We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells. These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules. PMID:7623796

  1. Gene Evolutionary Trajectories and GC Patterns Driven by Recombination in Zea mays

    PubMed Central

    Sundararajan, Anitha; Dukowic-Schulze, Stefanie; Kwicklis, Madeline; Engstrom, Kayla; Garcia, Nathan; Oviedo, Oliver J.; Ramaraj, Thiruvarangan; Gonzales, Michael D.; He, Yan; Wang, Minghui; Sun, Qi; Pillardy, Jaroslaw; Kianian, Shahryar F.; Pawlowski, Wojciech P.; Chen, Changbin; Mudge, Joann

    2016-01-01

    Recombination occurring during meiosis is critical for creating genetic variation and plays an essential role in plant evolution. In addition to creating novel gene combinations, recombination can affect genome structure through altering GC patterns. In maize (Zea mays) and other grasses, another intriguing GC pattern exists. Maize genes show a bimodal GC content distribution that has been attributed to nucleotide bias in the third, or wobble, position of the codon. Recombination may be an underlying driving force given that recombination sites are often associated with high GC content. Here we explore the relationship between recombination and genomic GC patterns by comparing GC gene content at each of the three codon positions (GC1, GC2, and GC3, collectively termed GCx) to instances of a variable GC-rich motif that underlies double strand break (DSB) hotspots and to meiocyte-specific gene expression. Surprisingly, GCx bimodality in maize cannot be fully explained by the codon wobble hypothesis. High GCx genes show a strong overlap with the DSB hotspot motif, possibly providing a mechanism for the high evolutionary rates seen in these genes. On the other hand, genes that are turned on in meiosis (early prophase I) are biased against both high GCx genes and genes with the DSB hotspot motif, possibly allowing important meiotic genes to avoid DSBs. Our data suggests a strong link between the GC-rich motif underlying DSB hotspots and high GCx genes. PMID:27713757

  2. Recombinant HT.sub.m4 gene, protein and assays

    DOEpatents

    Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

    1996-01-01

    The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

  3. Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

    PubMed Central

    Selimyan, Roza; Gerstein, Rachel M.; Ivanova, Irina; Precht, Patricia; Subrahmanyam, Ramesh; Perlot, Thomas; Alt, Frederick W.; Sen, Ranjan

    2013-01-01

    Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (DH) and joining (JH) gene segments were methylated, DJH junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination. PMID:23382652

  4. Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly.

    PubMed

    Selimyan, Roza; Gerstein, Rachel M; Ivanova, Irina; Precht, Patricia; Subrahmanyam, Ramesh; Perlot, Thomas; Alt, Frederick W; Sen, Ranjan

    2013-01-01

    Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.

  5. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

    PubMed

    Hosted, T J; Baltz, R H

    1996-10-01

    Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

  6. Expression of the synthetic gene for human angiogenin in recombinant vaccinia virus

    SciTech Connect

    Netesova, N.A.; Petrov, V.S.; Cheshenko, N.V.

    1995-08-01

    The gene for angiogenin was cloned into vaccinia virus genome. The recombinant virus expressing angiogenin was obtained. The level of protein synthesis directed by the recombinant virus was analyzed by immunoblotting using monoclonal antibodies against human angiogenin. 15 refs., 2 figs.

  7. Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.

    PubMed

    Boshra, Hani; Cao, Jingxin; Babiuk, Shawn

    2016-01-01

    The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses.

  8. Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.

    PubMed

    Boshra, Hani; Cao, Jingxin; Babiuk, Shawn

    2016-01-01

    The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses. PMID:26458835

  9. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination*

    PubMed Central

    Zhang, Qiang; Chen, Qi-he; Fu, Ming-liang; Wang, Jin-ling; Zhang, Hong-bo; He, Guo-qing

    2008-01-01

    The bglS gene encoding endo-l,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1S), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-l,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer. PMID:18600782

  10. Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa).

    PubMed Central

    Chin, D B; Arroyo-Garcia, R; Ochoa, O E; Kesseli, R V; Lavelle, D O; Michelmore, R W

    2001-01-01

    Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion. PMID:11157000

  11. Recombination rates of Streptococcus pneumoniae isolates with both erm(B) and mef(A) genes.

    PubMed

    Lee, Ji-Young; Song, Jae-Hoon; Ko, Kwan Soo

    2010-08-01

    Erythromycin-resistant Streptococcus pneumoniae isolates containing both erm(B) and mef(A) genes have a higher rate of multidrug resistance (MDR). We investigated the relationships between the presence of erythromycin resistance determinants and the recombination rate. We determined the mutation and recombination frequencies of 46 S. pneumoniae isolates, which included 19 with both erm(B) and mef(A), nine with only erm(B), six with only mef(A), and 11 erythromycin-susceptible isolates. Mutation frequency values were estimated as the number of rifampin-resistant colonies as a proportion of total viable count. Genotypes and serotypes of isolates with the hyper-recombination phenotype were determined. Twelve S. pneumoniae isolates were hypermutable and four isolates were determined to have hyper-recombination frequency. Streptococcus pneumoniae isolates with both erm(B) and mef(A) genes did not show a high mutation frequency. In contrast, all isolates with a hyper-recombination phenotype contained both erm(B) and mef(A) genes. In addition, the recombination rate of isolates with both erm(B) and mef(A) genes was statistically higher than the rate of other isolates. The dual presence of erm(B) and mef(A) genes in some pneumococcal isolates may be associated with high recombination frequency. This may be one of the reasons for the frequent emergence of MDR in certain pneumococcal isolates.

  12. Reciprocal and Nonreciprocal Recombination at the Glucocerebrosidase Gene Region: Implications for Complexity in Gaucher Disease

    PubMed Central

    Tayebi, Nahid; Stubblefield, Barbara K.; Park, Joseph K.; Orvisky, Eduard; Walker, Jamie M.; LaMarca, Mary E.; Sidransky, Ellen

    2003-01-01

    Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms

  13. Recombinant HT{sub m4} gene, protein and assays

    DOEpatents

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  14. Clustering of Drosophila melanogaster Immune Genes in Interplay with Recombination Rate

    PubMed Central

    Wegner, K. Mathias

    2008-01-01

    Background Gene order in eukaryotic chromosomes is not random and has been linked to coordination of gene expression, chromatin structure and also recombination rate. The evolution of recombination rate is especially relevant for genes involved in immunity because host-parasite co-evolution could select for increased recombination rate (Red Queen hypothesis). To identify patterns left by the intimate interaction between hosts and parasites, I analysed the genomic parameters of the immune genes from 24 gene families/groups of Drosophila melanogaster. Principal Findings Immune genes that directly interact with the pathogen (i.e. recognition and effector genes) clustered in regions of higher recombination rates. Out of these, clustered effector genes were transcribed fastest indicating that transcriptional control might be one major cause for cluster formation. The relative position of clusters to each other, on the other hand, cannot be explained by transcriptional control per se. Drosophila immune genes that show epistatic interactions can be found at an average distance of 15.44±2.98 cM, which is considerably closer than genes that do not interact (30.64±1.95 cM). Conclusions Epistatically interacting genes rarely belong to the same cluster, which supports recent models of optimal recombination rates between interacting genes in antagonistic host-parasite co-evolution. These patterns suggest that formation of local clusters might be a result of transcriptional control, but that in the condensed genome of D. melanogaster relative position of these clusters may be a result of selection for optimal rather than maximal recombination rates between these clusters. PMID:18665272

  15. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

  16. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese. PMID:26341925

  17. Construction and identification of recombinant adenovirus carrying human TIMP-1shRNA gene.

    PubMed

    Sun, Y L; Xie, H; Lin, H L; Feng, Q; Liu, Y

    2015-01-16

    The aim of this study was to construct the recombinant adenovirus carrying human TIMP-1shRNA gene expression system for preliminary identification to lay the foundation for the further study of gene therapy. Using the Adeno-X system, the recombinant adenovirus plasmid pAdeno-X green fluorescent protein (GFP)-tissue inhibitor of metalloprotease (TIMP)-1 small hairpin (1shRNA) was constructed by including the target gene fragment of the TIMP-1shRNA shuttle plasmid pShuttle2-GFP-TIMP-1shRNA and the backbone plasmid pAdeno-X by homologous recombination in Escherichia coli. Recombinant plasmids were transfected into HEK293A cells to package the recombinant adenovirus rvAdeno-XGFP-TIMP-1shRNA. The recombinant adenovirus was identified by polymerase chain reaction, and the viral titer and infection efficiency were detected using GFP. Polymerase chain reaction and restriction endonuclease digestion demonstrated that rvAdeno-XGFP-TIMP-1shRNA had been successfully constructed, which has a strong ability to infect the kidney. The TIMP-1shRNA adenovirus expression vector was successfully constructed using homologous recombination methods.

  18. Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences.

    PubMed

    Smith, K A; Stallard, V; Roos, J M; Hart, C; Cormier, N; Cohen, L K; Roberts, B E; Payne, L G

    1993-01-01

    A simple yet powerful selection system was developed for the insertion of foreign genes in vaccinia virus. The selection system utilizes the vaccinia virus K1L (29K) host range gene which is located in HindIII M. This gene is necessary for growth in RK-13 cells but not in BSC40 or CV-1 cells. A vaccinia mutant (vAbT33) unable to grow on RK-13 cells was constructed having sequences at the 3' end of the K1L gene and the adjacent M2L gene deleted and replaced with the beta-galactosidase gene regulated by the BamHI F (F7L) promoter. A recombination plasmid containing the hepatitis B surface (HBs) antigen gene regulated by the M2L promoter and the complete sequence of the K1L gene was used to insert the HBs gene into vAbT33. The M2L negative K1L positive recombinant was easily isolated in two rounds of plaque purification by plating the virus on RK-13 cell monolayers. The K1L gene selection system allows the isolation of recombinants arising at frequencies as low as 1/100,000. It was noted that recombinants containing vaccinia sequence duplications (promoters) resulted in intragenomic recombinations that eliminated all sequences between the duplications. A second recombination plasmid was constructed that allowed insertion into the vaccinia genome without the loss of vaccinia coding sequences. This was achieved by insertion of the pseudorabies virus GIII gene regulated by the vaccinia H5R (40K) promoter between the translation and transcription stop signals at the 3' end of the K1L gene. The K1L gene transcription stop signal thus became the stop signal for the inserted GIII gene and an upstream transcription stop signal present in the H5R promoter fragment provided the stop signal for the K1L gene. This manipulation of the vaccinia genome had no effect on the accumulation or 5' end of the M2L gene transcripts. Although the insertion lengthened the 3' end and lowered the accumulation of K1L transcripts it altered neither the virulence nor the immunogenicity of the

  19. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  20. Positive correlation between evolutionary rate and recombination rate in Drosophila genes with male-biased expression.

    PubMed

    Zhang, Zhi; Parsch, John

    2005-10-01

    Previous studies have shown that genes that are expressed predominantly or exclusively in males tend to evolve rapidly in comparison to other genes. In most cases, however, it is unknown whether this rapid evolution is the result of increased positive (or sexual) selection on male-expressed traits or if it is due to a relaxation of selective constraints. To distinguish between these two possibilities, we analyzed the relationship between the nonsynonymous substitution rate (dN) and local recombination rate for 343 Drosophila genes that were classified as male, female, or nonsex biased in their expression. For the male-biased genes, a positive correlation between dN and recombination rate was observed. This can be explained by an increased rate of adaptive evolution in regions of higher recombination due to a reduction of Hill-Robertson interference. In contrast, the correlation between dN and recombination rate was negative for both female- and nonsex-biased genes, suggesting that these genes are primarily subject to purifying selection, which is expected to be less effective in regions of reduced recombination.

  1. Impact of karyotype organization on interlocus recombination between T cell receptor genes in Equidae.

    PubMed

    Drbalova, Jitka; Musilova, Petra; Kubickova, Svatava; Sebestova, Hana; Vahala, Jiri; Rubes, Jiri

    2014-01-01

    The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

  2. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  3. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia.

  4. Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus.

    PubMed

    Morrison, H G; Goldsmith, C S; Regnery, H L; Auperin, D D

    1991-03-01

    A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.

  5. Recombinant Gene Expression in vivo within Endothelial Cells of the Arterial Wall

    NASA Astrophysics Data System (ADS)

    Nabel, Elizabeth G.; Plautz, Gregory; Boyce, Frederick M.; Stanley, James C.; Nabel, Gary J.

    1989-06-01

    A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant β -galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing β -galactosidase, an indication that they were successfully implanted on the vessel wall.

  6. Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets.

    PubMed

    Nicholls, Linda I; Ainscow, Edward K; Rutter, Guy A

    2002-03-01

    Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.

  7. Adenovirus-mediated expression of orphan nuclear receptor NR4A2 targeting hepatic stellate cell attenuates liver fibrosis in rats

    PubMed Central

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Yang, Quanjun; Huang, Jinlu; Gan, Run; Guo, Cheng

    2016-01-01

    Liver fibrosis is a wound-healing response characterized with the accumulation of extracellular matrix (ECM). And hepatic stellate cells (HSCs) are the principal cell source of ECM. NR4A2 (Nurr1) is a member of orphan nuclear receptor NR4A family and acts as transcription factor. It participates in regulating cell differentiation, proliferation and apoptosis. We previously demonstrated that NR4A2 expression in fibrotic liver reduced significantly compared with normal liver and NR4A2 knockout in HSCs promoted ECM production. In the present study we explored the role of NR4A2 on liver fibrosis. Studies in cultured HSCs demonstrated that NR4A2 over-expression suppressed the activation of HSCs, such as ECM production and invasion ability. Moreover cell cycle was arrested, cell apoptosis was promoted and cell signaling pathway was influenced. Adenovirus-mediated delivery of NR4A2 in rats ameliorated significantly dimethylnitrosamine (DMN) induced liver fibrosis. The In vivo experiments produced results consistent with in vitro experiments. Taken together these results demonstrate NR4A2 enhancement attenuates liver fibrosis via suppressing the activation of HSCs and NR4A2 may be an ideal target for anti-fibrotic therapy. PMID:27646469

  8. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  9. Adenovirus-mediated expression of orphan nuclear receptor NR4A2 targeting hepatic stellate cell attenuates liver fibrosis in rats.

    PubMed

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Yang, Quanjun; Huang, Jinlu; Gan, Run; Guo, Cheng

    2016-01-01

    Liver fibrosis is a wound-healing response characterized with the accumulation of extracellular matrix (ECM). And hepatic stellate cells (HSCs) are the principal cell source of ECM. NR4A2 (Nurr1) is a member of orphan nuclear receptor NR4A family and acts as transcription factor. It participates in regulating cell differentiation, proliferation and apoptosis. We previously demonstrated that NR4A2 expression in fibrotic liver reduced significantly compared with normal liver and NR4A2 knockout in HSCs promoted ECM production. In the present study we explored the role of NR4A2 on liver fibrosis. Studies in cultured HSCs demonstrated that NR4A2 over-expression suppressed the activation of HSCs, such as ECM production and invasion ability. Moreover cell cycle was arrested, cell apoptosis was promoted and cell signaling pathway was influenced. Adenovirus-mediated delivery of NR4A2 in rats ameliorated significantly dimethylnitrosamine (DMN) induced liver fibrosis. The In vivo experiments produced results consistent with in vitro experiments. Taken together these results demonstrate NR4A2 enhancement attenuates liver fibrosis via suppressing the activation of HSCs and NR4A2 may be an ideal target for anti-fibrotic therapy. PMID:27646469

  10. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    PubMed

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-11-02

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

  11. A genome-wide identification of genes undergoing recombination and positive selection in Neisseria.

    PubMed

    Yu, Dong; Jin, Yuan; Yin, Zhiqiu; Ren, Hongguang; Zhou, Wei; Liang, Long; Yue, Junjie

    2014-01-01

    Currently, there is particular interest in the molecular mechanisms of adaptive evolution in bacteria. Neisseria is a genus of gram negative bacteria, and there has recently been considerable focus on its two human pathogenic species N. meningitidis and N. gonorrhoeae. Until now, no genome-wide studies have attempted to scan for the genes related to adaptive evolution. For this reason, we selected 18 Neisseria genomes (14 N. meningitidis, 3 N. gonorrhoeae and 1 commensal N. lactamics) to conduct a comparative genome analysis to obtain a comprehensive understanding of the roles of natural selection and homologous recombination throughout the history of adaptive evolution. Among the 1012 core orthologous genes, we identified 635 genes with recombination signals and 10 genes that showed significant evidence of positive selection. Further functional analyses revealed that no functional bias was found in the recombined genes. Positively selected genes are prone to DNA processing and iron uptake, which are essential for the fundamental life cycle. Overall, the results indicate that both recombination and positive selection play crucial roles in the adaptive evolution of Neisseria genomes. The positively selected genes and the corresponding amino acid sites provide us with valuable targets for further research into the detailed mechanisms of adaptive evolution in Neisseria.

  12. Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

    PubMed Central

    Liu, Jia; Song, Hongshuo; Liu, Donglai; Zuo, Tao; Lu, Fengmin; Zhuang, Hui; Gao, Feng

    2014-01-01

    Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations. PMID:25211143

  13. Recombination between elongation factor 1α genes from distantly related archaeal lineages

    PubMed Central

    Inagaki, Yuji; Susko, Edward; Roger, Andrew J.

    2006-01-01

    Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1α (EF-1α) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1α gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1α gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential “informational” genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing. PMID:16537397

  14. Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

    PubMed Central

    Johnson, L G; Boyles, S E; Wilson, J; Boucher, R C

    1995-01-01

    Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis. Images PMID:7533790

  15. Pathogen corruption and site-directed recombination at a plant disease resistance gene cluster.

    PubMed

    Nagy, Ervin D; Bennetzen, Jeffrey L

    2008-12-01

    The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site-leucine-rich repeat (NBS-LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the approximately 3.7-kb NBS-LRR genes. Because any unequal recombination located within the flanking NBS-LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS-LRR loci.

  16. Selection, Recombination, and Virulence Gene Diversity among Group B Streptococcal Genotypes▿ †

    PubMed Central

    Springman, A. Cody; Lacher, David W.; Wu, Guangxi; Milton, Nicole; Whittam, Thomas S.; Davies, H. Dele; Manning, Shannon D.

    2009-01-01

    Transmission of group B Streptococcus (GBS) from mothers to neonates during childbirth is a leading cause of neonatal sepsis and meningitis. Although subtyping tools have identified specific GBS phylogenetic lineages that are important in neonatal disease, little is known about the genetic diversity of these lineages or the roles that recombination and selection play in the generation of emergent genotypes. Here, we examined genetic variation, selection, and recombination in seven multilocus sequence typing (MLST) loci from 94 invasive, colonizing, and bovine strains representing 38 GBS sequence types and performed DNA sequencing and PCR-based restriction fragment length polymorphism analysis of several putative virulence genes to identify gene content differences between genotypes. Despite the low level of diversity in the MLST loci, a neighbor net analysis revealed a variable range of genetic exchange among the seven clonal complexes (CCs) identified, suggesting that recombination is partly responsible for the diversity observed between genotypes. Recombination is also important for several virulence genes, as some gene alleles had evidence for lateral gene exchange across divergent genotypes. The CC-17 lineage, which is associated with neonatal disease, is relatively homogeneous and therefore appears to have diverged independently with an exclusive set of virulence characteristics. These data suggest that different GBS genetic backgrounds have distinct virulence gene profiles that may be important for disease pathogenesis. Such profiles could be used as markers for the rapid detection of strains with an increased propensity to cause neonatal disease and may be considered useful vaccine targets. PMID:19581371

  17. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    NASA Astrophysics Data System (ADS)

    Golubnitchaya-Labudová, O.; Portele, A.; Vaçata, V.; Lubec, G.; Rink, H.; Höfer, M.

    1997-10-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the insert of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9.

  18. Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

    PubMed

    Wei, S; Zan, L S; Wang, H B; Cheng, G; Du, M; Jiang, Z; Hausman, G J; McFarland, D C; Dodson, M V

    2013-01-01

    Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

  19. Does lack of recombination enhance asymmetric evolution among duplicate genes? Insights from the Drosophila melanogaster genome.

    PubMed

    Clément, Yves; Tavares, Raquel; Marais, Gabriel A B

    2006-12-30

    Gene duplication has different outcomes: pseudogenization (death of one of the two copies), gene amplification (both copies remain the same), sub-functionalization (both copies are required to perform the ancestral function) and neo-functionalization (one copy acquires a new function). Asymmetric evolution (one copy evolves faster than the other) is usually seen as a signature of neo-functionalization. However, it has been proposed that sub-functionalization could also generate asymmetric evolution among duplicate genes when they experience different local recombination rates. Indeed, the low recombination copy is expected to evolve faster because of Hill-Robertson effects. Here we tested this idea with about 100 pairs of young duplicates from the Drosophila melanogaster genome. Looking only at young duplicates allowed us to compare recombination rates and evolutionary rates on a similar time-scale contrary to previous work. We found that dispersed pairs tend to evolve more asymmetrically than tandem ones. Among dispersed copies, the low recombination copy tends to be the fast-evolving one. We also tested the possibility that all this was explained by a confounding factor (expression level) but found no evidence for it. In conclusion, our results do support the idea that asymmetric evolution among duplicates is enhanced by restricted recombination. However, further work is needed to clearly distinguish between sub-functionalization and neo-functionalization for the asymmetrically-evolving duplicate pairs that we found.

  20. Direct evidence of recombination in the recA gene of Aeromonas bestiarum.

    PubMed

    Sanglas, Ariadna; Albarral, Vicenta; Farfán, Maribel; Lorén, J Gaspar; Fusté, M Carmen

    2016-03-01

    Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248 bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures.

  1. Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

    PubMed Central

    Scala, Stefania; Portella, Giuseppe; Fedele, Monica; Chiappetta, Gennaro; Fusco, Alfredo

    2000-01-01

    High mobility group I (HMGI) proteins are overexpressed in several human malignant tumors. We previously demonstrated that inhibition of HMGI synthesis prevents thyroid cell transformation. Here, we report that an adenovirus carrying the HMGI(Y) gene in an antisense orientation (Ad-Yas) induced programmed cell death of two human thyroid anaplastic carcinoma cell lines (ARO and FB-1), but not normal thyroid cells. The Ad-Yas virus led to death of lung, colon, and breast carcinoma cells. A control adenovirus carrying the lacZ gene did not inhibit the growth of either normal or neoplastic cells. Ad-Yas treatment of tumors induced in athymic mice by ARO cells caused a drastic reduction in tumor size. Therefore, suppression of HMGI(Y) protein synthesis by an HMGI(Y) antisense adenoviral vector may be a useful treatment strategy in a variety of human malignant neoplasias, in which HMGI(Y) gene overexpression is a general event. PMID:10759549

  2. A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

    SciTech Connect

    Gong, Y.; Li, X.M.; Shapiro, L.J.

    1994-09-01

    Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand, and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.

  3. Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

    PubMed Central

    Serra-Moreno, Ruth; Acosta, Sandra; Hernalsteens, Jean Pierre; Jofre, Juan; Muniesa, Maite

    2006-01-01

    Background The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. Results Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. Conclusion This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer. PMID:16984631

  4. Adenovirus-Mediated Over-Expression of Nrf2 Within Mesenchymal Stem Cells (MSCs) Protected Rats Against Acute Kidney Injury

    PubMed Central

    Mohammadzadeh-Vardin, Mohammad; Habibi Roudkenar, Mehryar; Jahanian-Najafabadi, Ali

    2015-01-01

    Purpose: Recent developments in the field of cell therapy have led to a renewed interest in treatment of acute kidney injury (AKI). However, the early death of transplanted mesenchymal stem cells (MSCs) in stressful microenvironment of a recipient tissue is a major problem with this kind of treatment. The objective of this study was to determine whether overexpression of a cytoprotective factor, nuclear factor erythroid-2 related factor 2 (Nrf2), in MSCs could protect rats against AKI. Methods: The Nrf2 was overexpressed in MSCs by recombinant adenoviruses, and the MSCs were implanted to rats suffering from cisplatin-induced AKI. Results: The obtained results showed that transplantation with the engineered MSCs ameliorates cisplatin-induced AKI. Morphologic features of the investigated kidneys showed that transplantation with the MSCs in which Nrf2 had been overexpressed significantly improved the complications of AKI. Conclusion: These findings suggested that the engineered MSCs might be a good candidate to be further evaluated in clinical trials. However, detailed studies must be performed to investigate the possible carcinogenic effect of Nrf2 overexpression. PMID:26236658

  5. Identification of a recent recombination event within the human beta-globin gene cluster.

    PubMed Central

    Gerhard, D S; Kidd, K K; Kidd, J R; Egeland, J A; Housman, D E

    1984-01-01

    In a detailed study of inheritance of DNA sequence polymorphism in a large reference pedigree, an individual was identified with an apparent genetic recombination event within the human beta-globin gene cluster. Analysis of the haplotypes of relevant individuals within this pedigree suggested that the meiotic crossing-over event is likely to have occurred within a 19.8-kilobase-pair region of the beta-globin gene cluster. Analysis of other DNA markers closely linked to the beta-globin gene cluster--segment 12 of chromosome 11 (D11S12) and loci for insulin, the cellular oncogene c-Ha-ras, and preproparathyroid hormone--confirmed that a crossover event must have occurred within the region of chromosome 11 between D11S12 and the beta-globin gene cluster. It is suggested that the event observed has occurred within a DNA region compatible with recombinational "hot spots" suggested by population studies. PMID:6096866

  6. Homologous recombination within the capsid gene of porcine circovirus type 2 subgroup viruses via natural co-infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several studies had reported homologous recombination between porcine circovirus type 2 (PCV2)-group 1 (Gp1) and -group 2 (Gp2) viruses. Interestingly, the recombination events described thus far mapped either within the Rep gene sequences or the sequences flanking the Rep gene region. Previously, ...

  7. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the present study was to gain new insights into the evolution, homologous recombination and selection pressures imposed on the porcine torovirus (PToV), by examining changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerge...

  8. Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

    ERIC Educational Resources Information Center

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

  9. Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo.

    PubMed

    Chen, Weizao; Liu, Mingqiu; Jiao, Ye; Yan, Weiyao; Wei, Xuefeng; Chen, Jiulian; Fei, Liang; Liu, Yang; Zuo, Xiaoping; Yang, Fugui; Lu, Yonggan; Zheng, Zhaoxin

    2006-04-01

    Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD. PMID:16537624

  10. Adenovirus-mediated delivery of an anti-V antigen monoclonal antibody protects mice against a lethal Yersinia pestis challenge.

    PubMed

    Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Senina, Svetlana; Perlin, David; Crystal, Ronald G; Boyer, Julie L

    2009-04-01

    Pneumonic plague, caused by inhalation of Yersinia pestis, represents a major bioterrorism threat for which no vaccine is available. Based on the knowledge that genetic delivery of monoclonal antibodies (MAbs) with adenovirus (Ad) gene transfer vectors results in rapid, high-level antibody expression, we evaluated the hypothesis that Ad-mediated delivery of a neutralizing antibody directed against the Y. pestis V antigen would protect mice against a Y. pestis challenge. MAbs specific for the Y. pestis V antigen were generated, and the most effective in protecting mice against a lethal intranasal Y. pestis challenge was chosen for further study. The coding sequences for the heavy and light chains were isolated from the corresponding hybridoma and inserted into a replication-defective serotype 5 human Ad gene transfer vector (AdalphaV). Western analysis of AdalphaV-infected cell supernatants demonstrated completely assembled antibodies reactive with V antigen. Following AdalphaV administration to mice, high levels of anti-V antigen antibody titers were detectable as early as 1 day postadministration, peaked by day 3, and remained detectable through a 12-week time course. When animals that received AdalphaV were challenged with Y. pestis at day 4 post-AdalphaV administration, 80% of the animals were protected, while 0% of control animals survived (P < 0.01). Ad-mediated delivery of a V antigen-neutralizing antibody is an effective therapy against plague in experimental animals and could be developed as a rapidly acting antiplague therapeutic.

  11. Gene expression analysis in MCF-7 breast cancer cells treated with recombinant bromelain.

    PubMed

    Fouz, Nour; Amid, Azura; Hashim, Yumi Zuhanis Has-Yun

    2014-08-01

    The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p<0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.

  12. The breast cancer susceptibility gene, BRCA2: at the crossroads between DNA replication and recombination?

    PubMed Central

    Venkitaraman, A R

    2000-01-01

    The identification and cloning of the familial breast cancer susceptibility gene, BRCA2, has excited much interest in its biological functions. Here, evidence is reviewed that the protein encoded by BRCA2 has an essential role in DNA repair through its association with mRad51, a mammalian homologue of bacterial and yeast proteins involved in homologous recombination. A model is proposed that the critical requirement for BRCA2 in cell division and the maintenance of chromosome stability stems from its participation in recombinational processes essential for DNA replication. PMID:10724455

  13. [Constructing recombinant plasmid pSH-CUP and knockout of acid trehalase gene in baker's yeast].

    PubMed

    He, Dongqin; Xiao, Dongguang; Lv, Ye

    2008-02-01

    The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(delta ATH1, G418(r)), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kan(r) gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.

  14. Progress with Recombinant Adeno-Associated Virus Vectors for Gene Therapy of Alpha-1 Antitrypsin Deficiency.

    PubMed

    Gruntman, Alisha M; Flotte, Terence R

    2015-06-01

    The pathway to a clinical gene therapy product often involves many changes of course and strategy before obtaining successful results. Here we outline the methodologies, both clinical and preclinical, that went into developing a gene therapy approach to the treatment of alpha-1 antitrypsin deficiency lung disease using muscle-targeted recombinant adeno-associated virus. From initial gene construct development in mouse models through multiple rounds of safety and biodistribution studies in rodents, rabbits, and nonhuman primates to ultimate human trials, this review seeks to provide insight into what clinical translation entails and could thereby inform the process for future investigators.

  15. Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector.

    PubMed Central

    Shimada, T; Fujii, H; Mitsuya, H; Nienhuis, A W

    1991-01-01

    We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets. Images PMID:1885765

  16. Measuring Immune Responses to recombinant AAV Gene Transfer

    PubMed Central

    Martino, Ashley T.; Herzog, Roland W.; Anegon, Ignacio; Adjali, Oumeya

    2013-01-01

    Following AAV-based gene transfer, the occurrence of adaptive immune responses specific to the vector or the transgene product is a major roadblock to successful clinical translation. These responses include antibodies against the AAV capsid, which can be neutralizing and therefore prevent the ability to repeatedly administer the vector, and CD8+ cytotoxic T lymphocytes, which can eliminate transduced cells. In addition, humans may have both humoral and cellular pre-existing immunity, as a result from natural infection with parent virus or related serotypes. The need for assays to detect and measure these anti-capsid immune responses in humans and in experimental animals is profound. Here, ELISPOT, immunocapture (ELISA), and neutralization assays are explained and provided in detail. Furthermore, such techniques can readily be adapted to monitor and quantify immune responses against therapeutic transgene products encoded by the vector genome. PMID:22034034

  17. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    PubMed Central

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior. PMID:27148349

  18. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    PubMed

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  19. Region-specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene.

    PubMed

    Li, Y F; Numata, M; Wahls, W P; Smith, G R

    1997-03-01

    Mutations in the rec11 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies by as much as a factor of 300 on chromosome III but less than a factor of 4 in the intervals tested on chromosomes I and II. To gain insight into the function of this region- (or chromosome-) specific activator of recombination, we have cloned and sequenced the rec11 gene. Meiotic crosses with rec11 disruption mutations placed the rec11 gene 6 cM from ade6 on chromosome III. Transcripts of rec11 accumulated transiently at 2-3 h after induction of melosis in a pat1-114 (Ts) mutant. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of these transcripts revealed eight introns. The spliced RNA is predicted to encode a polypeptide of 923 amino acids with only very limited homology to reported proteins. The transient accumulation of rec11 transcripts and the phenotype of rec11 mutations suggest that the novel rec11 gene product acts early in meiosis to activate recombination preferentially on chromosome III. PMID:9076725

  20. Genetic diversity and recombination analysis in the coat protein gene of Banana bract mosaic virus.

    PubMed

    Balasubramanian, V; Selvarajan, R

    2014-06-01

    Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of the bract mosaic disease (BBrMD) that causes serious yield losses in banana and plantain in India and the Philippines. In this study, global genetic diversity and molecular evolution of BBrMV based on the capsid protein (CP) gene were investigated. Multiple alignments of CP gene of 49 BBrMV isolates showed nucleotide (nt) and amino acid (aa) identity of 79-100 and 80-100 %, respectively. Phylogenetic analysis revealed that except two Indians isolates (TN14 and TN16), all isolates clustered together. Eleven recombination events were detected using Recombination Detection Program. Codon-based maximum-likelihood methods revealed that most of the codons in the CP gene were under negative or neutral selection except for codons 28, 43, and 92 which were under positive selection. Gene flow between BBrMV populations of banana and cardamom was relatively frequent but not between two different populations of banana infecting isolates identified in this study. This is the first report on genetic diversity, and evolution of BBrMV isolates based on recombination and phylogenetic analysis in India. PMID:24691817

  1. Genetic diversity and recombination analysis in the coat protein gene of Banana bract mosaic virus.

    PubMed

    Balasubramanian, V; Selvarajan, R

    2014-06-01

    Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of the bract mosaic disease (BBrMD) that causes serious yield losses in banana and plantain in India and the Philippines. In this study, global genetic diversity and molecular evolution of BBrMV based on the capsid protein (CP) gene were investigated. Multiple alignments of CP gene of 49 BBrMV isolates showed nucleotide (nt) and amino acid (aa) identity of 79-100 and 80-100 %, respectively. Phylogenetic analysis revealed that except two Indians isolates (TN14 and TN16), all isolates clustered together. Eleven recombination events were detected using Recombination Detection Program. Codon-based maximum-likelihood methods revealed that most of the codons in the CP gene were under negative or neutral selection except for codons 28, 43, and 92 which were under positive selection. Gene flow between BBrMV populations of banana and cardamom was relatively frequent but not between two different populations of banana infecting isolates identified in this study. This is the first report on genetic diversity, and evolution of BBrMV isolates based on recombination and phylogenetic analysis in India.

  2. A system for assaying homologous recombination at the endogenous human thymidine kinase gene

    SciTech Connect

    Benjamin, M.B.; Little, J.B. ); Potter, H. ); Yandell, D.W. Massachusetts Eye and Ear Infirmary, Boston Harvard Medical School, Boston, MA )

    1991-08-01

    A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

  3. Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

    PubMed Central

    Bonaldo, Myrna C; Mello, Samanta M; Trindade, Gisela F; Rangel, Aymara A; Duarte, Adriana S; Oliveira, Prisciliana J; Freire, Marcos S; Kubelka, Claire F; Galler, Ricardo

    2007-01-01

    Background The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor. Results YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus. Conclusion This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection. PMID

  4. Construction of a mouse hepatitis virus recombinant expressing a foreign gene.

    PubMed

    Fischer, F; Stegen, C F; Koetzner, C A; Masters, P S

    1998-01-01

    The genome of the coronavirus mouse hepatitis virus (MHV) contains genes which have been shown to be nonessential for viral replication and which could, in principle, be used as sites for the introduction of foreign sequences. We have inserted heterologous genetic material into gene 4 of MHV in order (i) to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the N gene; (ii) to develop further genetic tools for mutagenesis of structural genes other than N; and (iii) to examine the feasibility of using MHV as an expression vector. A DI-like donor RNA vector containing the MHV S gene and all genes distal to S was constructed. Initially, a derivative of this was used to insert a 19-nucleotide tag into the start of ORF 4a of MHV-A59 using the N gene deletion mutant A1b4 as the recipient virus. Subsequently, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4. This heterologous gene was shown to be expressed by recombinant viruses but not at levels sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected displacement of the mobility, relative to those of wild-type, of all subgenomic mRNAs larger than mRNA5. An unexpected result of the Northern analysis was the observation that GFP recombinants also produced an RNA species the same size as that of wild-type mRNA4. RT-PCR analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from a cluster of 10 different sites, none of which possessed a canonical intergenic sequence. The finding of these aberrant mRNAs, all of nearly the same size as wild-type mRNA4, suggests that long range structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

  5. Inhibition of hepatocellular carcinoma growth by adenovirus-mediated expression of human telomerase reverse transcriptase COOH-27 terminal polypeptide in mice

    PubMed Central

    HE, LEI; GONG, HAN-XIAN; LI, XIANG-PEN; WANG, YI-DONG; LI, YI; HUANG, JUN-JIAN; XIE, DAN; KUNG, HSIANG-FU; PENG, YING

    2013-01-01

    A 27-kDa C-terminal fragment of human telomerase reverse transcriptase, hTERTC27, has previously been reported to inhibit the growth and tumorigenicity of HeLa human cervical cancer cells and U87-MG human glioblastoma multiforme cells. However, the antitumor effects of hTERTC27 in hepatoma and its underlying mechanisms are unclear. In the current study, the therapeutic effect of hTERTC27, mediated by recombinant adenovirus, in hepatocellular carcinoma (HCC) was explored in vitro and in vivo to investigate the possible mechanisms. The results indicated that recombinant adenovirus carrying hTERTC27 (rAdv-hTERTC27) effectively inhibited the growth and induced apoptosis of the Hepa 1–6 HCC cells. Dendritic cells transduced with rAdv-hTERTC27 were highly effective at inducing antigen-specific T cell proliferation and increasing the activated cytotoxicity of T cells against Hepa 1–6 cells. HCC was inhibited significantly when a single dose of 5×107 pfu rAdv-hTERTC27 was administered intravenously. In summary, the results of this study demonstrated that rAdv-hTERTC27 may serve as a reagent for intravenous administration when combined with telomerase-based gene therapy and immunotherapy for cancer. PMID:24137404

  6. Recombination products suggest the frequent occurrence of aberrant gene replacement in the moss Physcomitrella patens.

    PubMed

    Wendeler, Edelgard; Zobell, Oliver; Chrost, Bozena; Reiss, Bernd

    2015-02-01

    In gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. Gene replacement in the moss Physcomitrella patens is extremely efficient, but often large amounts of additional DNA are integrated at the target locus. A detailed analysis of recombination junctions of PpCOL2 gene knockout mutants shows that the integrated DNA can be highly rearranged. Our data suggest that the replaced sequences were excised by HR and became integrated back into the genome by non-homologous end-joining (NHEJ). RAD51-mediated strand-invasion and subsequent strand-exchange is central to the two-end invasion pathway, the major gene replacement pathway in yeast. In this pathway, integration is initiated by the free ends of a single replacement vector-derived donor molecule which then integrates as an entity. Gene replacement in P. patens is entirely RAD51-dependent suggesting the existence of a pathway mechanistically similar to two-end invasion. However, invasion of the two ends does not seem to be stringently coordinated in P. patens. Actually, often only one fragment end became integrated by HR, or one-sided integration of two independent donor fragments occurred simultaneously leading to a double-strand break that is subsequently sealed by NHEJ and thus causes the observed rearrangements.

  7. A recombinant varicella vaccine harboring a respiratory syncytial virus gene induces humoral immunity.

    PubMed

    Murakami, Kouki; Matsuura, Masaaki; Ota, Megumi; Gomi, Yasuyuki; Yamanishi, Koichi; Mori, Yasuko

    2015-11-01

    The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is highly efficient and causes few adverse events; therefore, it is used worldwide. We previously constructed recombinant vOka (rvOka) harboring the mumps virus gene. Immunizing guinea pigs with rvOka induced the production of neutralizing antibodies against the mumps virus and VZV. Here, we constructed recombinant vOka viruses containing either the respiratory syncytial virus (RSV) subgroup A fusion glycoprotein (RSV A-F) gene or RSV subgroup B fusion glycoprotein (RSV B-F) gene (rvOka-RSV A-F or rvOka-RSV B-F). Indirect immunofluorescence and Western blot analyses confirmed the expression of each recombinant RSV protein in virus-infected cells. Immunizing guinea pigs with rvOka-RSV A-F or rvOka-RSV B-F led to the induction of antibodies against RSV proteins. These results suggest that the current varicella vaccine genome can be used to generate custom-made vaccine vectors to develop the next generation of live vaccines.

  8. Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

    PubMed

    Halligan, B D; Desiderio, S V

    1987-10-01

    Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

  9. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis

    PubMed Central

    Piriya, P. Sobana; Vasan, P. Thirumalai; Padma, V. S.; Vidhyadevi, U.; Archana, K.; Vennison, S. John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production. PMID:22919503

  10. Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis.

    PubMed

    Piriya, P Sobana; Vasan, P Thirumalai; Padma, V S; Vidhyadevi, U; Archana, K; Vennison, S John

    2012-01-01

    The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production.

  11. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin.

    PubMed

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L

    2013-01-01

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

  12. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  13. Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

    PubMed Central

    Mosig, Gisela; Gewin, John; Luder, Andreas; Colowick, Nancy; Vo, Daniel

    2001-01-01

    Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses. PMID:11459968

  14. Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene.

    PubMed

    Kahraman, Huseyin; Aytan, Emel; Kurt, Ash Giray

    2011-09-01

    The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.

  15. CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.

    PubMed Central

    Vedel, M; Nicolas, A

    1999-01-01

    We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. PMID:10101154

  16. Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

    NASA Astrophysics Data System (ADS)

    Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

    2007-05-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

  17. Divergent genes in potential inoculant Sinorhizobium strains are related to DNA replication, recombination, and repair.

    PubMed

    Penttinen, Petri; Greco, Dario; Muntyan, Victoria; Terefework, Zewdu; De Lajudie, Philippe; Roumiantseva, Marina; Becker, Anke; Auvinen, Petri; Lindström, Kristina

    2016-06-01

    To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains.

  18. Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

    PubMed Central

    Baker, M D; Read, L R

    1992-01-01

    We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell. Images PMID:1406631

  19. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  20. Gene CATCHR--gene cloning and tagging for Caenorhabditis elegans using yeast homologous recombination: a novel approach for the analysis of gene expression.

    PubMed

    Sassi, Holly E; Renihan, Stephanie; Spence, Andrew M; Cooperstock, Ramona L

    2005-01-01

    Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression. PMID:16254074

  1. Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    PubMed Central

    Hong, Ran; Bai, Weiya; Zhai, Jianwei; Liu, Wei; Li, Xinyan; Zhang, Jiming; Cui, Xiaoxian; Zhao, Xue; Ye, Xiaoli; Deng, Qiang; Tiollais, Pierre; Wen, Yumei

    2013-01-01

    Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. PMID:23552416

  2. Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11.

    PubMed

    Chowdhury, Emdadull H; Mikami, Osamu; Murata, Hideo; Sultana, Parvin; Shimada, Nobuaki; Yoshioka, Miyako; Guruge, Keerthi S; Yamamoto, Sachiko; Miyazaki, Shigeru; Yamanaka, Noriko; Nakajima, Yasuyuki

    2004-02-01

    The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.

  3. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  4. Ethanol production by recombinant Escherichia coli carrying genes from Zymomonas mobilis

    SciTech Connect

    Lawford, H.G.; Rousseau, J.D.

    1991-12-31

    Efficient utilization of lignocellulosic feedstocks offers an opportunity to reduce the cost of producing fuel ethanol. The fermentation performance characteristics of recombinant Escherichia coli ATCC 11303 carrying the {open_quotes}PET plasmid{close_quotes} (pLO1297) with the lac operon controlling the expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase 11 (adhB) genes cloned from Zymomonas mobilis CP4 were assessed in batch and continuous processes with sugar mixtures designed to mimic process streams from lignocellulosic hydrolysis systems.

  5. Intragenomic heterogeneity and intergenomic recombination among Vibrio parahaemolyticus 16S rRNA genes.

    PubMed

    Harth, Erika; Romero, Jaime; Torres, Rafael; Espejo, Romilio T

    2007-08-01

    Vibrio parahaemolyticus is a marine bacterium bearing 11 copies of ribosomal operons. In some strains, such as RIMD2210633, the genome includes identical copies of 16S rRNA genes (rrs). However, it is known that other strains of the species, such as strains ATCC 17802 and RIMD2210856, show conspicuous intragenomic rrs heterogeneity. The extent and diversity of the rrs heterogeneity in V. parahaemolyticus were studied in further detail by characterization of the rrs copies in environmental isolates belonging to 21 different genotype groups. Thirteen of these groups showed intragenomic heterogeneity, containing altogether 16 sequences differing within a 25 bp segment of their rrs. These sequences grouped into four clusters differing in at least four nucleotide sites. Some isolates contained rrs alleles from up to three different clusters. Each segment sequence conserved the stem-loop characteristic of the 16S rRNA structure of this 25 bp sequence. The double-stranded stem sequence was quite variable, but almost every variation had a compensatory change to maintain seven to eight paired bases. Conversely, the single-strand loop sequence was conserved. The results may be explained as a consequence of recombination among rrs evolving in different bacteria. The results suggest that intergenomic rrs recombination is very high in V. parahaemolyticus and that it occurs solely among Vibrio species. This high rrs homologous intergenomic recombination could be an effective mechanism to maintain intragenomic rrs cohesion, mediating the dispersal of the most abundant rrs version among the 11 intragenomic loci. PMID:17660428

  6. Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.

    PubMed

    Ohi, S

    2000-07-01

    With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.

  7. Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    PubMed Central

    Hwang, In Sun; Ahn, Il-Pyung

    2016-01-01

    Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi. PMID:27298592

  8. Recombinant AAV-directed gene therapy for type I glycogen storage diseases

    PubMed Central

    Chou, JY; Mansfield, BC

    2011-01-01

    Introduction Glycogen storage disease (GSD) type Ia and Ib are disorders of impaired glucose homeostasis affecting the liver and kidney. GSD-Ib also affects neutrophils. Current dietary therapies cannot prevent long-term complications. In animal studies, recombinant adeno-associated virus (rAAV) vector-mediated gene therapy can correct or minimize multiple aspects of the disorders, offering hope for human gene therapy. Areas covered A summary of recent progress in rAAV-mediated gene therapy for GSD-I; strategies to improve rAAV-mediated gene delivery, transduction efficiency and immune avoidance; and vector refinements that improve expression. Expert opinion rAAV-mediated gene delivery to the liver can restore glucose homeostasis in preclinical models of GSD-I, but some long-term complications of the liver and kidney remain. Gene therapy for GSD-Ib is less advanced than for GSD-Ia and only transient correction of myeloid dysfunction has been achieved. A question remains whether a single rAAV vector can meet the expression efficiency and tropism required to treat all aspects of GSD-I, or if a multi-prong approach is needed. An understanding of the strengths and weaknesses of rAAV vectors in the context of strategies to achieve efficient transduction of the liver, kidney, and hematopoietic stem cells is required for treating GSD-I. PMID:21504389

  9. Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane.

    PubMed

    Noda, Ken-ichi; Watanabe, Kimiko; Maruhashi, Kenji

    2003-07-01

    Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.

  10. In vivo gene therapy of murine melanoma mediated by recombinant vaccinia virus encoding human IL-2 gene.

    PubMed

    Wan, T; Cao, X; Ju, D; Aces, B

    1997-04-01

    Direct gene transfer into somatic tissue iii vivo is a developing technology with potential application for cancer gene therapy. In this study, recombinant vaccinia virus encoding human IL-2 gene (rVV-IL-2) was used as a candidate vector in mediating iii vivo gene therapy. After rVV-IL-2 was expanded in VERO cells for 72 h, high titer (10(8)-10(10) PFU/ml) rVV-IL-2 were harvested. When 10(6) murine melanoma cells (F16-F10) were infected with rVV-IL-2, about 200 U/ml IL-2 activity was detected in the supernatants at 8 h, and the up-regulation of ICAM-1 and MHC-I expressions on the melanoma cells were observed. The treatment of murine melanoma model by local injection of rVV-IL-2 into the tumor site showed that rVV-IL-2 transfection significantly inhibited the tumor growth and prolonged the survival time of tumor-bearing mice. The splenocytes from rVV-IL-2 treated mice showed higher cytotoxicities of NK, LAK and CTL in comparison with those from the controls. These results suggest that in vivo transfection mediated by rVV-IL-2 has potential effectiveness in enhancing host immunity and would be a useful approach to cancer gene therapy. PMID:21533434

  11. The joint effects of background selection and genetic recombination on local gene genealogies.

    PubMed

    Zeng, Kai; Charlesworth, Brian

    2011-09-01

    Background selection, the effects of the continual removal of deleterious mutations by natural selection on variability at linked sites, is potentially a major determinant of DNA sequence variability. However, the joint effects of background selection and genetic recombination on the shape of the neutral gene genealogy have proved hard to study analytically. The only existing formula concerns the mean coalescent time for a pair of alleles, making it difficult to assess the importance of background selection from genome-wide data on sequence polymorphism. Here we develop a structured coalescent model of background selection with recombination and implement it in a computer program that efficiently generates neutral gene genealogies for an arbitrary sample size. We check the validity of the structured coalescent model against forward-in-time simulations and show that it accurately captures the effects of background selection. The model produces more accurate predictions of the mean coalescent time than the existing formula and supports the conclusion that the effect of background selection is greater in the interior of a deleterious region than at its boundaries. The level of linkage disequilibrium between sites is elevated by background selection, to an extent that is well summarized by a change in effective population size. The structured coalescent model is readily extendable to more realistic situations and should prove useful for analyzing genome-wide polymorphism data.

  12. In vivo and in vitro analyses of recombinant baculoviruses lacking a functional cg30 gene.

    PubMed

    Passarelli, A L; Miller, L K

    1994-02-01

    The cg30 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes two sequence motifs, a zinc finger-like motif and a leucine zipper, found in other polypeptides known to be involved in gene regulation. To gain insight into the function of the cg30 product, CG30, we constructed and characterized recombinant viruses lacking a functional cg30 gene. We found that cg30 mutants had no striking phenotype in cell lines derived from Spodoptera frugiperda or Trichoplusia ni or in T. ni larvae. Although cg30 is known to be transcribed as an early monocistronic RNA and as the second cistron of an abundant late bicistronic RNA, production of a CG30-beta-galactosidase fusion protein was observed mainly at early times postinfection. Viruses containing cg30 had a subtle growth advantage over those lacking cg30 after several viral passages in cell culture. We employed transient expression assays to determine whether cg30 and pe-38, an AcMNPV gene that encodes a polypeptide with zinc finger-like and leucine zipper motifs similar to those of cg30, have redundant functions. Although pe-38 may have a role in AcMNPV gene expression, there was no indication that cg30 and pe-38 are functionally redundant.

  13. Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall.

    PubMed

    Ye, Y W; Landau, C; Willard, J E; Rajasubramanian, G; Moskowitz, A; Aziz, S; Meidell, R S; Eberhart, R C

    1998-01-01

    The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation-precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing betaGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous

  14. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population

    PubMed Central

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-01-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment. PMID:27485384

  15. The effects of recombination, mutation and selection on the evolution of the Rp1 resistance genes in grasses.

    PubMed

    Jouet, Agathe; McMullan, Mark; van Oosterhout, Cock

    2015-06-01

    Plant immune genes, or resistance genes, are involved in a co-evolutionary arms race with a diverse range of pathogens. In agronomically important grasses, such R genes have been extensively studied because of their role in pathogen resistance and in the breeding of resistant cultivars. In this study, we evaluate the importance of recombination, mutation and selection on the evolution of the R gene complex Rp1 of Sorghum, Triticum, Brachypodium, Oryza and Zea. Analyses show that recombination is widespread, and we detected 73 independent instances of sequence exchange, involving on average 1567 of 4692 nucleotides analysed (33.4%). We were able to date 24 interspecific recombination events and found that four occurred postspeciation, which suggests that genetic introgression took place between different grass species. Other interspecific events seemed to have been maintained over long evolutionary time, suggesting the presence of balancing selection. Significant positive selection (i.e. a relative excess of nonsynonymous substitutions (dN /dS >1)) was detected in 17-95 codons (0.42-2.02%). Recombination was significantly associated with areas with high levels of polymorphism but not with an elevated dN /dS ratio. Finally, phylogenetic analyses show that recombination results in a general overestimation of the divergence time (mean = 14.3%) and an alteration of the gene tree topology if the tree is not calibrated. Given that the statistical power to detect recombination is determined by the level of polymorphism of the amplicon as well as the number of sequences analysed, it is likely that many studies have underestimated the importance of recombination relative to the mutation rate.

  16. Signs of neutralization in a redundant gene involved in homologous recombination in Wolbachia endosymbionts.

    PubMed

    Badawi, Myriam; Giraud, Isabelle; Vavre, Fabrice; Grève, Pierre; Cordaux, Richard

    2014-09-17

    Genomic reduction in bacterial endosymbionts occurs through large genomic deletions and long-term accumulation of mutations. The latter process involves successive steps including gene neutralization, pseudogenization, and gradual erosion until complete loss. Although many examples of pseudogenes at various levels of degradation have been reported, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in nonessential genes, for example, those involved in redundant functions. Here, we report an example of gene neutralization in the homologous recombination (HR) pathway of Wolbachia, a bacterial endosymbiont of arthropods and nematodes. The HR pathway is often depleted in endosymbiont genomes, but it is apparently intact in some Wolbachia strains. Analysis of 12 major HR genes showed that they have been globally under strong purifying selection during the evolution of Wolbachia strains hosted by arthropods, supporting the evolutionary importance of the HR pathway for these Wolbachia genomes. However, we detected signs of recent neutralization of the ruvA gene in a subset of Wolbachia strains, which might be related to an ancestral, clade-specific amino acid change that impaired DNA-binding activity. Strikingly, RuvA is part of the RuvAB complex involved in branch migration, whose function overlaps with the RecG helicase. Although ruvA is experiencing neutralization, recG is under strong purifying selection. Thus, our high phylogenetic resolution suggests that we identified a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome.

  17. Signs of Neutralization in a Redundant Gene Involved in Homologous Recombination in Wolbachia Endosymbionts

    PubMed Central

    Badawi, Myriam; Giraud, Isabelle; Vavre, Fabrice; Grève, Pierre; Cordaux, Richard

    2014-01-01

    Genomic reduction in bacterial endosymbionts occurs through large genomic deletions and long-term accumulation of mutations. The latter process involves successive steps including gene neutralization, pseudogenization, and gradual erosion until complete loss. Although many examples of pseudogenes at various levels of degradation have been reported, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in nonessential genes, for example, those involved in redundant functions. Here, we report an example of gene neutralization in the homologous recombination (HR) pathway of Wolbachia, a bacterial endosymbiont of arthropods and nematodes. The HR pathway is often depleted in endosymbiont genomes, but it is apparently intact in some Wolbachia strains. Analysis of 12 major HR genes showed that they have been globally under strong purifying selection during the evolution of Wolbachia strains hosted by arthropods, supporting the evolutionary importance of the HR pathway for these Wolbachia genomes. However, we detected signs of recent neutralization of the ruvA gene in a subset of Wolbachia strains, which might be related to an ancestral, clade-specific amino acid change that impaired DNA-binding activity. Strikingly, RuvA is part of the RuvAB complex involved in branch migration, whose function overlaps with the RecG helicase. Although ruvA is experiencing neutralization, recG is under strong purifying selection. Thus, our high phylogenetic resolution suggests that we identified a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome. PMID:25230723

  18. Recombineering-based dissection of flanking and paralogous Hox gene functions in mouse reproductive tracts.

    PubMed

    Raines, Anna M; Adam, Mike; Magella, Bliss; Meyer, Sara E; Grimes, H Leighton; Dey, Sudhansu K; Potter, S Steven

    2013-07-01

    Hox genes are key regulators of development. In mammals, the study of these genes is greatly confounded by their large number, overlapping functions and interspersed shared enhancers. Here, we describe the use of a novel recombineering strategy to introduce simultaneous frameshift mutations into the flanking Hoxa9, Hoxa10 and Hoxa11 genes, as well as their paralogs on the HoxD cluster. The resulting Hoxa9,10,11 mutant mice displayed dramatic synergistic homeotic transformations of the reproductive tracts, with the uterus anteriorized towards oviduct and the vas deferens anteriorized towards epididymis. The Hoxa9,10,11 mutant mice also provided a genetic setting that allowed the discovery of Hoxd9,10,11 redundant reproductive tract patterning function. Both shared and distinct Hox functions were defined. Hoxd9,10,11 play a crucial role in the regulation of uterine immune function. Non-coding non-polyadenylated RNAs were among the key Hox targets, with dramatic downregulation in mutants. We observed Hox cross-regulation of transcription and splicing. In addition, we observed a surprising anti-dogmatic apparent posteriorization of the uterine epithelium. In caudal regions of the uterus, the normal simple columnar epithelium flanking the lumen was replaced by a pseudostratified transitional epithelium, normally found near the more posterior cervix. These results identify novel molecular functions of Hox genes in the development of the male and female reproductive tracts.

  19. Temperature influences β-carotene production in recombinant Saccharomyces cerevisiae expressing carotenogenic genes from Phaffia rhodozyma.

    PubMed

    Shi, Feng; Zhan, Wubing; Li, Yongfu; Wang, Xiaoyuan

    2014-01-01

    Red yeast Phaffia rhodozyma is a prominent microorganism able to synthesize carotenoid. Here, three carotenogenic cDNAs of P. rhodozyma CGMCC 2.1557, crtE, crtYB and crtI, were cloned and introduced into Saccharomyces cerevisiae INVSc1. The recombinant Sc-EYBI cells could synthesize 258.8 ± 43.8 μg g(-1) dry cell weight (DCW) of β-carotene when growing at 20 °C, about 59-fold higher than in those growing at 30 °C. Additional expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from S. cerevisiae (Sc-EYBIH) increased the β-carotene level to 528.8 ± 13.3 μg g(-1) DCW as cells growing at 20 °C, 27-fold higher than cells growing at 30 °C, although cells grew faster at 30 °C than at 20 °C. Consistent with the much higher β-carotene level in cells growing at 20 °C, transcription level of three crt genes and cHMG1 gene in cells growing at 20 °C was a little higher than in those growing at 30 °C. Meanwhile, expression of three carotenogenic genes and accumulation of β-carotene promoted cell growth. These results reveal the influence of temperature on β-carotene biosynthesis and may be helpful for improving β-carotene production in recombinant S. cerevisiae. PMID:23861041

  20. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    PubMed

    Przystałowska, Hanna; Zeyland, Joanna; Szymanowska-Powałowska, Daria; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol₁,₃-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming

  1. New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat.

    PubMed

    Dong, Lingli; Zhang, Xiaofei; Liu, Dongcheng; Fan, Huajie; Sun, Jiazhu; Zhang, Zhongjuan; Qin, Huanju; Li, Bin; Hao, Shanting; Li, Zhensheng; Wang, Daowen; Zhang, Aimin; Ling, Hong-Qing

    2010-01-01

    The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding. PMID:20975830

  2. New Insights into the Organization, Recombination, Expression and Functional Mechanism of Low Molecular Weight Glutenin Subunit Genes in Bread Wheat

    PubMed Central

    Fan, Huajie; Sun, Jiazhu; Zhang, Zhongjuan; Qin, Huanju; Li, Bin; Hao, Shanting; Li, Zhensheng; Wang, Daowen; Zhang, Aimin; Ling, Hong-Qing

    2010-01-01

    The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding. PMID:20975830

  3. Half-life measurements of chemical inducers for recombinant gene expression

    PubMed Central

    2014-01-01

    Background Inducible promoters are widely spread genetic tools for triggering, tuning and optimizing the expression of recombinant genes in engineered biological systems. Most of them are controlled by the addition of a specific exogenous chemical inducer that indirectly regulates the promoter transcription rate in a concentration-dependent fashion. In order to have a robust and predictable degree of control on promoter activity, the degradation rate of such chemicals should be considered in many applications like recombinant protein production. Results In this work, we use whole-cell biosensors to assess the half-life of three commonly used chemical inducers for recombinant Escherichia coli: Isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (ATc) and N-(3-oxohexanoyl)-L-homoserine lactone (HSL). A factorial study was conducted to investigate the conditions that significantly contribute to the decay rate of these inducers. Temperature has been found to be the major factor affecting ATc, while medium and pH have been found to highly affect HSL. Finally, no significant degradation was observed for IPTG among the tested conditions. Conclusions We have quantified the decay rate of IPTG, ATc and HSL in many conditions, some of which were not previously tested in the literature, and the main effects affecting their degradation were identified via a statistics-based framework. Whole-cell biosensors were successfully used to conduct this study, yielding reproducible measurements via simple multiwell-compatible assays. The knowledge of inducer degradation rate in several contexts has to be considered in the rational design of synthetic biological systems for improving the predictability of induction effects, especially for prolonged experiments. PMID:24485151

  4. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    PubMed

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.

  5. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. ); Schild, D. )

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  6. Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing.

    PubMed

    Girardet, A; Lien, S; Leeflang, E P; Beaufrère, L; Tuffery, S; Munier, F; Arnheim, N; Claustres, M; Pellestor, F

    1999-01-01

    In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma. PMID:10196709

  7. Strain variation, based on the hemagglutinin gene, in Norwegian ISA virus isolates collected from 1987 to 2001: indications of recombination.

    PubMed

    Devold, M; Falk, K; Dale, B; Krossøy, B; Biering, E; Aspehaug, V; Nilsen, F; Nylund, A

    2001-11-01

    Infectious salmon anemia (ISA) is caused by a virus that probably belongs to the Orthomyxoviridae and was first recorded in Norway in 1984. The disease has since spread along the Norwegian coast and has later been found in Canada, Scotland, the Faroe Islands, Chile, and the USA. This study presents sequence variation of the hemagglutinin gene from 37 ISA virus isolates, viz. one isolate from Scotland, one from Canada and 35 from Norway. The hemagglutinin gene contains a highly polymorphic region (HPR), which together with the rest of the gene sequence provides a good tool for studies of epizootics. The gene shows temporal and geographical sequence variation, where certain areas are dominated by distinct groups of isolates. Evidence of transmission of ISA virus isolates within and between regions is given. It is suggested that the hemagglutinin gene from different isolates may recombine. Possible recombination sites are found within the HPR and in the 5'-end flanking region close to the HPR. PMID:11775793

  8. Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

    PubMed Central

    Humphries, P; Old, R; Coggins, L W; McShane, T; Watson, C; Paul, J

    1978-01-01

    Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids. Images PMID:347404

  9. Analysis of the CYP21A2 gene with intergenic recombination and multiple gene deletions in the RCCX module.

    PubMed

    Chang, Shwu-Fen; Lee, Hsien-Hsiung

    2011-01-01

    The most frequent bimodular RCCX module of the RP1-C4A-CYP21A1P-TNXA-RP2-C4B-CYP21A2-TNXB gene sequence is located on chromosome 6p21.3. To determine RCCX alterations, we used the polymerase chain reaction (PCR) product containing the tenascin B (TNXB) and CYP21A2 genes with TaqI digestion and Southern blot analysis with AseI and NdeI endonuclease digestion of genomic DNA from congenital adrenal hyperplasia patients with common mutations resulting from an intergenic conversion of CYP21A1P, such as an I2 splice, I172N, V281L, F306-L307insT, Q318X, and R356W, and dual mutations of I236N/V237E in the CYP21A2 gene. The results showed that a 3.7-kb fragment of the CYP21A2 gene was detected in each case, and 21.6- and 11.3-kb DNA fragments were found in the RCCX region by a Southern blot analysis with these corresponding mutations. However, the IVS2-12A/C- > G (I2 splice) haplotype in combination with the 707-714delGAGACTAC (without the P30L mutation) mutation produced a 3.2-kb TaqI fragment in the PCR product analysis and a specific 9.3-kb fragment by the Southern blot method. Therefore, we concluded that the rearrangement in the RCCX region resulting from processing of either an intergenic recombination or multiple gene deletions can be identified by the PCR analysis and Southern blot method based on a fragment-distinguishing configuration without a family study.

  10. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    PubMed

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  11. A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene.

    PubMed

    Gadea, Gilles; Bos, Sandra; Krejbich-Trotot, Pascale; Clain, Elodie; Viranaicken, Wildriss; El-Kalamouni, Chaker; Mavingui, Patrick; Desprès, Philippe

    2016-10-01

    Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766(NIID), the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.

  12. Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

    PubMed Central

    TAKAHASHI, Hitomi; TSUNAZAKI, Makoto; HAMANO, Takashi; TAKAHASHI, Masashi; OKUDA, Kiyoshi; INUMARU, Shigeki; OKANO, Akira; GESHI, Masaya; HIRAKO, Makoto

    2013-01-01

    ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ. PMID:24212505

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

    PubMed

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

    2002-02-26

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

  14. Genetic engineering and heterologous expression of the disorazol biosynthetic gene cluster via Red/ET recombineering

    PubMed Central

    Tu, Qiang; Herrmann, Jennifer; Hu, Shengbiao; Raju, Ritesh; Bian, Xiaoying; Zhang, Youming; Müller, Rolf

    2016-01-01

    Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The successful reconstitution and expression of the genetic sequences encoding for these promising cytotoxic compounds will allow combinatorial biosynthesis to generate novel disorazol derivatives for further bioactivity evaluation. PMID:26875499

  15. Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes.

    PubMed

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S; Boeke, Jef D

    2015-02-10

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10(-10)), consistent with their orthogonal nature and the individual escape frequencies of <10(-6). Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance. PMID:25624482

  16. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

    PubMed Central

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S.; Boeke, Jef D.

    2015-01-01

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer’s yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational “safeguard” control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10−10), consistent with their orthogonal nature and the individual escape frequencies of <10−6. Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance. PMID:25624482

  17. Overproduction of polygalacturonase by Penicillium griseoroseum recombinant strains and functional analysis by targeted disruption of the pgg2 gene.

    PubMed

    Teixeira, Janaina Aparecida; Ribeiro, João Batista; Gonçalves, Daniel Bonoto; de Queiroz, Marisa Vieira; de Araújo, Elza Fernandes

    2013-03-01

    Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production. PMID:23354503

  18. Phylogenetic conservation of the 3' cryptic recombination signal sequence (3'cRSS) in the VH genes of jawed vertebrates.

    PubMed

    Sun, Yi; Liu, Zhancai; Li, Zhaoyong; Lian, Zhengxing; Zhao, Yaofeng

    2012-01-01

    The VH replacement process is a RAG-mediated secondary recombination in which the variable region of a rearranged VHDJH is replaced by a different germline VH gene. In almost all human and mouse VH genes, two sequence features appear to be crucial for VH replacement. First, an embedded heptamer, which is located near the 3' end of the rearranged VH gene, serves as a cryptic recombination signal sequence (3'cRSS) for the VH replacement process. Second, a short stretch of nucleotides located downstream of the 3'cRSS serve as a footprint of the original VH region, frequently encoding charged amino acids. In this review, we show that both of these two features are conserved in the VH genes of all jawed vertebrates, which suggests that the VH replacement process may be a conserved mechanism.

  19. Genetic variability and potential recombination events in the HC-Pro gene of sugarcane streak mosaic virus.

    PubMed

    Bagyalakshmi, K; Parameswari, B; Chinnaraja, C; Karuppaiah, R; Ganesh Kumar, V; Viswanathan, R

    2012-07-01

    Sugarcane streak mosaic virus (SCSMV), a member of the family Potyviridae, is an important viral pathogen affecting sugarcane production in India. The variability in the nucleotide (nt) and amino acid (aa) sequences of helper component proteinase (HC-Pro) of SCSMV isolates from India was investigated and compared with those of previously published virus isolates from different Asian countries. Comparison of all of the sequenced virus isolates revealed a high level of diversity in the HC-Pro gene (72-97% nt sequence identity; 83-99% aa sequence identity), and the Indian isolates were found to be the most divergent (up to 12% variation at the amino acid level). Phylogenetic analysis revealed clustering of 16 SCSMV isolates into two groups. Group I included isolates from India and Pakistan, and group II consisted of isolates from Japan and Indonesia. Recombination analysis revealed nine potentially significant recombination events, and putative recombination sites were identified throughout the HC-Pro gene. Analysis of selection pressure indicated that the HC-Pro gene of SCSMV is under strong negative selection. It is likely that recombination, along with strong negative selection, enhances the speed of elimination of deleterious mutations in the HC-Pro gene.

  20. An intramolecular recombination mechanism for the formation of the rRNA gene palindrome of Tetrahymena thermophila

    SciTech Connect

    Butler, D.K.; Yasuda, L.E.; Yao, Meng-Chao

    1995-12-01

    This report discusses the formation of rRNA gene palindrome in Tetrahymena thermophila and the involvement of intramolecular recombination. This, along with the authors` previous study, is the first to define a molecular pathway of palindrome formation. 48 refs., 6 figs.

  1. Molecular mapping of four blast resistance genes using recombinant inbred lines of 93-11 and nipponbare

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular mapping of new blast resistance genes is important for developing resistant rice cultivars using marker-assisted selection. In this study, 259 recombinant inbred lines (RILs) were developed from a cross between Nipponbare and 93-11, and were used to construct a 1165.8-cM linkage map with 1...

  2. Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

    PubMed Central

    Kanavaros, P; Farcet, J P; Gaulard, P; Haioun, C; Divine, M; Le Couedic, J P; Lefranc, M P; Reyes, F

    1991-01-01

    Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. Images PMID:1991851

  3. Cloning, expression of a peroxiredoxin gene from Acinetobacter sp. SM04 and characterization of its recombinant protein for zearalenone detoxification.

    PubMed

    Yu, Yuanshan; Wu, Hui; Tang, Yuqian; Qiu, Liping

    2012-03-20

    Zearalenone (ZEN) is a Fusarium mycotoxin, which has been associated with hyperestrogenism and other reproductive disorders in farm animals. ZEN-contaminated grains as well as its by-products had engendered numerous economic losses to farm animals' production, so the detoxification of ZEN-contaminated grains and its by-products would be necessary and beneficial. In this study, a peroxiredoxin (Prx) gene from Acinetobacter sp. SM04 was cloned, and over-expressed in Escherichia coli BL21 (DE3). The Prx gene of Acinetobacter sp. SM04 encodes a protein of 187 amino acids residues and NCBI BLAST program analysis of deduced amino acids shows high identity with 2-Cys Prx family. Interestingly, recombinant Prx show efficient ability to degrade ZEN using H(2)O(2). Results of MCF-7 cell proliferation assay also found ZEN were oxidized into little estrogenic metabolites by purified recombinant Prx plus H(2)O(2). Further, model experiments on decontamination of ZEN-contaminated corn using recombinant Prx were performed, and results found nearly 90% of ZEN was degraded when crushed ZEN-contaminated corn samples (nearly 1,000 μg ZEN per kg grain) were treated with purified recombinant Prx plus 0.09% (m/m) H(2)O(2) for 6h at 30°C. In addition, the optimum pH and temperature of purified recombinant Prx for ZEN degradation were 9.0 and 70°C respectively.

  4. Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus.

    PubMed

    Lee, Lucy E; Witter, R L; Reddy, S M; Wu, P; Yanagida, N; Yoshida, S

    2003-01-01

    Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

  5. A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene

    PubMed Central

    Shi, Yi; Ji, Wei; Jia, Hao; Zhou, Yongming; Wen, Honghua; Zhao, Honglan; Liu, Huaxing; Li, Hong; Wang, Qihui; Wu, Ying; Wang, Liang; Liu, Di; Liu, Guang; Yu, Hongjie; Holmes, Edward C.; Lu, Lin; Gao, George F.

    2016-01-01

    The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals. Herein, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3’-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia. Therefore, here we report a putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, providing insights into the fundamental mechanisms of viral evolution. PMID:27676249

  6. Gene structure, recombinant expression and functional characterization of grass carp leptin.

    PubMed

    Li, Guan-Gui; Liang, Xu-Fang; Xie, Qiuling; Li, Guangzhao; Yu, Ying; Lai, Kaaseng

    2010-03-01

    Leptin is an important hormone for the regulation of food intake, energy expenditure and reproduction in mammals, but information regarding its role in teleosts remains scant. In the present study, the gene structures of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) leptins were characterized. Recombinant grass carp leptin (rgc-LEP) was expressed in Escherichia coli and purified, and identified by mass spectrometric analysis. A strong anorexic effect on food intake was observed in grass carp on the first day after intraperitoneal (IP) injection of rgc-LEP, but not during the following days. Body weight of the leptin group (LEP group) and the pair-fed group (PF group) showed no difference throughout the experimental period. The acute and chronic effects on the expression of key genes correlating to food intake, energy expenditure, lipid metabolism and digestion were further characterized by real-time PCR. Accordingly, the mRNA levels of neuropeptide Y (NPY), Stearoyl-CoA desaturase 1 (SCD1) and lipoprotein lipase (LPL) were significantly reduced whereas the mRNA levels of uncoupling protein 2 (UCP2), bile salt-activated lipase (BSAL) and fatty acid elongase (ELO) were significantly elevated on the first day after injection. No effect on the expression of these genes (except LPL) was observed on day 13. In contrast to the down-regulation by exogenous leptin in mammals, the mRNA level of grass carp leptin was elevated 5.76-fold on the first day after rgc-LEP treatment. Our results suggest that leptin has an acute effect on the regulation of food intake, energy expenditure and lipid metabolism in grass carp, but the effect can be rapidly counteracted through mechanisms that are currently unknown.

  7. Overexpressing target helper genes enhances secretion and glycosylation of recombinant proteins in Pichia pastoris under simulated microgravity.

    PubMed

    Huangfu, Jie; Xu, Yinghua; Li, Chun; Li, Jun

    2016-10-01

    In this study, the potential helper genes were identified through the data analysis of transcriptomic and proteomic profiling in recombinant Pichia pastoris cultured under simulated microgravity (SMG). Co-expressing of four genes PRX1, YAP1, AHA1, and YPT6, involved in the oxidative stress response and protein folding, exhibited promising helper factor effects on the recombinant protein yields in engineered P. pastoris, respectively. When two of the above genes were co-expressed simultaneously, β-glucuronidase (PGUS) specific activity was further increased by 30.3-50.6 % comparing with that of single helper gene, particularly when the oxidative stress response and protein folding genes were both present in the combinations. In addition, co-expressing co-chaperone AHA1 and transcription factor YAP1 not only enhanced PGUS secretion, but also affected its glycosylation. Thus, through deep "omics" analysis of SMG effects, our results provided combined impact of new helper factors to improve the efficacy of recombinant protein secretion and glycosylation in engineered P. pastoris. PMID:27535143

  8. SEX LINKAGE, SEX-SPECIFIC SELECTION, AND THE ROLE OF RECOMBINATION IN THE EVOLUTION OF SEXUALLY DIMORPHIC GENE EXPRESSION

    PubMed Central

    Connallon, Tim; Clark, Andrew G.

    2010-01-01

    Sex-biased genes -- genes that are differentially expressed within males and females -- are nonrandomly distributed across animal genomes, with sex chromosomes and autosomes often carrying markedly different concentrations of male- and female-biased genes. These linkage patterns are often gene- and lineage-dependent, differing between functional genetic categories and between species. While sex-specific selection is often hypothesized to shape the evolution of sex-linked and autosomal gene content, population genetics theory has yet to account for many of the gene- and lineage-specific idiosyncrasies emerging from the empirical literature. With the goal of improving the connection between evolutionary theory and a rapidly growing body of genome-wide empirical studies, we extend previous population genetics theory of sex-specific selection by developing and analyzing a biologically informed model that incorporates sex linkage, pleiotropy, recombination, and epistasis, factors that are likely to vary between genes and between species. Our results demonstrate that sex-specific selection and sex-specific recombination rates can generate, and are compatible with, the gene- and species-specific linkage patterns reported in the genomics literature. The theory suggests that sexual selection may strongly influence the architectures of animal genomes, as well as the chromosomal distribution of fixed substitutions underlying sexually dimorphic traits. PMID:20874735

  9. Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: an unconventional combat with resistant pests.

    PubMed

    Kiani, Sarfraz; Mohamed, Bahaeldeen Babiker; Shehzad, Kamran; Jamal, Adil; Shahid, Muhammad Naveed; Shahid, Ahmad Ali; Husnain, Tayyab

    2013-07-10

    Plants transformed with single Bt gene are liable to develop insect resistance and this has already been reported in a number of studies carried out around the world where Bt cotton was cultivated on commercial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat with resistant larvae. This approach seems valid as various Bt genes possess different binding domains which could delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult under field conditions to develop homozygous plants expressing all Bt genes equally after many generations without undergoing recombination effects. A number of researches claiming to transform plants from three to seven transgenes in a single plant were reported during the last decade but none has yet applied for patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modified for improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy to develop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expression of hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfully applied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our transgenic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory and field bioassays gave promising results against American bollworm (Heliothis armigera), pink bollworm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reported to have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed by ELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While, localization of recombinant Cry1Ac-RB protein in

  10. The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

    PubMed Central

    Shimada, Midori; Nabeshima, Kentaro; Tougan, Takahiro; Nojima, Hiroshi

    2002-01-01

    During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Δ, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Δ was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17+-dependent manner. dmc1Δ also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression. PMID:12032093

  11. The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast.

    PubMed

    Shimada, Midori; Nabeshima, Kentaro; Tougan, Takahiro; Nojima, Hiroshi

    2002-06-01

    During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression. PMID:12032093

  12. Gene targeting and transgene stacking using intra genomic homologous recombination in plants.

    PubMed

    Kumar, Sandeep; Barone, Pierluigi; Smith, Michelle

    2016-01-01

    Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

  13. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    NASA Technical Reports Server (NTRS)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  14. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production

    PubMed Central

    Duwadi, Kishor; Chen, Ling; Menassa, Rima; Dhaubhadel, Sangeeta

    2015-01-01

    Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER), suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves. PMID:26148064

  15. A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge.

    PubMed

    Liu, Xiaohui; Yang, Youtian; Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F; Niu, Xuefeng; Guo, Xiaofeng

    2014-01-01

    The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines. PMID:24498294

  16. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  17. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  18. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters

    PubMed Central

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F.

    2015-01-01

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this “dead” cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters. PMID:26150486

  19. Recombinational cloning of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor A3(2).

    PubMed

    Zhang, Ran; Xia, Haiyang; Xu, Qingyu; Dang, Fujun; Qin, Zhongjun

    2013-08-01

    The model organism Streptomyces coelicolor A3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure, we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters.

  20. Efficient expression of truncated recombinant cadmium-metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis in Escherichia coli.

    PubMed

    Shuja, Rukhsana Nighat; Taimuri, Shuja Uddin Ahmad; Shakoori, Farah Rauf; Shakoori, Abdul Rauf

    2013-12-01

    Truncated recombinant metallothionein GST-fusion protein has been successfully expressed in Escherichia coli. The previously identified novel Cd-inducible metallothionein (TMCd1) gene from the locally isolated ciliate, Tetrahymena tropicalis lahorensis, was inserted into a pET-41a vector, in frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail. Truncated recombinant GST fusion protein has been purified by affinity column chromatography using glutathione sepharose. After enzymatic cleavage of GST tail with enterokinase, the truncated TMCd1 MT shows molecular weight of 11.5 kDa, corresponding to the expected value. This is the first successful report of expression of cadmium metallothionein gene of a ciliate, T. t. lahorensis, reported from this part of the world, in E. coli. This study will further help in characterization of metallothionein protein of this ciliate.

  1. Construction of recombinant Escherichia coli strains for secretory expression of artificial genes for human granulocyte-macrophage colony stimulating factor

    SciTech Connect

    Petrovskaya, L.E.; Ruzin, A.V.; Shingarova, L.N.; Korobko, V.G.

    1995-11-01

    A number of recombinant plasmids for expression of artificial genes encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. A hybrid gene was obtained that contains a sequence encoding the leader peptide and a tandem of two IgG-binding domains of protein A from Staphylococcus aureus coupled, through an enteropepdidase linker, to a synthetic gmcsf gene. The construction enables Escherichia coli to carry out biosynthesis of the hybrid protein and its subsequent transport into the periplasmic space of bacteria. Another hybrid gene, combining sequences for the signal peptide of the E. coli outer membrane protein OmpA and GM-CSF, was obtained using polymerase chain reaction. The localization of the mature protein produced by the hybrid gene was found to depend on the strength of the promoter used. 39 refs., 6 figs.

  2. Re-Designed Recombinant Hepatitis B Virus Vectors Enable Efficient Delivery of Versatile Cargo Genes to Hepatocytes with Improved Safety

    PubMed Central

    Bai, Weiya; Cui, Xiaoxian; Chen, Ruidong; Tao, Shuai; Hong, Ran; Zhang, Jiming; Zhang, Junqi; Wang, Yongxiang; Xie, Youhua; Liu, Jing

    2016-01-01

    Hepatitis B virus (HBV) takes humans as its sole natural host, and productive infection in vivo is restricted exclusively to hepatocytes in the liver. Consequently, HBV-derived viral vectors are attractive candidates for liver-targeting gene therapies. Previously, we developed a novel recombinant HBV vector, designated 5c3c, from a highly replicative clinical isolate. 5c3c was demonstrated to be capable of efficiently delivering protein or RNA expression into infected primary tupaia hepatocytes (PTH), but the design of 5c3c imposes stringent restrictions on inserted sequences, which have limited its wider adoption. In this work, we addressed issues with 5c3c by re-designing the insertion strategy. The resultant vector, designated 5dCG, was more replicative than parental 5c3c, imposed no specific restrictions on inserted sequences, and allowed insertion of a variety of cargo genes without significant loss of replication efficiency. 5dCG-based recombinant HBV effectively delivered protein and RNA expression into infected PTH. Furthermore, due to the loss of functional core ORF, 5dCG vectors depend on co-infecting wild type HBV for replication and efficient expression of cargo genes. Development of the improved 5dCG vector makes wider applications of recombinant HBV possible, while dependence on co-infecting wild type HBV results in improved safety for certain in vivo applications. PMID:27171107

  3. Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

    SciTech Connect

    Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi; Mathews, Lydia; Lucas, William T.; Murphy, Cynthia G.; Felber, Barbara K.; Pavlakis, George N.; Deluca, Neal A.; Knipe, David M. . E-mail: david_knipe@hms.harvard.edu

    2007-01-20

    Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.

  4. Do genetic recombination and gene density shape the pattern of DNA elimination in rice long terminal repeat retrotransposons?

    PubMed

    Tian, Zhixi; Rizzon, Carene; Du, Jianchang; Zhu, Liucun; Bennetzen, Jeffrey L; Jackson, Scott A; Gaut, Brandon S; Ma, Jianxin

    2009-12-01

    In flowering plants, the accumulation of small deletions through unequal homologous recombination (UR) and illegitimate recombination (IR) is proposed to be the major process counteracting genome expansion, which is caused primarily by the periodic amplification of long terminal repeat retrotransposons (LTR-RTs). However, the full suite of evolutionary forces that govern the gain or loss of transposable elements (TEs) and their distribution within a genome remains unclear. Here, we investigated the distribution and structural variation of LTR-RTs in relation to the rates of local genetic recombination (GR) and gene densities in the rice (Oryza sativa) genome. Our data revealed a positive correlation between GR rates and gene densities and negative correlations between LTR-RT densities and both GR and gene densities. The data also indicate a tendency for LTR-RT elements and fragments to be shorter in regions with higher GR rates; the size reduction of LTR-RTs appears to be achieved primarily through solo LTR formation by UR. Comparison of indica and japonica rice revealed patterns and frequencies of LTR-RT gain and loss within different evolutionary timeframes. Different LTR-RT families exhibited variable distribution patterns and structural changes, but overall LTR-RT compositions and genes were organized according to the GR gradients of the genome. Further investigation of non-LTR-RTs and DNA transposons revealed a negative correlation between gene densities and the abundance of DNA transposons and a weak correlation between GR rates and the abundance of long interspersed nuclear elements (LINEs)/short interspersed nuclear elements (SINEs). Together, these observations suggest that GR and gene density play important roles in shaping the dynamic structure of the rice genome.

  5. [Transcatheter delivery of recombinant adenovirus vector containing exogenous aquaporin gene in treatment of Sjögren's syndrome].

    PubMed

    He, Hong; Zhang, Jieqiong; Fan, Yan; Sun, Xiaoshuang; Zhu, Yuhao

    2016-01-01

    Sjögren's syndrome is a kind of autoimmune disease, whose main clinical symptoms are dry mouth, dry eye and chronic parotid glandular inflammation. The conservative treatments include artificial tears or saliva,oral administration of corticosteroids,and immunosuppressantsl with limited effectiveness. Along with the development of molecular biology, vast attentions are being paid to researches on gene therapy for Sjögren's syndrome, hopefully to bring gospel to patients with Sjögren's syndrome. This article reviews the recent research progresses on transcatheter delivery of recombinant adenovirus vector with aquaporin gene in experimental treatment of Sjögren's syndrome. PMID:27045247

  6. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris.

    PubMed

    Hohenblum, Hubertus; Gasser, Brigitte; Maurer, Michael; Borth, Nicole; Mattanovich, Diethard

    2004-02-20

    The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source. PMID:14755554

  7. Expression of cytokine and apoptosis-related genes in bovine peripheral blood mononuclear cells stimulated with Brucella abortus recombinant proteins.

    PubMed

    Im, Young Bin; Jung, Myunghwan; Shin, Min-Kyoung; Kim, Suk; Yoo, Han Sang

    2016-02-11

    Brucellosis is a clinically and economically important disease. Therefore, eradication programs of the disease have been implemented in several countries. One hurdle in these programs is the detection of infected animals at the early stage. Although the protein antigens as diagnostic antigens have recently received attention, the exact mechanisms at the beginning of immune responses are not yet known. Therefore, genes encoding five B. abortus cellular proteins were cloned and the expressed recombinant proteins were purified. The expression of several cytokine genes (IL-1β, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α, and iNOS) was analyzed in bovine peripheral blood mononuclear cells (bPBMC) after stimulation with the recombinant proteins. Three apoptosis-related genes, Bax, Bcl-2, and TLR4, were also included in the analysis to find out the adverse effects of the proteins to the cells. Each protein induced different patterns of cytokine expression depending on the stimulation time and antigen dose. Expression of IL-6, IL-12p40, and IFN-γ was induced with all of the proteins while IL-1β, IL-4, TNF-α, and iNOS gene expression was not. Expression of apoptosis-related genes was not altered except TLR4. These results suggest that the cellular antigens of B. abortus induce both humoral and cellular immunity via the production of IL-6, IL-12p40, and IFN-γ in bPBMC without exerting any adverse effects on the cells.

  8. Polymorphism, recombination and alternative unscrambling in the DNA polymerase alpha gene of the ciliate Stylonychia lemnae (Alveolata; class Spirotrichea).

    PubMed Central

    Ardell, David H; Lozupone, Catherine A; Landweber, Laura F

    2003-01-01

    DNA polymerase alpha is the most highly scrambled gene known in stichotrichous ciliates. In its hereditary micronuclear form, it is broken into >40 pieces on two loci at least 3 kb apart. Scrambled genes must be reassembled through developmental DNA rearrangements to yield functioning macronuclear genes, but the mechanism and accuracy of this process are unknown. We describe the first analysis of DNA polymorphism in the macronuclear version of any scrambled gene. Six functional haplotypes obtained from five Eurasian strains of Stylonychia lemnae were highly polymorphic compared to Drosophila genes. Another incompletely unscrambled haplotype was interrupted by frameshift and nonsense mutations but contained more silent mutations than expected by allelic inactivation. In our sample, nucleotide diversity and recombination signals were unexpectedly high within a region encompassing the boundary of the two micronuclear loci. From this and other evidence we infer that both members of a long repeat at the ends of the loci provide alternative substrates for unscrambling in this region. Incongruent genealogies and recombination patterns were also consistent with separation of the two loci by a large genetic distance. Our results suggest that ciliate developmental DNA rearrangements may be more probabilistic and error prone than previously appreciated and constitute a potential source of macronuclear variation. From this perspective we introduce the nonsense-suppression hypothesis for the evolution of ciliate altered genetic codes. We also introduce methods and software to calculate the likelihood of hemizygosity in ciliate haplotype samples and to correct for multiple comparisons in sliding-window analyses of Tajima's D. PMID:14704164

  9. Adenovirus-mediated co-expression of ING4 and PTEN cooperatively enhances their antitumor activity in human hepatocellular carcinoma cells.

    PubMed

    Rakshit, Nargis; Yang, Sijun; Zhou, Wei; Xu, Yi; Deng, Chenghui; Yang, Jiecheng; Yu, Huijun; Wei, Wenxiang

    2016-08-01

    Both inhibitor of growth 4 (ING4) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are well known as tumor suppressors that are closely related to tumor occurrence and progression. It was reported that ING4 and PTEN showed synergistic antitumor activities in nasopharyngeal carcinoma cells. The two tumor suppressors demonstrated synergistic effect on growth inhibition and apoptosis activation. In this study, we investigated their therapeutic potential in hepatocellular carcinoma (HCC) cells. Recombinant adenoviruses co-expressing ING4 and PTEN (Ad-ING4-PTEN) were constructed, and the antitumor effect on SMMC-7721 and HepG2 HCC cells was evaluated. Ad-ING4-PTEN cooperatively inhibited cell growth, stimulated apoptosis, and suppressed invasion in both HCC cells, and regulated cell cycle in SMMC-7721. Further studies showed that the combination of ING4 and PTEN by Ad-ING4-PTEN cooperatively enhanced the alteration of the expression of cell cycle-related proteins (p53, p21, and cyclin D1) and apoptotic factors (Bad, Bcl-2, Bcl-XL, and Bax), which are involved in the regulation of cell cycle and the activation of apoptotic pathways, leading to the synergistic antitumor effect. These results indicate that the combination of ING4 and PTEN may provide an effective therapeutic strategy for HCC.

  10. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

    PubMed

    Stieger, Knut; Colle, Marie-Anne; Dubreil, Laurence; Mendes-Madeira, Alexandra; Weber, Michel; Le Meur, Guylène; Deschamps, Jack Yves; Provost, Nathalie; Nivard, Delphine; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne

    2008-05-01

    Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

  11. Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1).

    PubMed

    Yang, A H; Yeh, K W

    2005-06-01

    A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5'-/3'-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST-CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 microg recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150-200 microg/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.

  12. A Genetic Analysis of the Drosophila mcm5 Gene Defines a Domain Specifically Required for Meiotic Recombination

    PubMed Central

    Lake, Cathleen M.; Teeter, Kathy; Page, Scott L.; Nielsen, Rachel; Hawley, R. Scott

    2007-01-01

    Members of the minichromosome maintenance (MCM) family have pivotal roles in many biological processes. Although originally studied for their role in DNA replication, it is becoming increasingly apparent that certain members of this family are multifunctional and also play roles in transcription, cohesion, condensation, and recombination. Here we provide a genetic dissection of the mcm5 gene in Drosophila that demonstrates an unexpected function for this protein. First, we show that homozygotes for a null allele of mcm5 die as third instar larvae, apparently as a result of blocking those replication events that lead to mitotic divisions without impairing endo-reduplication. However, we have also recovered a viable and fertile allele of mcm5 (denoted mcm5A7) that specifically impairs the meiotic recombination process. We demonstrate that the decrease in recombination observed in females homozygous for mcm5A7 is not due to a failure to create or repair meiotically induced double strand breaks (DSBs), but rather to a failure to resolve those DSBs into meiotic crossovers. Consistent with their ability to repair meiotically induced DSBs, flies homozygous for mcm5A7 are fully proficient in somatic DNA repair. These results strengthen the observation that members of the prereplicative complex have multiple functions and provide evidence that mcm5 plays a critical role in the meiotic recombination pathway. PMID:17565942

  13. Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1).

    PubMed

    Yang, A H; Yeh, K W

    2005-06-01

    A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5'-/3'-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST-CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 microg recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150-200 microg/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium. PMID:15647900

  14. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    PubMed

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  15. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.

    PubMed

    Wang, Dan; Mou, Haiwei; Li, Shaoyong; Li, Yingxiang; Hough, Soren; Tran, Karen; Li, Jia; Yin, Hao; Anderson, Daniel G; Sontheimer, Erik J; Weng, Zhiping; Gao, Guangping; Xue, Wen

    2015-07-01

    CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9. PMID:26086867

  16. Hepatitis B virus (HBV) X gene diversity and evidence of recombination in HBV/HIV co-infected persons.

    PubMed

    Martin, Christina M; Welge, Jeffrey A; Blackard, Jason T

    2011-07-01

    The high frequency of mutation during hepatitis B virus (HBV) infection has resulted in 8 genotypes (A-H) with varying effects on disease severity and treatment efficacy. However, analysis of intrapatient HBV diversity is limited, especially during HIV co-infection. Therefore, a preliminary study was performed to analyze HBV X gene diversity in 17 HBV/HIV co-infected individuals. Phylogenetic analysis revealed HBV genotype A in 13 individuals (76.5%) or genotype E in 1 individual (5.9%). Additionally, 3 individuals were dually infected with HBV genotypes A and G (17.6%). Overall, higher genetic distance and entropy were observed in the X region and overlapping polymerase (Pol(X)) regions when compared to the PreS, S, and overlapping polymerase (Pol(PS) and Pol(S)) regions analyzed in the same patients as part of a previous study. In addition, multiple viral variants from 2 individuals with dual HBV infection did not group with either genotype A or G by phylogenetic analysis, indicating possible recombination. SimPlot bootscan analysis confirmed recombination breakpoints within the X gene in both individuals. Recombination between HBV genotypes may represent an important evolutionary strategy that enhances overall pathogenic potential and/or alters the downstream effects of the HBV X protein.

  17. Semiconservative DNA replication is initiated at a single site in recombination-deficient gene 32 mutants of bacteriophage T4.

    PubMed Central

    Dannenberg, R; Mosig, G

    1981-01-01

    We have investigated, by electron microscopy, replicative intermediate produced early after infection of Escherichia coli with two phage T4 gene 32 mutants (amA453 and tsG26) which replicate their parental DNA but are defective in secondary replications and in moderating the activities of recombination nucleases. Under conditions completely restrictive for progeny production, both of these mutant produced replicative intermediates, each containing a single internal loop. Both branches of these loops were double stranded; i.e., both leading and lagging strands were synthesized. The replicative intermediates of these mutants qualitatively and quantitatively resembled early replicating wild-type T4 chromosomes after solitary infection of E. coli. However, in contrast to intracellular wild-type T4 DNA isolated from multiple infection, the mutant DNAs showed neither multiple branches nor multiple tandem loops. These results demonstrate that a truncated gene 32 protein which consists of less than one-third of the wild-type T4 helix-destabilizing protein can facilitate the functions of T4 replication proteins, specifically those of T4 DNA polymerase and priming proteins. Our results also support the hypothesis that the generation of multiple tandem loops or branches in vegetative T4 DNA depends on recombination (Mosig et al., in B. Alberts, ed., Mechanistic Studies of DNA Replication and Genetic Recombination, p. 527-543, Academic Press, Inc., New York, 1980). Images PMID:7321104

  18. Protective Immunity against Eimeria acervulina following In Ovo Immunization with a Recombinant Subunit Vaccine and Cytokine Genes

    PubMed Central

    Ding, Xicheng; Lillehoj, Hyun S.; Quiroz, Marco A.; Bevensee, Erich; Lillehoj, Erik P.

    2004-01-01

    A purified recombinant protein from Eimeria acervulina (3-1E) was used to vaccinate chickens in ovo against coccidiosis both alone and in combination with expression plasmids encoding the interleukin 1 (IL-1), IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or gamma interferon (IFN-γ) gene. When used alone, vaccination with 100 or 500 μg of 3-1E resulted in significantly decreased oocyst shedding compared with that in nonvaccinated chickens. Simultaneous vaccination of the 3-1E protein with the IL-1, -15, -16, or -17 gene induced higher serum antibody responses than 3-1E alone. To evaluate protective intestinal immunity, vaccinated birds were challenged with live E. acervulina oocysts 14 days posthatch, and fecal-oocyst shedding and body weight gain were determined as parameters of coccidiosis. Chickens vaccinated with 3-1E protein showed significantly lower oocyst shedding and normal body weight gain than nonvaccinated and infected controls. Simultaneous immunization with 3-1E and the IL-2, -15, -17, or -18 or IFN-γ gene further reduced oocyst shedding compared with that achieved with 3-1E alone. These results provide the first evidence that in ovo vaccination with the recombinant 3-1E Eimeria protein induces protective intestinal immunity against coccidiosis, and this effect was enhanced by coadministration of genes encoding immunity-related cytokines. PMID:15557615

  19. Relative contributions of recombination and mutation to the diversification of the opa gene repertoire of Neisseria gonorrhoeae.

    PubMed

    Bilek, Nicole; Ison, Catherine A; Spratt, Brian G

    2009-03-01

    To understand the rates and mechanisms of Neisseria gonorrhoeae opa gene variation, the 11 opa genes were amplified independently so that an opa allelic profile could be defined for any isolate from the sequences at each locus. The opa allelic profiles from 14 unrelated isolates were all different, with no opa alleles shared between isolates. Examination of very closely related isolates from sexual contacts and sexual networks showed that these typically shared most opa alleles, and the mechanisms by which recent changes occurred at individual opa loci could be determined. The great majority of changes were due to recombination among existing alleles that duplicated an opa allele present at another locus or resulted in a mosaic of existing opa alleles. Single nucleotide changes or insertion/deletion of a single codon also occurred, but few of these events were assigned to mutation, the majority being assigned to localized recombination. Introduction of novel opa genes from coinfecting strains was rare, and all but one were observed in the same sexual network. Changes at opa loci occurred at a greater rate than those at the porin locus, and the opa11 locus changed more rapidly than other opa loci, almost always differing even between recent sexual contacts. Examination of the neighboring pilE gene showed that changes at opa11 and pilE often occurred together, although this linkage may not be a causal one.

  20. Relative contributions of recombination and mutation to the diversification of the opa gene repertoire of Neisseria gonorrhoeae.

    PubMed

    Bilek, Nicole; Ison, Catherine A; Spratt, Brian G

    2009-03-01

    To understand the rates and mechanisms of Neisseria gonorrhoeae opa gene variation, the 11 opa genes were amplified independently so that an opa allelic profile could be defined for any isolate from the sequences at each locus. The opa allelic profiles from 14 unrelated isolates were all different, with no opa alleles shared between isolates. Examination of very closely related isolates from sexual contacts and sexual networks showed that these typically shared most opa alleles, and the mechanisms by which recent changes occurred at individual opa loci could be determined. The great majority of changes were due to recombination among existing alleles that duplicated an opa allele present at another locus or resulted in a mosaic of existing opa alleles. Single nucleotide changes or insertion/deletion of a single codon also occurred, but few of these events were assigned to mutation, the majority being assigned to localized recombination. Introduction of novel opa genes from coinfecting strains was rare, and all but one were observed in the same sexual network. Changes at opa loci occurred at a greater rate than those at the porin locus, and the opa11 locus changed more rapidly than other opa loci, almost always differing even between recent sexual contacts. Examination of the neighboring pilE gene showed that changes at opa11 and pilE often occurred together, although this linkage may not be a causal one. PMID:19114493

  1. Sequence analysis of the gene for a novel superantigen produced by Yersinia pseudotuberculosis and expression of the recombinant protein

    SciTech Connect

    Yasuhiko, Ito; Abe, Jun; Kohsaka, Takao

    1995-06-01

    We previously reported that the Gram-negative bacterium Yersinia pseudotuberculosis produces a superantigen (YPM, Y. pseudotuberculosis-derived mitogen) that expands T cells bearing V{beta}s 3, 9, 13.1, and 13.2 in an MHC class II-dependent manner. Based on the previously determined N-terminal 23 amino acids of YPM (T-D-Y-D-N-T-L-N-S-I-P-S-L-R-I-P-N-I-A-T-Y-T-G- (one-letter code)), we cloned the ypm gene and analyzed the nucleotide sequence. The gene encodes a 151-amino acid protein with a 20-amino acid signal peptide at its N terminus. The recombinant YPM expressed by the cloned gene exerted a mitogenic activity on human PBMC at a concentration of approximately 1 pg/ml. T cells bearing V{beta} 13.3 were preferentially expanded as well as T cells bearing the same V{beta} repertoires stimulated by native YPM. T cells were stimulated by the recombinant YPM in the presence of either fixed or unfixed HLA class II-transfected mouse fibroblasts. Furthermore, sequence diversity in the junctional region of the TCR {beta}-chain containing the V{beta}3 element could be observed after stimulation by the recombinant YPM. These results indicate that YPM belongs to the category of superantigens and should be included as a novel member. The amino acid sequence of the mature protein showed no significant homology to other superantigens derived from Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes. This observation, together with the substantially smaller m.w. suggest that ypm must have evolved from a different ancestral gene. 67 refs., 7 figs., 5 tabs.

  2. Phylogenetic analysis of VP1 gene sequences of waterfowl parvoviruses from the Mainland of China revealed genetic diversity and recombination.

    PubMed

    Wang, Shao; Cheng, Xiao-Xia; Chen, Shao-Ying; Lin, Feng-Qiang; Chen, Shi-Long; Zhu, Xiao-Li; Wang, Jin-Xiang; Huang, Mei-Qing; Zheng, Min

    2016-03-01

    To determine the origin and evolution of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) in the Mainland of China, phylogenetic and recombination analyses in the present study were performed on 32 complete VP1 gene sequences from China and other countries. Based on the phylogenetic analysis of the VP1 gene, GPV strains studied here from Mainland China (PRC) could be divided into three genotypes, namely PRC-I, PRC-II and PRC-III. Genotype PRC-I is indigenous to Mainland China. Only one GPV strain from Northeast China was of Genotype PRC-II and was thought to be imported from Europe. Genotype PRC-III, which was the most isolated genotype during 1999-2012, is related to GPVs in Taiwan and has been the predominant pathogen responsible for recent Derzy's disease outbreaks in Mainland China. Current vaccine strains used in Mainland China belong to Genotype PRC-I that is evolutionary distant from Genotypes PRC-II and PRC-III. In comparison, MDPV strains herein from Mainland China are clustered in a single group which is closely related to Taiwanese MDPV strains, and the full-length sequences of the VP1 gene of China MDPVs are phylogenetic closely related to the VP1 sequence of a Hungarian MDPV strain. Moreover, We also found that homologous recombination within VP1 gene plays a role in generating genetic diversity in GPV evolution. The GPV GDFSh from Guangdong Province appears to be the evolutionary product of a recombination event between parental GPV strains GD and B, while the major parent B proved to be a reference strain for virulent European GPVs. Our findings provide valuable information on waterfowl parvoviral evolution in Mainland China.

  3. Ethanol production by recombinant Escherichia coli carrying genes from Zymomonas mobilis.

    PubMed

    Lawford, H G; Rousseau, J D

    1991-01-01

    Efficient utilization of lignocellulosic feedstocks offers an opportunity to reduce the cost of producing fuel ethanol. The fermentation performance characteristics of recombinant Escherichia coli ATCC 11303 carrying the "PET plasmid" (pLOI297) with the lac operon controlling the expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes cloned from Zymomonas mobilis CP4 (Alterthum & Ingram, 1989) were assessed in batch and continuous processes with sugar mixtures designed to mimic process streams from lignocellulosic hydrolysis systems. Growth was pseudoexponential at a rate (generation time) of 1.28 h at pH 6.8 and 1.61 h at pH 6.0. The molar growth yields for glucose and xylose were 17.28 and 7.65 g DW cell/mol, respectively (at pH 6.3 and 30 degrees C), suggesting that the net yield of ATP from xylose metabolism is only 50% compared to glucose. In pH-stat batch fermentations (Luria broth with 6% sugar, pH 6.3), glucose was converted to ethanol 4-6 times faster than xylose, but the glucose conversion rate was much less than can be achieved with comparable cell densities of Zymomonas. Sugar-to-ethanol conversion efficiencies in nutrient-rich, complex LB medium were near theoretical at 98 and 88% for glucose and xylose, respectively. The yield was 10-20% less in a defined-mineral-salts medium. Acetate at a concentration of 0.1M (present in lignocellulosic hydrolysates from thermochemical processing) inhibited glucose utilization (about 50%) much more than xylose, and caused a decrease in product yield of about 30% for both sugars. With phosphate-buffered media (pH 7), glucose was a preferred substrate in mixtures with a ratio of hexose to pentose of 2.3 to 1. Xylose was consumed after glucose, and the product yield was less (0.37 g/g). Under steady-state conditions of continuous culture, the specific productivity ranged from 0.76-1.24 g EtOH/g cell/h, and the maximum volumetric productivity, 2.5 g EtOH/L/h, was achieved with a rich

  4. Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

    PubMed Central

    Carlon, Marianne S.; Vidović, Dragana; Dooley, James; da Cunha, Marina Mori; Maris, Michael; Lampi, Youlia; Toelen, Jaan; Van den Haute, Chris; Baekelandt, Veerle; Deprest, Jan; Verbeken, Erik; Liston, Adrian; Gijsbers, Rik

    2014-01-01

    Abstract Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α1-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months. PMID:24548076

  5. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    PubMed

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  6. Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes

    SciTech Connect

    van Endert, P.M.; Lopez, M.T.; Patel, S.D.; McDevitt, H.O. ); Monaco, J.J. )

    1992-12-01

    Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.

  7. Mutational analysis of the Drosophila DNA repair and recombination gene mei-9.

    PubMed Central

    Yildiz, Ozlem; Kearney, Hutton; Kramer, Benjamin C; Sekelsky, Jeff J

    2004-01-01

    Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift. PMID:15166153

  8. Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

    NASA Astrophysics Data System (ADS)

    Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

    1985-01-01

    Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

  9. Growth properties and vaccine efficacy of recombinant pseudorabies virus defective in glycoprotein E and thymidine kinase genes.

    PubMed

    Wu, Ching-Ying; Liao, Chih-Ming; Chi, Jiun-Ni; Chien, Maw-Sheng; Huang, Chienjin

    2016-07-10

    Pseudorabies virus (PRV) is an alphaherpesvirus that causes pseudorabies (PR), an economically important viral disease of pigs. Marker vaccines were widely used in PR prevention and eradication programs. The purpose of this study was to construct a novel recombinant virus with deletions at defined regions in the glycoprotein E (gE) and thymine kinase (TK) genes by homologous recombination. This study also evaluated the safety and efficacy of the virus for a live attenuated marker vaccine. No significant difference was observed in virus replication between gE gene-deleted (gE(-)), gE/TK double gene-deleted (gE(-)TK(-)), and wild-type PRV by growth curve analysis. However, gE(-)TK(-) PRV was completely attenuated in mice. To evaluate the immunogenicity of gE(-)TK(-) PRV, four 12-week-old specific-pathogen-free pigs per group were immunized intramuscularly with viral titers of 1×10(4), 1×10(5), or 1×10(6) TCID50, followed by intranasal challenge infection with virulent PRV (1×10(8) TCID50) at 3 weeks post vaccination. The gE(-)TK(-) PRV-vaccinated pigs displayed no general adverse effects after immunization and had protective immune responses after PRV challenge. Thus, gE(-)TK(-) PRV was safe and efficacious and might be a potential candidate for a live attenuated marker vaccine against PRV. PMID:27164258

  10. Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

    PubMed

    Browne, H M; Churcher, M J; Stanley, M A; Smith, G L; Minson, A C

    1988-06-01

    The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

  11. NAC1, a POZ/BTB protein present in the adult mammalian brain, triggers apoptosis after adenovirus-mediated overexpression in PC-12 cells.

    PubMed

    Korutla, Laxminarayana; Neustadter, Jason H; Fournier, Keith M; Mackler, Scott A

    2003-05-01

    POZ/BTB proteins influence cellular development and in some examples act as oncoproteins. However, several POZ/BTB transcription factors have been found in terminally differentiated neurons, where their functions remain unknown. One example is NAC1, a constitutively-expressed protein that can regulate behaviors associated with cocaine use. The present study represents an initial attempt to understand the actions of NAC1 within neurons by using adenoviral-mediated gene transfer into differentiated PC-12 cells. Cell survival in PC-12 cells overexpressing NAC1 was greatly reduced compared with cells infected by a control Ad-GFP. The morphological appearance of the dying cells was consistent with programmed cell death. Fragmentation of genomic DNA occurred in PC-12 cells infected with adenoviruses encoding NAC1 but not control viruses. NAC1 over expression was followed by the down regulation of the anti-apoptotic proteins Bcl-2 and Bcl-2-xl. Concurrently, levels of the pro-apoptotic proteins Bax and p53 increased following NAC1 overexpression. These observations suggest that NAC1expression in PC-12 cells induces apoptosis by altering the expression of these upstream mediators of the execution phase of programmed cell death. These findings raise the possibility that aberrantly regulated NAC1 expression in the mammalian brain may contribute to programmed cell death.

  12. Mapping stripe rust resistance genes in a Brundage x Coda winter wheat recombinant inbred line population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recombinant inbred line (RIL) mapping population developed from a cross between winter wheat (Triticum aestivum L.) cultivars Coda and Brundage was evaluated for reaction to stripe rust (caused by Puccinia striiformis f. sp. tritici). Two hundred and sixty eight RIL from the population were evalua...

  13. Registration of a rice gene mapping population of Lemont X Jasmine 85 recombinant inbred lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A mapping population developed from a cross of rice (Oryza sativa L.) tropical japonica cultivar ‘Lemont’ and indica cultivar ‘Jasmine 85’ was developed to facilitate genetic studies for important agronomic traits. The indica- and japonica-based rice recombinant inbred line (RIL) mapping population ...

  14. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model.

    PubMed

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4(+)CD25(+) regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4(+) or CD8(+) cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3(DTR) mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  15. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

    PubMed Central

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4+CD25+ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4+ or CD8+ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  16. Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.

    PubMed Central

    Arredondo-Peter, R; Hargrove, M S; Sarath, G; Moran, J F; Lohrman, J; Olson, J S; Klucas, R V

    1997-01-01

    Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2. PMID:9390447

  17. Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses.

    PubMed

    Kim, Il-Hwan; Kwon, Hyuk-Joon; Lee, Su-Hyung; Kim, Dae-Yong; Kim, Jae-Hong

    2015-01-30

    To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity of rPR8-NS(0028) might be associated with the lower virus titer and IFN-β level in the lungs of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028. In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous monitoring of their mammalian pathogenicity.

  18. Recombination Rate Variation Modulates Gene Sequence Evolution Mainly via GC-Biased Gene Conversion, Not Hill-Robertson Interference, in an Avian System.

    PubMed

    Bolívar, Paulina; Mugal, Carina F; Nater, Alexander; Ellegren, Hans

    2016-01-01

    The ratio of nonsynonymous to synonymous substitution rates (ω) is often used to measure the strength of natural selection. However, ω may be influenced by linkage among different targets of selection, that is, Hill-Robertson interference (HRI), which reduces the efficacy of selection. Recombination modulates the extent of HRI but may also affect ω by means of GC-biased gene conversion (gBGC), a process leading to a preferential fixation of G:C ("strong," S) over A:T ("weak," W) alleles. As HRI and gBGC can have opposing effects on ω, it is essential to understand their relative impact to make proper inferences of ω. We used a model that separately estimated S-to-S, S-to-W, W-to-S, and W-to-W substitution rates in 8,423 avian genes in the Ficedula flycatcher lineage. We found that the W-to-S substitution rate was positively, and the S-to-W rate negatively, correlated with recombination rate, in accordance with gBGC but not predicted by HRI. The W-to-S rate further showed the strongest impact on both dN and dS. However, since the effects were stronger at 4-fold than at 0-fold degenerated sites, likely because the GC content of these sites is farther away from its equilibrium, ω slightly decreases with increasing recombination rate, which could falsely be interpreted as a consequence of HRI. We corroborated this hypothesis analytically and demonstrate that under particular conditions, ω can decrease with increasing recombination rate. Analyses of the site-frequency spectrum showed that W-to-S mutations were skewed toward high, and S-to-W mutations toward low, frequencies, consistent with a prevalent gBGC-driven fixation bias.

  19. Sequence analysis of the fusion protein gene from infectious salmon anemia virus isolates: evidence of recombination and reassortment.

    PubMed

    Devold, M; Karlsen, M; Nylund, A

    2006-07-01

    Studies of infectious salmon anemia virus (ISAV; genus Isavirus, family Orthomyxoviridae) haemagglutinin-esterase (HE) gene sequences have shown that this gene provides a tool for genotyping and, hence, a tool to follow the dissemination of ISAV. The problem with using only the HE gene is that ISAV has a segmented genome and one segment may not tell the whole story about the origin and history of ISAV from outbreaks. To achieve a better genotyping system, the present study has focused on segment 5, the fusion (F) protein gene, which contains sequence variation at about the same level as the HE gene. The substitution rates of the HE and F gene sequences, based on 54 Norwegian ISAV isolates, are 6.1(+/-0.3)x10(-6) and 8.6(+/-5.0)x10(-5) nt per site per year, respectively. The results of phylogenetic analysis of the two gene segments have been compared and, with the exception of a few cases of reassortment, they tell the same story about the ISAV isolates. A combination of the two segments is recommended as a tool for future genotyping of ISAV. Inserts (INs) of 8-11 aa may occur close to the cleavage site of the precursor F(0) protein in some ISAV isolates. The nucleotide sequence of two of these INs shows 100% sequence identity to parts of the 5' end of the F protein gene, whilst the third IN is identical to a part of the nucleoprotein gene. This shows that recombination is one of the evolutionary mechanisms shaping the genome of ISAV. The possible importance of the INs with respect to virulence remains uncertain. PMID:16760406

  20. Development of wheat-Aegilops speltoides recombinants and simple PCR-based markers for stem rust resistance genes on the 2S#1 chromosome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild relatives of wheat are important but underutilized resources for new rust resistance genes because linked negative traits often hinder deployment of these genes in commercial wheats. Here we report reduced alien chromatin recombinants derived from E.R. Sears wheat-Aegilops speltoides translocat...

  1. Development of wheat-Aegilops speltoides recombinants and simple PCR-based markers for stem rust resistance genes on the 2S#1 chromosome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild relatives of wheat are important but underutilized resources for new rust resistance genes because linked negative traits often hinder deployment of these genes in commercial wheats. Here we report reduced alien chromatin recombinants derived from E.R. Sears' wheat-Aegilops speltoides transloca...

  2. Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae.

    PubMed

    Duffy, M F; Whithear, K G; Noormohammadi, A H; Markham, P F; Catton, M; Leydon, J; Browning, G F

    1999-04-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.

  3. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. PMID:25743073

  4. Construction of a conditional lethal Salmonella mutant via genetic recombination using the ara system and asd gene.

    PubMed

    Kim, Sam Woong; Kang, Ho Young; Hur, Jin; Gal, Sang Wan; Bang, Woo Young; Cho, Kwang-Keun; Kim, Chul Wook; Bahk, Jeong Dong; Lee, John Hwa

    2011-11-01

    In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned into a plasmid carrying a p15A replication origin to create the recombinant plasmid pMMP55. The growth of Salmonella MMP10 harboring pMMP55 was dependent on the presence of arabinose. In the presence of arabinose, the Asd deficiency due to chromosomal deletion of asd in the Salmonella host was complemented by the asd gene transcribed and translated under the P(araBAD) promoter and araBAD Shine-Dalgarno (SD) sequence in pMMP55. Growth inhibition of the strain was demonstrated by arabinose depletion in M9 minimal medium, indicating that the strain were unable to grow in an arabinose-limited environment. In addition, the analysis of a 50% lethal dose (LD50) using mice revealed that the strain MMP10 exhibited attenuation by approximately 100-fold relative to that of the unmodified strain. In conclusion, these data suggest that the araC P(araBAD)::asd system developed in this study can be used to construct conditional lethal Salmonella mutants for application as safe, live-attenuated Salmonella vaccines.

  5. Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system.

    PubMed

    Lwa, Teng Rhui; Tan, Chuan Hao; Lew, Qiao Jing; Chu, Kai Ling; Tan, Janice; Lee, Yih Yean; Chao, Sheng-Hao

    2010-04-15

    Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells. PMID:20188772

  6. MHC Class IIB Exon 2 Polymorphism in the Grey Partridge (Perdix perdix) Is Shaped by Selection, Recombination and Gene Conversion

    PubMed Central

    Bryjová, Anna; Albrecht, Tomáš; Bryja, Josef

    2013-01-01

    Among bird species, the most studied major histocompatibility complex (MHC) is the chicken MHC. Although the number of studies on MHC in free-ranging species is increasing, the knowledge on MHC variation in species closely related to chicken is required to understand the peculiarities of bird MHC evolution. Here we describe the variation of MHC class IIB (MHCIIB) exon 2 in a population of the Grey partridge (Perdix perdix), a species of high conservation concern throughout Europe and an emerging galliform model in studies of sexual selection. We found 12 alleles in 108 individuals, but in comparison to other birds surprisingly many sites show signatures of historical positive selection. Individuals displayed between two to four alleles both on genomic and complementary DNA, suggesting the presence of two functional MHCIIB loci. Recombination and gene conversion appear to be involved in generating MHCIIB diversity in the Grey partridge; two recombination breakpoints and several gene conversion events were detected. In phylogenetic analysis of galliform MHCIIB, the Grey partridge alleles do not cluster together, but are scattered through the tree instead. Thus, our results indicate that the Grey partridge MHCIIB is comparable to most other galliforms in terms of copy number and population polymorphism. PMID:23935938

  7. Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia.

    PubMed

    López, Eva; Casasnovas, Carlos; Giménez, Javier; Matilla-Dueñas, Antoni; Sánchez, Ivelisse; Volpini, Víctor

    2015-04-01

    Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779 + 746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5' breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3' breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases. PMID:25398481

  8. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    SciTech Connect

    Zacharewski, T.

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  9. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.

    PubMed Central

    Fisher, K J; Gao, G P; Weitzman, M D; DeMatteo, R; Burda, J F; Wilson, J M

    1996-01-01

    Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy. PMID:8523565

  10. Site-specific recombination-based genetic system for reporting transient or low-level gene expression.

    PubMed

    Casavant, N Carol; Beattie, Gwyn A; Phillips, Gregory J; Halverson, Larry J

    2002-07-01

    We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 microM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment.

  11. The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture.

    PubMed

    Wahls, W P; Wallace, L J; Moore, P D

    1990-02-01

    Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented. PMID:2405255

  12. Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination

    PubMed Central

    Zhang, Lin; Zhao, Guoping; Ding, Xiaoming

    2011-01-01

    We describe a site-specific recombination-based tandem assembly (SSRTA) method for reconstruction of biological parts in synthetic biology. The system was catalyzed by Streptomyces phage φBT1 integrase, which belongs to the large serine recombinase subfamily. This one-step approach was efficient and accurate, and able to join multiple DNA molecules in vitro in a defined order. Thus, it could have applications in constructing metabolic pathways and genetic networks. PMID:22355658

  13. Gene conversion causing human inherited disease: evidence for involvement of non-B-DNA-forming sequences and recombination-promoting motifs in DNA breakage and repair

    PubMed Central

    Chuzhanova, Nadia; Chen, Jian-Min; Bacolla, Albino; Patrinos, George P.; Férec, Claude; Wells, Robert D.; Cooper, David N.

    2009-01-01

    A variety of DNA sequence motifs including inverted repeats, minisatellites, and the χ recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically-based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 non-overlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could in turn serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (p<0.01) in a truncated version of the χ-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome. PMID:19431182

  14. Genetic mapping on chromosome 20 near the MODY gene: Is there suppression of recombination?

    SciTech Connect

    Howard, T.D.; Rothschild, C.B.; Bowden, D.W.

    1994-09-01

    Maturity onset diabetes of the young (MODY) is a rare, autosomal dominant form of non-insulin-dependent diabetes mellitus. We are evaluating a chromosome 20-linked form of MODY in a large, well-characterized pedigree known as the R-W family. DNA from 97 family members, including spouses, have been genotyped with nine microsatellite markers and four RFLPs from 20q12-13.1. The highest likelihood order of loci in this 9.8 cM (sex-average) interval is: cen-ADA-D20S119-D20S17-PPGB-D20S178-D20S197-D20S213-D20S16A-D20S22-D20S16B-qter. Two polymorphic loci separable by recombination comprise D20S16 (called A and B here). The highest twopoint LOD scores were observed with D20S16 (Zmax = 17.0, {theta} = 0.001) and ADA (Zmax = 16.4, {theta} = 0.001). Multipoint analysis limits MODY to a sex-average interval of approximately 10 cM (calculated from CEPH family data) defined by ADA and D20S16. The maximum multipoint LOD score observed in this region was 17.22. No recombination events have been observed within this 10 cM interval in affected individuals. The probability of this occurring by chance is P = (1-{theta}){sup n}, where n = number of meioses. With the present data there are 57 (equivalent) informative meioses, so the probability of finding no recombination within the 10 cM inteval is 0.002. In the R-W family, however, there is a 3:1 ratio of male:female meioses. When this is taken into consideration, and the sex-specific genetic distances are used (15.9 cM for female map, 6.8 cM for male map), the probability of observing no recombination in this region is still quite low: P = 0.004, suggesting the possibility that recombination is suppressed in this family.

  15. Change of Gene Structure and Function by Non-Homologous End-Joining, Homologous Recombination, and Transposition of DNA

    PubMed Central

    Goettel, Wolfgang; Messing, Joachim

    2009-01-01

    An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization

  16. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

    PubMed

    Goettel, Wolfgang; Messing, Joachim

    2009-06-01

    An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization

  17. Construction of a recombinant-BCG containing the LMP2A and BZLF1 genes and its significance in the Epstein-Barr virus positive gastric carcinoma.

    PubMed

    Xue, Qing-Jie; Dai, Jun; Li, Xiu-Zhen; Zhu, Wei; Si, Chuan-Ping; Chen, Ting

    2014-10-01

    The signal peptide Ag85B of Bacillus Chalmette-Guerin (BCG) was used to construct a recombinant plasmid of BCG. The BCG-Ag85B gene and fused EBV LMP2A and BZLF1 genes were amplified and successively inserted into the Escherichia coli-BCG shuttle-vector pMV261. The recombinant plasmids were then amplified in E. coli DH5α and transformed into competent BCG. The expression of BZLF1 and LMP2A fusion proteins in recombinant-BCG (rBCG) was shown by Western blot. After the injection of recombinant-BCG into mice, antibodies against the fusion protein BZLF1 and LMP2A were measured by ELISA, and the cellular immune effects were determined by the lactate dehydrogenate (LDH) release assays. The results confirmed that the cloned genes of BCG-Ag85B and Z2A were correctly inserted into the vector pMV261. The recombinant plasmid pMVZ2A expressed Z2A in BCG effectively after transformation. The rBCG proteins were recognized by the BZLF1 (LMP2A) antibody. An ELISA demonstrated that rBCG could stimulate the generation of antibody against the fusion protein. The fusion gene was constructed successfully, and the rBCG induced humoral and cellular immune response in mice.

  18. Climate-Driven Reshuffling of Species and Genes: Potential Conservation Roles for Species Translocations and Recombinant Hybrid Genotypes.

    PubMed

    Scriber, Jon Mark

    2013-01-01

    Comprising 50%-75% of the world's fauna, insects are a prominent part of biodiversity in communities and ecosystems globally. Biodiversity across all levels of biological classifications is fundamentally based on genetic diversity. However, the integration of genomics and phylogenetics into conservation management may not be as rapid as climate change. The genetics of hybrid introgression as a source of novel variation for ecological divergence and evolutionary speciation (and resilience) may generate adaptive potential and diversity fast enough to respond to locally-altered environmental conditions. Major plant and herbivore hybrid zones with associated communities deserve conservation consideration. This review addresses functional genetics across multi-trophic-level interactions including "invasive species" in various ecosystems as they may become disrupted in different ways by rapid climate change. "Invasive genes" (into new species and populations) need to be recognized for their positive creative potential and addressed in conservation programs. "Genetic rescue" via hybrid translocations may provide needed adaptive flexibility for rapid adaptation to environmental change. While concerns persist for some conservationists, this review emphasizes the positive aspects of hybrids and hybridization. Specific implications of natural genetic introgression are addressed with a few examples from butterflies, including transgressive phenotypes and climate-driven homoploid recombinant hybrid speciation. Some specific examples illustrate these points using the swallowtail butterflies (Papilionidae) with their long-term historical data base (phylogeographical diversity changes) and recent (3-decade) climate-driven temporal and genetic divergence in recombinant homoploid hybrids and relatively recent hybrid speciation of Papilio appalachiensis in North America. Climate-induced "reshuffling" (recombinations) of species composition, genotypes, and genomes may become

  19. Segregation of recessive phenotypes in somatic cell hybrids role of mitotic recombination, gene inactivation, and chromosome nondisjunction

    SciTech Connect

    Campbell, C.E.; Worton, R.G.

    1981-04-01

    Somatic cell hybrids heterozygous at the emetine resistance locus (emt/sup r//emt/sup +/) or the chromate resistance locus (chr/sup r//chr/sup +/) are known to segregate the recessive drug resistance phenotype at high frequency. The authors have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To allow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion 2p/sup -/) or a long-arm addition (2q/sup +/). Karotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt/sup +/ - or chr/sup +/-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci giving rise to homozygous resistant segregants; (iii) inactivation of the emt/sup +/ or chr/sup +/ alleles; and (iv) loss of the emt/sup +/ - or chr/sup +/-bearing chromosome with duplication of the homologous chromosome carrying the emt/sup r/ or chr/sup r/ allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.

  20. Germline and Somatic Mutations in Homologous Recombination Genes Predict Platinum Response and Survival in Ovarian, Fallopian Tube, and Peritoneal Carcinomas

    PubMed Central

    Pennington, Kathryn P.; Walsh, Tom; Harrell, Maria I.; Lee, Ming K.; Pennil, Christopher C.; Rendi, Mara H.; Thornton, Anne; Norquist, Barbara M.; Casadei, Silvia; Nord, Alexander S.; Agnew, Kathy J.; Pritchard, Colin C.; Scroggins, Sheena; Garcia, Rochelle L.; King, Mary-Claire; Swisher, Elizabeth M.

    2013-01-01

    Purpose Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination (HR) DNA repair genes is uncertain. Experimental Design Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the HR pathway. Results 31% of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 HR genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Non-serous ovarian carcinomas had similar rates of HR mutations to serous carcinomas (28% vs. 31%, p=0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic HR mutations was highly predictive of primary platinum sensitivity (p=0.0002) and improved overall survival (p=0.0006), with median overall survival 66 months in germline HR mutation carriers, 59 months in cases with a somatic HR mutation, and 41 months for cases without an HR mutation. Conclusions Germline or somatic mutations in HR genes are present in almost one-third of ovarian carcinomas, including both serous and non-serous histologies. Somatic BRCA1/2 mutations and mutations in other HR genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of HR mutations in non-serous carcinomas supports their inclusion in PARP inhibitor clinical trials. PMID:24240112

  1. Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes.

    PubMed

    Zhou, J; Crawford, L; McLean, L; Sun, X Y; Stanley, M; Almond, N; Smith, G L

    1990-09-01

    The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

  2. Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'' of Fission Yeast

    PubMed Central

    Klar, AJS.; Bonaduce, M. J.

    1991-01-01

    Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 ``hot spot'' for transposition should be contrasted with the ``cold spot'' of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination. PMID:1783290

  3. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow.

    PubMed

    Yu, Huan; Meng, Jiao; Xu, Jian; Liu, Tong-Xian; Wang, Dun

    2015-01-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm. PMID:26296090

  4. p21 is dispensable for AID-mediated class switch recombination and mutagenesis of immunoglobulin genes during somatic hypermutation.

    PubMed

    Shansab, Maryam; Selsing, Erik

    2011-03-01

    In B cells, activation-induced cytidine deaminase (AID) induces somatic hypermutation (SHM) at rearranged immunoglobulin (Ig) variable (V) regions. Previous studies have shown that both monoubiquitination of proliferating cell nuclear antigen (PCNA) and translesional DNA polymerase activity are important for inducing mutagenesis during SHM. Regulation of PCNA ubiquitination by p21, also known as Cdkn1a and p21(Cip1/Waf1), is an important mechanism that controls mutation loads in mammalian cells. In this study, we have assessed whether p21 has an in vivo function in regulating mutagenesis in B cells by analyzing SHM frequency in p21-deficient mice. Our results show that p21 is dispensable for SHM. This suggests that, during SHM of Ig genes, p21 does not act to regulate mutagenesis load. We also show that p21 transcript levels are the same in both wildtype and AID-deficient B cells during B cell activation, and that AID-mediated class switch recombination (CSR) is not affected by p21 deficiency; thereby indicating that p21 regulation in B cells is not altered by AID-induced DNA damage and that p21 has no affect on AID-dependent Ig gene diversification. Our results suggest that regulation of p21 in activated B cells is probably more important for maintaining proper cell cycle progression as opposed to promoting SHM of Ig genes.

  5. Cloning of Bacillus licheniformis xylanase gene and characterization of recombinant enzyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hemicellulose is a major component of lignocellulose biomass. Complete enzymatic degradation of this substrate requires several different activities, including xylanase. We isolated a strain of Bacillus licheniformis from a hot springs environment that exhibited xylanase activity. A gene encoding...

  6. A vaccinia virus double recombinant expressing the F and H genes of rinderpest virus protects cattle against rinderpest and causes no pock lesions.

    PubMed Central

    Giavedoni, L; Jones, L; Mebus, C; Yilma, T

    1991-01-01

    Rinderpest is a highly contagious viral disease of ruminants with greater than 95% morbidity and mortality. We have constructed an infectious vaccinia virus recombinant that expresses both the fusion (F) gene and the hemagglutinin (H) gene of rinderpest virus. The Wyeth strain of vaccinia virus was used for the construction of the recombinant. Cattle vaccinated with the recombinant virus were 100% protected from challenge inoculation with greater than 1000 times the lethal dose of rinderpest virus. No transmission of recombinant vaccinia virus from vaccinated animals to contact animals was observed. The lyophilized form of vaccinia virus is thermostable and allows circumvention of the logistical problems associated with the distribution and administration of vaccines in the arid and hot regions of Asia and Africa. The insertional inactivation of both the thymidine kinase and the hemagglutinin genes of vaccinia virus led to increased attenuation of the virus; this was manifested by the lack of detectable pock lesions in vaccinated animals. This approach may have wide application in the development of safe and efficacious recombinant vaccines for humans and animals. This becomes quite relevant with the concern of the use of vaccinia virus in a population with high incidence of the human immunodeficiency virus. Images PMID:1896447

  7. ama1 Genes of Sympatric Plasmodium vivax and P. falciparum from Venezuela Differ Significantly in Genetic Diversity and Recombination Frequency

    PubMed Central

    Ord, Rosalynn L.; Tami, Adriana; Sutherland, Colin J.

    2008-01-01

    Background We present the first population genetic analysis of homologous loci from two sympatric human malaria parasite populations sharing the same human hosts, using full-length sequences of ama1 genes from Plasmodium vivax and P. falciparum collected in the Venezuelan Amazon. Methodology/Principal Findings Significant differences between the two species were found in genetic diversity at the ama1 locus, with 18 distinct haplotypes identified among the 73 Pvama1 sequences obtained, compared to 6 unique haplotypes from 30 Pfama1 sequences, giving overall diversity estimates of h = 0.9091, and h = 0.538 respectively. Levels of recombination were also found to differ between the species, with P. falciparum exhibiting very little recombination across the 1.77kb sequence. In contrast, analysis of patterns of nucleotide substitutions provided evidence that polymorphisms in the ama1 gene of both species are maintained by balancing selection, particularly in domain I. The two distinct population structures observed are unlikely to result from different selective forces acting upon the two species, which share both human and mosquito hosts in this setting. Rather, the highly structured P. falciparum population appears to be the result of a population bottleneck, while the much less structured P. vivax population is likely to be derived from an ancient pool of diversity, as reflected in a larger estimate of effective population size for this species. Greatly reduced mosquito transmission in 1997, due to low rainfall prior to the second survey, was associated with far fewer P. falciparum infections, but an increase in P. vivax infections, probably due to hypnozoite activation. Conclusions/Significance The relevance of these findings to putative competitive interactions between these two important human pathogen species is discussed. These results highlight the need for future control interventions to employ strategies targeting each of the parasite species present

  8. Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene.

    PubMed Central

    Schweizer, M; Case, M E; Dykstra, C C; Giles, N H; Kushner, S R

    1981-01-01

    The early reactions in the catabolism of quinic acid in Neurospora crassa are controlled by at least four genes which are clustered on linkage group VII. Three of the loci (qa-2, qa-4, and qa-3) encode enzymes that convert quinic acid to protocatechuic acid. The fourth gene (qa-1) encodes a positive regulatory protein which, in the presence of quinic acid, leads to the de novo synthesis of the other proteins in the qa cluster. This communication describes a series of recombinant plasmids that span 36.5 kilobases of linkage group VII and contain the coding sequences for qa-2, qa-4, qa-3, and the qa-1 regulatory protein. The plasmids were obtained by partial digestion of wild-type N. crassa DNA with EcoRI and ligation into the cosmid cloning vehicle pHC79. Two independently derived plasmids (pMSK331 and pMSK335), each containing 36.5-kilobase inserts, were shown by transformation back into N. crassa to contain the entire qa gene cluster. A preliminary physical organization of the gene cluster is presented. An improved procedure for the transformation of N. crassa with plasmid DNA is also described. PMID:6272290

  9. [Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein].

    PubMed

    Yu, Xing-Long; Tu, Chang-Chun; Xu, Xing-Ran; Zhang, Mao-Lin; Chen, Yi-Xiang; Liu, Bo-Hua

    2003-07-01

    Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The

  10. Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

    PubMed Central

    Storz, Jay F.; Natarajan, Chandrasekhar; Cheviron, Zachary A.; Hoffmann, Federico G.; Kelly, John K.

    2012-01-01

    Spatially varying selection on a given polymorphism is expected to produce a localized peak in the between-population component of nucleotide diversity, and theory suggests that the chromosomal extent of elevated differentiation may be enhanced in cases where tandemly linked genes contribute to fitness variation. An intriguing example is provided by the tandemly duplicated β-globin genes of deer mice (Peromyscus maniculatus), which contribute to adaptive differentiation in blood–oxygen affinity between high- and low-altitude populations. Remarkably, the two β-globin genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion and alleles of the same functional type are in perfect coupling-phase linkage disequilibrium (LD). Here we report a multilocus analysis of nucleotide polymorphism and LD in highland and lowland mice with different genetic backgrounds at the β-globin genes. The analysis of haplotype structure revealed a paradoxical pattern whereby perfect LD between the two β-globin paralogs (which are separated by 16.2 kb) is maintained in spite of the fact that LD within both paralogs decays to background levels over physical distances of less than 1 kb. The survey of nucleotide polymorphism revealed that elevated levels of altitudinal differentiation at each of the β-globin genes drop away quite rapidly in the external flanking regions (upstream of the 5′ paralog and downstream of the 3′ paralog), but the level of differentiation remains unexpectedly high across the intergenic region. Observed patterns of diversity and haplotype structure are difficult to reconcile with expectations of a two-locus selection model with multiplicative fitness. PMID:22042573

  11. Genetic diversity of spike, 3a, 3b and e genes of infectious bronchitis viruses and emergence of new recombinants in Korea.

    PubMed

    Mo, Mei-Lan; Hong, Seung-Min; Kwon, Hyuk-Joon; Kim, Il-Hwan; Song, Chang-Seon; Kim, Jae-Hong

    2013-01-31

    The nucleotide sequences of a region including S1, S2, 3a, 3b and E genes of twenty-seven infectious bronchitis virus (IBV) isolates in Korea between 1990-2011 were determined and phylogenetic and computational recombination analyses were conducted. The sizes of coding regions of some genes varied among IBV isolates due to deletion or insertion of nucleotides; the nucleotide similarities of S1, S2, 3a, 3b and E genes among the 27 isolates were 75.9%-100.0%, 85%-100.0%, 64.0%-100.0%, 60.4%-100.0% and 83.1%-100.0%, respectively. According to phylogenetic analysis of S1 gene, the 27 isolates were divided into five genotypes, Mass, Korean-I (K-I), QX-like, KM91-like and New cluster 1. The phylogenetic trees based on the S2, 3a, 3b, E genes and S1-S2-3a-3b-E (S1-E) region nucleotide sequences did not closely follow the clustering based on the S1 sequence. The New cluster 1 prevalent during 2009 and 2010 was not found in 2011 but QX-like viruses became prevalent in 2011. The recombination analysis revealed two new S gene recombinants, 11036 and 11052 which might have been derived from recombinations between the New cluster 1 and QX-like viruses and between the K-I and H120 (vaccine) viruses, respectively. In conclusion, multiple IBV genotypes have co-circulated; QX-like viruses have recurred and new recombinants have emerged in Korea. This has enriched molecular epidemiology information of IBV and is useful for the control of IB in Korea.

  12. Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.

    PubMed

    Du, Deyao; Wang, Lu; Tian, Yuqing; Liu, Hao; Tan, Huarong; Niu, Guoqing

    2015-03-04

    Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.

  13. Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces

    PubMed Central

    Du, Deyao; Wang, Lu; Tian, Yuqing; Liu, Hao; Tan, Huarong; Niu, Guoqing

    2015-01-01

    Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces. PMID:25737113

  14. 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.

  15. 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.

  16. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

    PubMed

    Thomason, L C; Calendar, R; Ow, D W

    2001-08-01

    The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

  17. Differential targeting of feline photoreceptors by recombinant adeno-associated viral vectors: implications for preclinical gene therapy trials.

    PubMed

    Minella, A L; Mowat, F M; Willett, K L; Sledge, D; Bartoe, J T; Bennett, J; Petersen-Jones, S M

    2014-10-01

    The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

  18. The female-specific Cs-ACS1G gene of cucumber. A case of gene duplication and recombination between the non-sex-specific 1-aminocyclopropane-1-carboxylate synthase gene and a branched-chain amino acid transaminase gene.

    PubMed

    Knopf, Ronit Rimon; Trebitsh, Tova

    2006-09-01

    Cucumber (Cucumis sativus L.) is a monoecious plant in which female sex expression (gynoecy) is controlled by the Female (F) locus that can be modified by other sex-determining genes as well as by environmental and hormonal factors. As in many other cucurbits, ethylene is the major plant hormone regulating female sex expression. Previously we isolated the Cs-ACS1 (ACS, 1-aminocyclopropane-1-carboxylate synthase) gene that encodes the rate-limiting enzyme in the ethylene biosynthetic pathway. We proposed that Cs-ACS1 is present in a single copy in monoecious (ffMM) plants whereas gynoecious plants (FFMM) contain an additional copy Cs-ACS1G that was mapped to the F locus. To study the origin of Cs-ACS1G, we cloned and analyzed both the gynoecious-specific Cs-ACS1G gene and the non-sex-specific Cs-ACS1 gene. Our results indicate that Cs-ACS1G is the result of a relatively recent gene duplication and recombination, between Cs-ACS1 and a branched-chain amino acid transaminase (BCAT) gene. Taking into consideration that the Cs-ACS1G gene was mapped to the F locus, we propose that this duplication event gave rise to the F locus and to gynoecious cucumber plants. Computer analysis of the 1 kb region upstream of the transcription initiation site revealed several putative cis-acting regulatory elements that can potentially confer the responsiveness of Cs-ACS1G to developmental and hormonal factors and thereby control female sex determination in cucumber. These findings lead us to a model explaining the action of Cs-ACS1 and Cs-ACS1G in cucumber floral sex determination. PMID:16887844

  19. A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation, reduced recombination of nuclear genes, and altered transmission of chloroplast genes.

    PubMed

    Rosen, H; Newman, S M; Boynton, J E; Gillham, N W

    1991-01-01

    Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt+ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt- parent, arise at a low frequency. Mutations at the mt(+)-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt- parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically with the mat-3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt+ parent.

  20. Rules of donor preference in saccharomyces mating-type gene switching revealed by a competition assay involving two types of recombination.

    PubMed

    Wu, X; Wu, C; Haber, J E

    1997-10-01

    Mating type (MAT) switching in Saccharomyces cerevisiae is initiated by a double-strand break (DSB) created at MAT by HO endonuclease. MATa cells activate the entire left arm of chromosome III; thus MATa preferentially recombines with the silent donor HML. In contrast, MAT alpha cells inactivate the left arm, including HML, and thus preferentially recombine with HMR, 100 kb to the right of MAT. We present a novel competition assay, in which the DSB at MAT can be repaired either by MAT switching or by single-strand annealing (SSA) between two URA3 genes flanking MAT. With preferred donors, MATa or MAT alpha switching occurs 65-70% of the time in competition with SSA. When HML is deleted, 40% of MATa cells recombine with the "wrong" donor HMR; however, when HMR is deleted, only 18% of MAT alpha cells recombine with HML. In interchromosomal switching, with donors on chromosome III and MAT on chromosome V, MATa retains its strong preference for HML and switching is efficient, when the chromosome III recombination enhancer is present. However, MAT alpha donor preference is lost and interchromosomal switching is very inefficient. These experiments demonstrate the utility of using competition between two outcomes to measure the relative efficiency of recombination.

  1. Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination.

    PubMed

    Zhu, Hongmei; Liu, Jun; Cui, Chenchen; Song, Yujie; Ge, Hengtao; Hu, Linyong; Li, Qian; Jin, Yaping; Zhang, Yong

    2016-01-01

    Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG). Here, we employed transcription activator-like effector nuclease (TALEN)-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA) genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs) while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine. PMID:27258157

  2. Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.

    PubMed

    Böttcher, Romy; Hollmann, Manuel; Merk, Karin; Nitschko, Volker; Obermaier, Christina; Philippou-Massier, Julia; Wieland, Isabella; Gaul, Ulrike; Förstemann, Klaus

    2014-06-01

    The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5'-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.

  3. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    PubMed

    Dong, G; Vieille, C; Zeikus, J G

    1997-09-01

    The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.

  4. Debates, divisions, and decisions: recombinant DNA advisory committee (RAC) authorization of the first human gene transfer experiments.

    PubMed Central

    Carmen, I H

    1992-01-01

    Possibly the most far-reaching, controversial research currently being conducted in the international biological science community involves human gene therapy experimentation. In this paper, I report the dynamics of the political process which ultimately found the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health approving for the first time protocols of this genre. A full appreciation of the policy-making dialogue shows that significant participants perceived the process from very different vantage points regarding the way in which the American political system works and the way in which it ought to work. I argue that, if we are to understand how the RAC should proceed in orchestrating a human gene therapy policy agenda, then we must flesh out and critically analyze these competing vantage points. To that end, I postulate seven possible "action models" for characterizing how protocol assessments of the type at issue might be developed given the nature of our politics, reaching the conclusion that one of these models holds out the most promise for synthesizing efficaciously the key factors involved. In conclusion, I discuss how the RAC might profitably employ this preferred strategy in these and other cases. PMID:1734711

  5. Intragenic recombination generated two distinct Cf genes that mediate AVR9 recognition in the natural population of Lycopersicon pimpinellifolium

    PubMed Central

    Van der Hoorn, Renier A. L.; Kruijt, Marco; Roth, Ronelle; Brandwagt, Bas F.; Joosten, Matthieu H. A. J.; De Wit, Pierre J. G. M.

    2001-01-01

    Resistance gene Cf-9 of cultivated tomato (Lycopersicon esculentum) confers recognition of the AVR9 elicitor protein of the fungal pathogen Cladosporium fulvum. The Cf-9 locus, containing Cf-9 and four homologs (Hcr9s), originates from Lycopersicon pimpinellifolium (Lp). We examined naturally occurring polymorphism in Hcr9s that confer AVR9 recognition in the Lp population. AVR9 recognition occurs frequently throughout this population. In addition to Cf-9, we discovered a second gene in Lp, designated 9DC, which also confers AVR9 recognition. Compared with Cf-9, 9DC is more polymorphic, occurs more frequently, and is more widely spread throughout the Lp population, suggesting that 9DC is older than Cf-9. The sequences of Cf-9 and 9DC suggest that Cf-9 evolved from 9DC by intragenic recombination between 9DC and another Hcr9. The fact that the 9DC and Cf-9 proteins differ in 61 aa residues, and both mediate recognition of AVR9, shows that in nature Hcr9 proteins with the same recognitional specificity can vary significantly. PMID:11517316

  6. Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

    PubMed Central

    Cui, Chenchen; Song, Yujie; Ge, Hengtao; Hu, Linyong; Li, Qian; Jin, Yaping; Zhang, Yong

    2016-01-01

    Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG). Here, we employed transcription activator-like effector nuclease (TALEN)-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA) genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs) while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine. PMID:27258157

  7. Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis.

    PubMed

    Yang, Hui; Li, Shihao; Li, Fuhua; Yu, Kuijie; Yang, Fusheng; Xiang, Jianhai

    2016-01-01

    Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture. PMID:27517939

  8. Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis

    PubMed Central

    Yang, Hui; Li, Shihao; Li, Fuhua; Yu, Kuijie; Yang, Fusheng; Xiang, Jianhai

    2016-01-01

    Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture. PMID:27517939

  9. Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens.

    PubMed

    Vennema, H; de Groot, R J; Harbour, D A; Horzinek, M C; Spaan, W J

    1991-03-01

    Feline infectious peritonitis virus (FIPV) causes a mostly fatal, immunologically mediated disease in cats. Previously, we demonstrated that immunization with a recombinant vaccinia virus expressing the FIPV spike protein (S) induced early death after challenge with FIPV (Vennema et al., 1990, J. Virol. 64, 1407-1409). In this paper we describe similar immunizations with the FIPV membrane (M) and nucleocapsid (N) proteins. The genes encoding these proteins were cloned and sequenced. Comparison of the amino acid sequences with the corresponding sequences of porcine transmissible gastroenteritis virus revealed 84.7 and 77% identity for M and N, respectively. Vaccinia virus recombinants expressing the cloned genes induced antibodies in immunized kittens. Immunization with neither recombinant induced early death after challenge with FIPV, strongly suggesting that antibody-dependent enhancement is mediated by antibodies against S only. Immunization with the N protein recombinant had no apparent effect on the outcome of challenge. However, three of eight kittens immunized with the M protein recombinant survived the challenge, as compared to one of eight kittens of the control group.

  10. Population structure of Banana bract mosaic virus reveals recombination and negative selection in the helper component protease (HC-Pro) gene.

    PubMed

    Balasubramanian, V; Sukanya, R S; Anuradha, C; Selvarajan, R

    2014-12-01

    Banana bract mosaic virus (BBrMV) is a serious constraint in the production of banana and plantain in India. In this study, we have cloned, sequenced and analyzed the helper component proteinase (HC-Pro) gene of 22 isolates from India and compared with previously reported BBrMV isolates. Sequence identity of BBrMV isolates encoding HC-Pro gene, were 92-100 % both at the nucleotide (nt) and amino acid level. Phylogenetic analysis based on nt sequences of non recombinant isolates showed that TN15, TN9 and TN24 formed one cluster and all the remaining isolates formed into another cluster. Different functional motifs in the central region of HC-Pro gene of BBrMV isolates were found conserved. Four potential recombinants with a total of 15 breakpoints were mostly observed at the N and a few from C terminal regions. The codon based selection analysis revealed that most of the codons were under purifying or negative selection except a codon at position 74 which was under positive selection. It is likely that recombination identified in Indian BBrMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the HC-Pro gene. This study suggested that negative selection and recombination were important evolutionary factors driving the genetic diversification and population structure of Indian BBrMV isolates. To the best of our knowledge, this is the first report on the diversity analysis and occurrence of recombination in the HC-Pro gene of BBrMV. PMID:25674623

  11. Population structure of Banana bract mosaic virus reveals recombination and negative selection in the helper component protease (HC-Pro) gene.

    PubMed

    Balasubramanian, V; Sukanya, R S; Anuradha, C; Selvarajan, R

    2014-12-01

    Banana bract mosaic virus (BBrMV) is a serious constraint in the production of banana and plantain in India. In this study, we have cloned, sequenced and analyzed the helper component proteinase (HC-Pro) gene of 22 isolates from India and compared with previously reported BBrMV isolates. Sequence identity of BBrMV isolates encoding HC-Pro gene, were 92-100 % both at the nucleotide (nt) and amino acid level. Phylogenetic analysis based on nt sequences of non recombinant isolates showed that TN15, TN9 and TN24 formed one cluster and all the remaining isolates formed into another cluster. Different functional motifs in the central region of HC-Pro gene of BBrMV isolates were found conserved. Four potential recombinants with a total of 15 breakpoints were mostly observed at the N and a few from C terminal regions. The codon based selection analysis revealed that most of the codons were under purifying or negative selection except a codon at position 74 which was under positive selection. It is likely that recombination identified in Indian BBrMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the HC-Pro gene. This study suggested that negative selection and recombination were important evolutionary factors driving the genetic diversification and population structure of Indian BBrMV isolates. To the best of our knowledge, this is the first report on the diversity analysis and occurrence of recombination in the HC-Pro gene of BBrMV.

  12. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    PubMed

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  13. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

    PubMed Central

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H.; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A.

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  14. Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2*♦

    PubMed Central

    Zhang, Yu-Hang; Shetty, Keerthi; Surleac, Marius D.; Petrescu, Andrei J.; Schatz, David G.

    2015-01-01

    The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 μm) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa “mini” RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997–1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity. PMID:25745109

  15. Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression

    PubMed Central

    Kruzel, Marian L.; Actor, Jeffrey K.; MichałZimecki; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

    2014-01-01

    Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200 mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. PMID:24070904

  16. Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

    PubMed

    Kruzel, Marian L; Actor, Jeffrey K; Zimecki, Michał; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

    2013-12-01

    Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications.

  17. A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Virus Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine

    PubMed Central

    Ryder, Alex B.; Buonocore, Linda; Vogel, Leatrice; Nachbagauer, Raffael; Krammer, Florian

    2014-01-01

    ABSTRACT The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. IMPORTANCE Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell

  18. Gene Delivery Mediated by Recombinant Silk Proteins Containing Cationic and Cell Binding Motifs

    PubMed Central

    Numata, Keiji; Hamasaki, Juliana; Subramanian, Balajikarthick; Kaplan, David L

    2010-01-01

    Silk proteins are biodegradable and biocompatible, and can also be tailored to contain additional features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered both with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency. Ionic complexes of these silk-polylysine-RGD block copolymers with pDNA were prepared, characterized and utilized for gene delivery to HeLa cells and human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentialmeter, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide molar ratio. Samples with 30-lysine residues and 11 RGD sequences, prepared at the ratio of number of amines/phosphates from pDNA (N/P) of 2, had an average solution diameter of 186 nm and showed the highest transfection efficiency. The intracellular distribution of complexes of Cy5-labeled pDNA was investigated by confocal laser scanning microscopy. The Cy5-labeled pDNA was distributed near the cell membrane and around the nuclei, indicating that the pDNA was transferred near the nucleus. The results demonstrated the potential of bioengineered silk proteins with additional functional features as a new family of highly tailored gene delivery systems. PMID:20457191

  19. Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

    SciTech Connect

    Napp, S.J.; Da Silva, N.A. )

    1993-04-01

    Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product [beta]-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and [beta]-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both [beta]-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and urid-k mutations were very stable; after more than 50 generation of growth in complex medium, stability values of 99-100% were measured.

  20. Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis

    PubMed Central

    Zhang, Dong-Xiu; Lu, Hsiao-Ling; Liao, Xinggang; St. Leger, Raymond J.; Nuss, Donald L.

    2013-01-01

    Reverse-genetics analysis has played a significant role in advancing fungal biology, but is limited by the number of available selectable marker genes (SMGs). The Cre-loxP recombination system has been adapted for use in filamentous fungi to overcome this limitation. Expression of the Cre recombinase results in excision of an integrated SMG that is flanked by loxP sites, allowing a subsequent round of transformation with the same SMG. However, current protocols for regulated expression or presentation of Cre require multiple time-consuming steps. During efforts to disrupt four different RNA-dependent RNA polymerase genes in a single strain of the chestnut blight fungus Cryphonectria parasitica, we tested whether Cre could successfully excise loxP-flanked SMGs when provided in trans via anastomosis. Stable Cre-producing donor strains were constructed by transformation of wild-type C. parasitica strain EP155 with the Cre-coding domain under the control of a constitutive promoter. Excision of multiple loxP-flanked SMGs was efficiently achieved by simply pairing the Cre-donor strain and the loxP-flanked SMGs-transformed recipient strain and recovering mycelia from the margin of the recipient colony near the anastomosis zone. This method was shown to be as efficient as and much less time consuming than excision by transformation-mediated expression of Cre. It also allows unlimited recycling of loxP-flanked SMGs and the generation of disruption mutant strains that are free of any foreign gene. The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi. PMID:24007936

  1. Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum.

    PubMed

    Zhang, Chengsheng; Feng, Chao; Wang, Jing; Kong, Fanyu; Sun, Wenxiu; Wang, Fenglong

    2016-05-01

    Polygalacturonase inhibiting proteins (PGIPs) are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG) activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum), hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5'-untranslated region (UTR), and 209 bp 3'-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP's deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA), abscisic acid (ABA), salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3). Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding. PMID:27441281

  2. Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns

    PubMed Central

    Kushwaha, Amit Kumar; Sangwan, Neelam Singh; Trivedi, Prabodh Kumar; Negi, Arvind Singh; Misra, Laxminarain; Sangwan, Rajender Singh

    2013-01-01

    Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ∼60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression

  3. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  4. Cloning and expression analysis of recombination activating genes (RAG1/2) in red snapper (Lutjanus sanguineus).

    PubMed

    Zhang, X L; Lu, Y S; Jian, J C; Wu, Z H

    2012-04-01

    Recombination activating genes (RAG1 and RAG2), involved in the V(D)J recombination of immunoglobulin and T-cell receptor genes play a crucial role in the adaptive immune response in vertebrates. The expression of these genes was required for the proper development and maturity of lymphocytes so that they can be used as useful markers to evaluate the development of lymphoid organ. In this paper, the cDNA of RAG1 and RAG2 in red snapper, Lutjanus sanguineus were cloned by homological cloning and rapid amplification of cDNA ends (RACE) methods. Results showed the full length of RAG1 cDNA was 3944 bp, containing a 5' untranslated region (UTR) of 200 bp, a 3'-UTR of 561 bp and an open reading frame of 3183 bp encoding 1060 amino acids. Three important structural motifs, a RING/U-box domain, a RING/FYVE/PHD-type domain and a RAG Nonamer-binding domain were detected in the deduced amino acid sequence of RAG1 by InterProScan analysis. The full length of RAG2 cDNA was 2200 bp, consisting of a 141 bp 5'-UTR, a 457 bp 3'-UTR and an open reading frame of 1602 bp encoding 533 amino acids. Two important structural motifs, a Galactose oxidase/kelch, beta-propeller domain and a kelch-type beta-propeller domain were detected in the deduced amino acid sequence of RAG2 by InterProScan analysis. BLAST analysis revealed that the RAG1 and RAG2 in red snapper shared a high homology with other known RAG1 and RAG2 genes, while the greatest degree of identity was observed with Hippoglossus hippoglossus RAG1 at 82% and Takifugu rubripes RAG2 at 87%, respectively. The differential expressions of RAG1 and RAG2 in various tissues of red snapper were analyzed by fluorescent quantitative real-time PCR. The overall expression pattern of the two genes was quite similar. In healthy red snappers, the RAGs transcripts were mainly detected in thymus, following head kidney, spleen, intestine, liver and brain. After vaccinated with inactivated Vibrio alginolyticus 48 h later, the RAGs m

  5. Evaluation in broilers of the probiotic properties of Pichia pastoris and a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene.

    PubMed

    Gil de los Santos, João Rodrigo; Storch, Otávio Brod; Fernandes, Cristina Gevehr; Gil-Turnes, Carlos

    2012-05-01

    The probiotic properties of Pichia pastoris and of a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene were evaluated in broilers. One-day-old chicks randomly divided in four groups were fed with commercial feed devoid of antibacterials. The control group (1) received plain food, while the other groups were supplemented with either P. pastoris (2), the recombinant P. pastoris (3) or Bacillus cereus var. Toyoi (4). At day 49, live weights, feed efficiency and seroconversions were higher (P<0.05) in the supplemented groups than in the control groups. Group 3 showed the best results, while group 2 had lower weight gain than groups 3 and 4 although food conversion was better than in group 4. Seroconversions were not different (P>0.05) among the supplemented groups. Adverse reactions were not observed in histopathologic evaluation. We concluded that P. pastoris and the recombinant P. pastoris could be used as probiotics in broilers. PMID:22176763

  6. Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

    PubMed Central

    Potocki De Montalk, G.; Remaud-Simeon, M.; Willemot, R. M.; Planchot, V.; Monsan, P.

    1999-01-01

    The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75. PMID:9882648

  7. Iron-dependent hydrogenases of Entamoeba histolytica and Giardia lamblia: activity of the recombinant entamoebic enzyme and evidence for lateral gene transfer.

    PubMed

    Nixon, Julie E J; Field, Jessica; McArthur, Andrew G; Sogin, Mitchell L; Yarlett, Nigel; Loftus, Brendan J; Samuelson, John

    2003-02-01

    Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes. To understand better the biochemistry of the protist Fe-hydrogenases, we prepared a recombinant E. histolytica short Fe-hydrogenase and measured its activity in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic sequences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phylogenetic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation enzymes of these protists.

  8. Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    Poncet, S; Bernard, C; Dervyn, E; Cayley, J; Klier, A; Rapoport, G

    1997-01-01

    Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control. PMID:9361428

  9. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences

    SciTech Connect

    Cai, Yuping; Wolk, C.P. )

    1990-06-01

    Use of the sacB gene provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, they mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

  10. Conditional Gene Expression/Deletion Systems for Marchantia polymorpha Using its Own Heat-Shock Promoter and Cre/loxP-Mediated Site-Specific Recombination.

    PubMed

    Nishihama, Ryuichi; Ishida, Sakiko; Urawa, Hiroko; Kamei, Yasuhiro; Kohchi, Takayuki

    2016-02-01

    The liverwort Marchantia polymorpha is an emerging model plant suitable for addressing, using genetic approaches, various evolutionary questions in the land plant lineage. Haploid dominancy in its life cycle facilitates genetic analyses, but conversely limits the ability to isolate mutants of essential genes. To overcome this issue and to be employed in cell lineage, mosaic and cell autonomy analyses, we developed a system that allows conditional gene expression and deletion using a promoter of a heat-shock protein (HSP) gene and the Cre/loxP site-specific recombination system. Because the widely used promoter of the Arabidopsis HSP18.2 gene did not operate in M. polymorpha, we identified a promoter of an endogenous HSP gene, MpHSP17.8A1, which exhibited a highly inducible transient expression level upon heat shock with a low basal activity level. Reporter genes fused to this promoter were induced globally in thalli under whole-plant heat treatment and also locally using a laser-assisted targeted heating technique. By expressing Cre fused to the glucocorticoid receptor under the control of the MpHSP17.8A1 promoter, a low background, sufficiently inducible control for loxP-mediated recombination could be achieved in M. polymorpha. Based on these findings, we developed a Gateway technology-based binary vector for the conditional induction of gene deletions. PMID:26148498

  11. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    NASA Astrophysics Data System (ADS)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  12. A novel horse α-defensin: gene transcription, recombinant expression and characterization of the structure and function

    PubMed Central

    Bruhn, Oliver; Regenhard, Petra; Michalek, Matthias; Paul, Sven; Gelhaus, Christoph; Jung, Sascha; Thaller, Georg; Podschun, Rainer; Leippe, Matthias; Grötzinger, Joachim; Kalm, Ernst

    2007-01-01

    Defensins are a predominant class of antimicrobial peptides, which act as endogenous antibiotics. Defensins are classified into three distinct sub-families: θ-, β-, and α-defensins. Synthesis of α-defensin has been confirmed only in primates and glires to date and is presumably unique for a few tissues, including neutrophils and Paneth cells of the small intestine. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. In the present study, we report the characterization of the equine α-defensin DEFA (defensin α) 1. Transcription analysis revealed that the transcript of the gene is present in the small intestine only. An alignment with known α-defensins from primates and glires displayed a homology with Paneth-cell-specific α-defensins. DEFA1 was recombinantly expressed in Escherichia coli and subsequently analysed structurally by CD and molecular modelling. To examine the antimicrobial properties, a radial diffusion assay was performed with 12 different micro-organisms and the LD90 (lethal dose killing ≥90% of target organism) and MBC (minimal bactericidal concentration) values were examined. DEFA1 showed an antimicrobial activity against different Gram-positive and Gram-negative bacteria and against the yeast Candida albicans. Using viable bacteria in combination with a membrane-impermeable fluorescent dye, as well as depolarization of liposomes as a minimalistic system, it became evident that membrane permeabilization is at least an essential part of the peptide's mode of action. PMID:17620056

  13. Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt.

    PubMed

    Meshaal, Safa; El Hawary, Rabab; Elsharkawy, Marwa; Mousa, Reem K; Farid, Reem J; Abd Elaziz, Dalia; Alkady, Radwa; Galal, Nermeen; Massaad, Michel J; Boutros, Jeannette; Elmarsafy, Aisha

    2015-06-01

    The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

  14. Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy.

    PubMed

    Meena, Balakrishnan; Anburajan, Lawrance; Vinithkumar, Nambali Valsalan; Shridhar, Divya; Raghavan, Rangamaran Vijaya; Dharani, Gopal; Kirubagaran, Ramalingam

    2016-09-30

    l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5mM of isopropyl-β-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37°C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy.

  15. Application to Photocatalytic H2 Production of a Whole-Cell Reaction by Recombinant Escherichia coli Cells Expressing [FeFe]-Hydrogenase and Maturases Genes.

    PubMed

    Honda, Yuki; Hagiwara, Hidehisa; Ida, Shintaro; Ishihara, Tatsumi

    2016-07-01

    A photocatalytic H2 production system using an inorganic-bio hybrid photocatalyst could contribute to the efficient utilization of solar energy, but would require the development of a new approach for preparing a H2 -forming biocatalyst. In the present study, we constructed a recombinant strain of Escherichia coli expressing the genes encoding the [FeFe]-hydrogenase and relevant maturases from Clostridium acetobutylicum NBRC 13948 for use as a biocatalyst. We investigated the direct application of a whole-cell of the recombinant E. coli. The combination of TiO2 , methylviologen, and the recombinant E. coli formed H2 under light irradiation, demonstrating that whole cells of the recombinant E. coli could be employed for photocatalytic H2 production without any time-consuming and costly manipulations (for example, enzyme purification). This is the first report of the direct application of a whole-cell reaction of recombinant E. coli to photocatalytic H2 production. PMID:27194524

  16. Case of Localized Recombination in 23S rRNA Genes from Divergent Bradyrhizobium Lineages Associated with Neotropical Legumes

    PubMed Central

    Parker, Matthew A.

    2001-01-01

    Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii. PMID:11319084

  17. Case of localized recombination in 23S rRNA genes from divergent bradyrhizobium lineages associated with neotropical legumes.

    PubMed

    Parker, M A

    2001-05-01

    Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5' portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5' 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii.

  18. Novel recombinant adenoviral vector that targets the interleukin-13 receptor alpha2 chain permits effective gene transfer to malignant glioma.

    PubMed

    Ulasov, Ilya V; Tyler, Matthew A; Han, Yu; Glasgow, Joel N; Lesniak, Maciej S

    2007-02-01

    Transduction of malignant glioma with adenovirus serotype 5 (Ad5) vectors is limited by the low levels of coxsackievirus and adenovirus receptor (CAR) on tumor cells. However, malignant brain tumors have been found to overexpress a glioma-associated receptor, interleukin-13 receptor alpha2 chain (IL-13Ralpha2), a marker of both glial transformation and tumor grade. To selectively target Ad5 to IL-13Ralpha2, we constructed a replication-deficient adenoviral vector that possesses an IL-13 ligand presented by a T4 phage fibritin shaft, and designated the new virus LU-13. Western blot and sequence analyses confirmed proper trimerization and ligand presentation by the T4 fibritin shaft. Confocal microscopy analysis of primary glioma suspensions incubated with viral recombinants showed that LU-13 colocalized with IL-13Ralpha2. Luciferase transduction assays conducted in both primary and passaged glioma cell cultures exhibited at least 10-fold enhanced gene transduction. Moreover, the virus preferentially bound to glioma cells, as documented by increased adenoviral E4 DNA copy number. In vitro competition assays performed with anti-human IL-13 monoclonal antibody confirmed significant attenuation of LU-13 transduction. These results were further confirmed in vivo, where LU-13 showed a 300-fold increase in transgene expression. In summary, we describe here the development of a novel and targeted adenoviral vector that binds IL-13Ralpha2. Our findings confirm the ability of LU-13 to bind IL-13Ralpha2 and increase transgene expression, making it an attractive gene therapy vector for the treatment of malignant glioma in a clinical setting.

  19. Genetic correlates of gene expression in recombinant inbred strains: a relational model system to explore neurobehavioral phenotypes.

    PubMed

    Chesler, Elissa J; Wang, Jintao; Lu, Lu; Qu, Yanhua; Manly, Kenneth F; Williams, Robert W

    2003-01-01

    Full genome sequencing, high-density genotyping, expanding sets of microarray assays, and systematic phenotyping of neuroanatomical and behavioral traits are producing a wealth of data on the mouse central nervous system (CNS). These disparate resources are still poorly integrated. One solution is to acquire these data using a common reference population of isogenic lines of mice, providing a point of integration between the data types. Recombinant inbred (RI) mice, derived through inbreeding of progeny from an inbred cross, are a powerful tool for complex trait mapping and analysis of the challenging phenotypes of neuroscientific interest. These isogenic RI lines are a retrievable genetic resource that can be repeatedly studied using a wide variety of assays. Diverse data sets can be related through fixed and known genomes, using tools such as the interactive web-based system for complex trait analysis, www.WebQTL.org. In this report, we demonstrate the use of WebQTL to explore complex interactions among a wide variety of traits--from mRNA transcripts to the impressive behavioral and pharmacological variation among RI strains. The relational approach exploiting a common set of strains facilitates study of multiple effects of single genes (pleiotropy) without a priori hypotheses required. Here we demonstrate the power of this technique through genetic correlation of gene expression with a database of neurobehavioral phenotypes collected in these strains of mice through more than 20 years of experimentation. By repeatedly studying the same panel of mice, early data can be re-examined in light of technological advances unforeseen at the time of their initial collection.

  20. Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy

    PubMed Central

    Okada, Takashi; Takeda, Shin'ichi

    2013-01-01

    Various characteristics of adeno-associated virus (AAV)-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD). Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been developed and a number of studies with AAV vector-mediated muscle transduction were attempted. Notably, an intravenous limb perfusion transduction technique enables extensive transgene expression in the skeletal muscles without noticeable adverse events. Furthermore, cardiac transduction by the rAAV9-microdystrophin would be promising to prevent development of cardiac dysfunction. Recent achievements in transduction technology suggest that long-term transgene expression with therapeutic benefits in DMD treatment would be achieved by the rAAV-mediated transduction strategy with an adequate regimen to regulate host immune response. PMID:24276316

  1. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.

    PubMed Central

    Wu, H; Liu, X; Jaenisch, R

    1994-01-01

    A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation. Images PMID:8146196

  2. Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer.

    PubMed

    Kim, Soo-Yeon; Lee, Sang-Jin; Lim, Soo-Jeong

    2014-11-20

    Advancements in the use of adenoviral vectors in gene therapy have been limited by the need for specific receptors on targeted cell types, immunogenicity and hepatotoxicity following systemic administration. In an effort to overcome the current limitations of adenovirus-mediated gene transfer, cationic emulsions were explored as a vehicle to improve adenoviral vector-mediated gene transfer. Complexation of adenovirus with emulsions containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced the potency of adenoviral gene transfer as compared to DOTAP liposomes. Among the various emulsion formulations examined, those containing the iodized oil, Lipiodol, as an inner core and stabilized by DOTAP/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(poly-ethylene glycol)-5000 most efficiently enhanced adenovirus-mediated gene transfer. Optimized Lipiodol-containing emulsions appear to be more strongly associated with adenoviral particles, exhibiting higher complex stability compared to other formulations. They provide the adenovirus with an additional cellular entry mechanism through caveolae-dependent endocytosis, thereby increasing adenovirus entry into cells. Furthermore, adenovirus-emulsion complexation significantly reduced transgene expression in the liver following systemic administration. These findings indicate that emulsion complexation may be a promising strategy for overcoming many of the challenges associated with the use of adenoviruses in gene therapy. Additionally, the observation of increased transgene expression in lung together with reduced expression in liver demonstrates that the adenovirus-emulsion complex may act as a lung-targeting adenoviral gene delivery system.

  3. Identification of human immunodeficiency virus type 1 envelope genes recombinant between subtypes B and F in two epidemiologically linked individuals from Brazil.

    PubMed Central

    Sabino, E C; Shpaer, E G; Morgado, M G; Korber, B T; Diaz, R S; Bongertz, V; Cavalcante, S; Galvão-Castro, B; Mullins, J I; Mayer, A

    1994-01-01

    Sequence analysis of a human immunodeficiency virus type 1 env gene PCR amplified from a Brazilian woman's peripheral blood mononuclear cell DNA (sample RJIO1) showed that it was likely to have been derived from a double recombination event between human immunodeficiency virus type 1 subtypes B and F. The major portion of the gp120 coding sequence belonged to the B lineage, but a segment of the C2 to V3 region (approximately 135 nucleotides) clearly associated with sequences of the F lineage. The subtype F-like segment had 15 noncontiguous signature nucleotides in common with Brazilian subtype F sequences that were not found, or were rare, in subtype B sequences. In contrast, this same segment had only 3 signature nucleotides shared with subtype B sequences and not present in the Brazilian subtype F sequences. Phylogenetic analysis, amino acid signature pattern analysis, and the pattern of synonymous mutations all supported the hypothesis of a recombinational origin of the RJIO1 sequence. Related recombinant genes were also detected in peripheral blood mononuclear cell DNA obtained from the woman's recent sexual partner, indicating that the recombination event probably occurred at some previous time in the chain of virus transmission. Divergent viral sequences in the V3 region were found in the male sexual partner, while a relatively homogeneous viral population was detected in the woman, consistent with her recent infection. PMID:8083973

  4. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    SciTech Connect

    Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W.

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  5. High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

    PubMed

    Gaillet, Bruno; Gilbert, Rénald; Amziani, Rachid; Guilbault, Claire; Gadoury, Christine; Caron, Antoine W; Mullick, Alaka; Garnier, Alain; Massie, Bernard

    2007-01-01

    To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

  6. The Mitochondrial Genome of Paraminabea aldersladei (Cnidaria: Anthozoa: Octocorallia) Supports Intramolecular Recombination as the Primary Mechanism of Gene Rearrangement in Octocoral Mitochondrial Genomes

    PubMed Central

    Brockman, Stephanie A.; McFadden, Catherine S.

    2012-01-01

    Sequencing of the complete mitochondrial genome of the soft coral Paraminabea aldersladei (Alcyoniidae) revealed a unique gene order, the fifth mt gene arrangement now known within the cnidarian subclass Octocorallia. At 19,886 bp, the mt genome of P. aldersladei is the second largest known for octocorals; its gene content and nucleotide composition are, however, identical to most other octocorals, and the additional length is due to the presence of two large, noncoding intergenic regions. Relative to the presumed ancestral octocoral gene order, in P. aldersladei a block of three protein-coding genes (nad6–nad3–nad4l) has been translocated and inverted. Mapping the distribution of mt gene arrangements onto a taxonomically comprehensive phylogeny of Octocorallia suggests that all of the known octocoral gene orders have evolved by successive inversions of one or more evolutionarily conserved blocks of protein-coding genes. This mode of genome evolution is unique among Metazoa, and contrasts strongly with that observed in Hexacorallia, in which extreme gene shuffling has occurred among taxonomic orders. Two of the four conserved gene blocks found in Octocorallia are, however, also conserved in the linear mt genomes of Medusozoa and in one group of Demospongiae. We speculate that the rate and mechanism of gene rearrangement in octocorals may be influenced by the presence in their mt genomes of mtMutS, a putatively active DNA mismatch repair protein that may also play a role in mediating intramolecular recombination. PMID:22975720

  7. Relative expression of genes involved in the resistance/sensitivity of Enterococcus faecalis JH2-2 to recombinant divercin RV41.

    PubMed

    Calvez, Ségolène; Prevost, H; Drider, Djamel

    2008-10-01

    The relative expression of mptABCD operon, glpQ, pde, rpoN, mptR and gap-1 was studied by reverse transcription combined with the real time polymerase chain reaction to understand the role of each gene in resistance/sensitivity of Enterococcus faecalis to class IIa bacteriocins such as recombinant divercin V41 (DvnRV41). Comparative critical threshold methods in presence or absence of DvnRV41 were then used to determine the level of expression of each gene cited above. In the presence of DvnRV41, the rpoN and glpQ genes were down-regulated, mptR, mptC, gap-1 and pde genes were up-regulated, whilst expression of mptB and mptD genes remained unmodified.

  8. Immunoglobulin genes undergo legitimate repair in human B cells not only after cis- but also frequent trans-class switch recombination.

    PubMed

    Laffleur, B; Bardet, S M; Garot, A; Brousse, M; Baylet, A; Cogné, M

    2014-01-01

    Immunoglobulin (Ig) genes specifically recruit activation-induced deaminase (AID) for 'on-target' DNA deamination, initiating either variable (V) region somatic hypermutation, or double-strand break intermediates of class switch recombination (CSR). Such breaks overwhelmingly undergo legitimate intra-Ig repair rather than rare illegitimate and potentially oncogenic junctions outside of Ig loci. We show that in human B cells, legitimate synapsis and repair efficiently join Ig genes whether physically linked on one chromosome or located apart on both alleles. This indicates mechanisms faithfully recognizing and/or pairing loci with homology in structure and accessibility, thus licensing interchromosomal trans-CSR junctions while usually preventing illegitimate interchromosomal recombination with AID off-target genes. Physical linkage of IgH genes in cis on the same allele just increases the likelihood of legitimate repair by another fourfold. The strongest force driving CSR might thus be recognition of legitimate target genes. Formation of IgH intra-allelic loops along this process would then constitute a consequence rather than a pre-requisite of this gene-pairing process.

  9. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen SO7.

    PubMed

    Konjufca, Vjollca; Jenkins, Mark; Wang, Shifeng; Juarez-Rodriguez, Maria Dolores; Curtiss, Roy

    2008-12-01

    Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain chi8879 (Delta phoP233 Delta sptP1033::xylE Delta asdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain chi9242 (Delta phoP233 Delta asdA16 Delta araBAD23 Delta relA198::araC P(BAD) lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.

  10. Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

    PubMed Central

    Castro-Pérez, Edgardo; Soto-Soto, Emilio; Pérez-Carambot, Marizabeth; Dionisio-Santos, Dawling; Saied-Santiago, Kristian; Ortiz-Zuazaga, Humberto G.; Peña de Ortiz, Sandra

    2016-01-01

    An increasing body of evidence suggests that mechanisms related to the introduction and repair of DNA double strand breaks (DSBs) may be associated with long-term memory (LTM) processes. Previous studies from our group suggested that factors known to function in DNA recombination/repair machineries, such as DNA ligases, polymerases, and DNA endonucleases, play a role in LTM. Here we report data using C57BL/6 mice showing that the V(D)J recombination-activating gene 1 (RAG1), which encodes a factor that introduces DSBs in immunoglobulin and T-cell receptor genes, is induced in the amygdala, but not in the hippocampus, after context fear conditioning. Amygdalar induction of RAG1 mRNA, measured by real-time PCR, was not observed in context-only or shock-only controls, suggesting that the context fear conditioning response is related to associative learning processes. Furthermore, double immunofluorescence studies demonstrated the neuronal localization of RAG1 protein in amygdalar sections prepared after perfusion and fixation. In functional studies, intra-amygdalar injections of RAG1 gapmer antisense oligonucleotides, given 1 h prior to conditioning, resulted in amygdalar knockdown of RAG1 mRNA and a significant impairment in LTM, tested 24 h after training. Overall, these findings suggest that the V(D)J recombination-activating gene 1, RAG1, may play a role in LTM consolidation. PMID:26843989

  11. Evaluation of a novel strain of infectious bronchitis virus emerged as a result of spike gene recombination between two highly diverged parent strains.

    PubMed

    Hewson, Kylie A; Noormohammadi, Amir H; Devlin, Joanne M; Browning, Glenn F; Schultz, Bridie K; Ignjatovic, Jagoda

    2014-01-01

    The emergence of new variant strains of the poultry pathogen infectious bronchitis virus (IBV) is continually reported worldwide, owing to the labile nature of the large single-stranded RNA IBV genome. High resolution melt curve analysis previously detected a variant strain, N1/08, and the present study confirmed that this strain had emerged as a result of recombination between Australian subgroup 2 and 3 strains in the spike gene region, in a similar manner reported for turkey coronaviruses. The S1 gene for N1/08 had highest nucleotide similarity with subgroup 2 strains, which is interesting considering subgroup 2 strains have not been detected since the early 1990s. SimPlot analysis of the 7.2-kb 3' end of the N1/08 genome with the same region for other Australian reference strains identified the sites of recombination as immediately upstream and downstream of the S1 gene. A pathogenicity study in 2-week-old chickens found that N1/08 had similar pathogenicity for chicken respiratory tissues to that reported for subgroup 2 strains rather than subgroup 3 strains. The results of this study demonstrate that recombination is a mechanism utilized for the emergence of new strains of IBV, with the ability to alter strain pathogenicity in a single generation.

  12. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    PubMed

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  13. Replacement of Imu-Cmu intron by NeoR gene alters Imu germ-line expression but has no effect on V(D)J recombination.

    PubMed

    Haddad, Dania; Dougier, Hei-Lanne; Laviolette, Nathalie; Puget, Nadine; Khamlichi, Ahmed Amine

    2010-02-01

    The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3' regulatory region (3'RR). This polarized long-range effect of the 3'RR is disturbed upon insertion of NeoR gene in the IgH constant (C(H)) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Emu enhancer or Emu core enhancer (cEmu) by NeoR gene fully blocked V(D)J recombination and mu0 GL transcription which originates 5' of DQ52 and severely diminished Imu GL transcription derived from Emu/Imu promoter, suggesting a critical role for cEmu in the regulation of V(D)J recombination and of mu0 and Imu expression. Here we focus on the effect of NeoR gene on mu0 and Imu GL transcription in a mouse line in which the Imu-Cmu intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Emu. mu0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Imu transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Imu expression. Surprisingly, in stark contrast with the NeoR effect in the C(H) region, LPS-induced NeoR expression restored Imu transcript levels to normal. The data suggest that Emu enhancer may be the master control element that counteracts the down-regulatory "Neo effect" on Imu expression upon LPS

  14. Recombination and horizontal transfer of nodulation and ACC deaminase (acdS) genes within Alpha- and Betaproteobacteria nodulating legumes of the Cape Fynbos biome.

    PubMed

    Lemaire, Benny; Van Cauwenberghe, Jannick; Chimphango, Samson; Stirton, Charles; Honnay, Olivier; Smets, Erik; Muasya, A Muthama

    2015-11-01

    The goal of this work is to study the evolution and the degree of horizontal gene transfer (HGT) within rhizobial genera of both Alphaproteobacteria (Mesorhizobium, Rhizobium) and Betaproteobacteria (Burkholderia), originating from South African Fynbos legumes. By using a phylogenetic approach and comparing multiple chromosomal and symbiosis genes, we revealed conclusive evidence of high degrees of horizontal transfer of nodulation genes among closely related species of both groups of rhizobia, but also among species with distant genetic backgrounds (Rhizobium and Mesorhizobium), underscoring the importance of lateral transfer of symbiosis traits as an important evolutionary force among rhizobia of the Cape Fynbos biome. The extensive exchange of symbiosis genes in the Fynbos is in contrast with a lack of significant events of HGT among Burkholderia symbionts from the South American Cerrado and Caatinga biome. Furthermore, homologous recombination among selected housekeeping genes had a substantial impact on sequence evolution within Burkholderia and Mesorhizobium. Finally, phylogenetic analyses of the non-symbiosis acdS gene in Mesorhizobium, a gene often located on symbiosis islands, revealed distinct relationships compared to the chromosomal and symbiosis genes, suggesting a different evolutionary history and independent events of gene transfer. The observed events of HGT and incongruence between different genes necessitate caution in interpreting topologies from individual data types.

  15. Gene therapy of Hunter syndrome: evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS).

    PubMed

    Friso, A; Tomanin, R; Zanetti, A; Mennuni, C; Calvaruso, F; La Monica, N; Marin, O; Zacchello, F; Scarpa, M

    2008-10-01

    Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.

  16. A major recombination hotspot in the XqYq pseudoautosomal region gives new insight into processing of human gene conversion events.

    PubMed

    Sarbajna, Shriparna; Denniff, Matthew; Jeffreys, Alec J; Neumann, Rita; Soler Artigas, María; Veselis, Amelia; May, Celia A

    2012-05-01

    Recombination plays a fundamental role in meiosis. Non-exchange gene conversion (non-crossover, NCO) may facilitate homologue pairing, while reciprocal crossover (CO) physically connects homologues so they orientate appropriately on the meiotic spindle. In males, X-Y homologous pairing and exchange occurs within the two pseudoautosomal regions (PARs) together comprising <5% of the human sex chromosomes. Successful meiosis depends on an obligatory CO within PAR1, while the nature and role of exchange within PAR2 is unclear. Here, we describe the identification and characterization of a typical ~1 kb wide recombination hotspot within PAR2. We find that both COs and NCOs are strongly modulated in trans by the presumed chromatin remodelling protein PRDM9, and in cis by a single nucleotide polymorphism (SNP) located at the hotspot centre that appears to influence recombination initiation and which causes biased gene conversion in SNP heterozygotes. This, the largest survey to date of human NCOs reveals for the first time substantial inter-individual variation in the NCO:CO ratio. Although the extent of biased transmission at the central marker in COs is similar across men, it is highly variable among NCO recombinants. This suggests that cis-effects are mediated not only through recombination initiation frequencies varying between haplotypes but also through subsequent processing, with the potential to significantly intensify meiotic drive of hotspot-suppressing alleles. The NCO:CO ratio and extent of transmission distortion among NCOs appear to be inter-related, suggesting the existence of two NCO pathways in humans. PMID:22291443

  17. Localization of genes influencing ethanol-induced conditioned place preference and locomotor activity in BXD recombinant inbred mice.

    PubMed

    Cunningham, C L

    1995-07-01

    Genetic differences in ethanol's ability to induce conditioned place preference were studied in 20 BXD Recombinant Inbred (RI) mouse strains and in the C57BL/6J and DBA/2J progenitor strains. Male mice from each strain were exposed to a Pavlovian conditioning procedure in which a distinctive floor stimulus (CS+) was paired four times with ethanol (2 g/kg). A different floor stimulus (CS-) was paired with saline. Control mice were injected only with saline. Floor preference testing without ethanol revealed significant genetic differences in conditioned place preference, with some strains spending nearly 80% time on the ethanol-paired floor while others spent only 50% (i.e., no preference). Control mice showed genetic differences in unconditioned preference for the floor cues, but unconditioned preference was not genetically correlated with conditioned preference. There were also substantial genetic differences in ethanol-stimulated activity, but contrary to psychomotor stimulant theory, ethanol-induced activity on conditioning trials was not positively correlated with strength of conditioned place preference. However, there was a significant negative genetic correlation (r = -0.42) between test session activity and preference. Quantitative trait loci (QTL) analyses showed strong associations (P < 0.01) between conditioned place preference and marker loci on chromosomes 4, 8, 9, 18 and 19. Weaker associations (0.01 < P < 0.05) were identified on several other chromosomes. Analysis also yielded several significant QTL for unconditioned preference, ethanol-stimulated activity, and sensitization. Overall, these data support the conclusion that genotype influences ethanol-induced conditioned place preference, presumably via genetic differences in sensitivity to ethanol's rewarding effects. Moreover, several chromosomal regions containing candidate genes of potential relevance to ethanol-induced conditioned place preference have been identified.

  18. Genetic mutation of recombination activating gene 1 in Dahl salt-sensitive rats attenuates hypertension and renal damage.

    PubMed

    Mattson, David L; Lund, Hayley; Guo, Chuanling; Rudemiller, Nathan; Geurts, Aron M; Jacob, Howard

    2013-03-15

    Hypertension and renal damage in Dahl SS rats are associated with increased infiltrating immune cells in the kidney. To examine the role of infiltrating immune cells in this disease process, a zinc finger nuclease targeting bases 672-706 of recombination-activating gene 1 (Rag1) was injected into the pronucleus of Dahl SS (SS/JrHsdMcwi) strain embryos and implanted in pseudopregnant females. This strategy yielded a rat strain with a 13-base frame-shift mutation in the target region of Rag1 and a deletion of immunoreactive Rag1 protein in the thymus. Flow cytometry demonstrated that the Rag1-null mutant rats have a significant reduction in T and B lymphocytes in the circulation and spleen. Studies were performed on SS and Rag1-null rats fed a 4.0% NaCl diet for 3 wk. The infiltration of T cells into the kidney following high-salt intake was significantly blunted in the Rag1-null rats (1.7 ± 0.6 × 10(5) cells/kidney) compared with the Dahl SS (5.6 ± 0.9 × 10(5) cells/kidney). Accompanying the reduction in infiltration of immune cells in the kidney, mean arterial blood pressure and urinary albumin excretion rate were significantly lower in Rag1-null mutants (158 ± 3 mmHg and 60 ± 16 mg/day, respectively) than in SS rats (180 ± 11 mmHg and 251 ± 37 mg/day). Finally, a histological analysis revealed that the glomerular and tubular damage in the kidneys of the SS rats fed a high-salt diet was also attenuated in the Rag1 mutants. These studies demonstrate the importance of renal infiltration of immune cells in the pathogenesis of hypertension and renal damage in Dahl SS rats.

  19. Antitumor effects of recombinant antivascular protein ABRaA-VEGF121 combined with IL-12 gene therapy.

    PubMed

    Ciomber, Agnieszka; Smagur, Andrzej; Mitrus, Iwona; Cichoń, Tomasz; Smolarczyk, Ryszard; Sochanik, Aleksander; Szala, Stanisław; Jarosz, Magdalena

    2014-04-01

    Development and neoplastic progression strongly rely on tumor microenvironment cells. Various kinds of cells that form such tumor milieu play substantial roles in angiogenesis and immunosuppression. Attempts to inhibit tumor vascularization alter tumor milieu and enhance immune response against the tumor. Anticancer therapeutic strategy bringing together antiangiogenic and immunostimulating agents has emerged as a promising approach. We here investigated whether therapy directed against preexisting vessels, combined with an immunomodulatory factor would be equally effective in arresting tumor growth. To this goal, we investigated the effectiveness of ABRaA-vascular endothelial growth factor isoform 121 (VEGF121), an antivascular drug constructed by us. It is a fusion protein composed of VEGF121, and abrin A chain (translation-inhibiting toxin). We used it in combination with interleukin (IL-12) gene therapy and tried to inhibit B16-F10 melanoma tumor growth. ABRaA-VEGF121 is a chimeric recombinant protein capable of destroying tumor vasculature and triggering necrosis in the vicinity of damaged vessels. IL-12 cytokine, in turn, activates both specific and non-specific immune responses. Our results demonstrate that combination of ABRaA-VEGF121 antivascular agent with immunostimulatory cytokine IL-12 indeed inhibits tumor growth more effectively than either agent alone, leading to complete cure of ca. 20 % mice. Post-therapeutic analysis of tumors excised from mice treated with combination therapy showed decreased numbers of blood microvessels in the tumor microenvironment, lowered numbers of regulatory T lymphocytes, as well as showed higher levels of CD4(+) and CD8(+) as compared to control mice. It seems that bringing together antivascular strategy and the action of immunostimulating agents indeed inhibits growth of tumors. PMID:24220932

  20. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  1. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  2. Genome-wide copy number variation analysis of a Branchio-Oto-Renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes

    PubMed Central

    Brophy, Patrick D.; Alasti, Fatemeh; Darbro, Benjamin W.; Clarke, Jason; Nishimura, Carla; Cobb, Bryan; Smith, Richard J.; Manak, J. Robert

    2013-01-01

    Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization (aCGH) on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants (CNVs) we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus in total 5 probands (14%) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR. PMID:23851940

  3. The isolation of a human Ig V lambda gene from a recombinant library of chromosome 22 and estimation of its copy number.

    PubMed Central

    Anderson, M L; Szajnert, M F; Kaplan, J C; McColl, L; Young, B D

    1984-01-01

    We report the first isolation and characterisation of a human Ig V lambda gene. The gene was isolated from a recombinant phage library of human chromosome 22 using a mouse Ig lambda cDNA as probe. DNA sequence analysis predicts a short leader peptide interrupted by an intron of 88 nucleotides, and a mature polypeptide of 96-97 amino acids which shares 61% homology with mouse V lambda I chains. Comparison with the amino acid sequence of known human lambda chains of all six subgroups shows agreement at 22/25 low variance positions. However the maximum homology with human chains is 49%, so we conclude that this sequence represents a new IgV lambda subgroup. The coding region is followed by the conserved heptamer, CACAGTG, and nonamer, ACATAAACC, sequences which have been implicated in V-J segment recombination. This gene has the hallmarks of an active V lambda gene including recently identified transcriptional controlling sequences. Probing genomic DNA with the subcloned V lambda gene detects a family of about 10 cross hybridizing members at low stringency and 2 at high stringency. There is limited polymorphism of the V lambda locus. Images PMID:6091030

  4. Recombinant baculovirus isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2007-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac or BaculoDirect, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay. Methods unique to the Bac-to-Bac system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  5. [Toward an unpublished method of detecting human retroviruses: activation of HIV-1 LacZ recombinant provirus by the tat gene product].

    PubMed

    Bonnerot, C; Savatier, N; Nicolas, J F

    1988-01-01

    For a few of retroviruses, the level of synthesis of viral proteins is greatly increased in the presence of a transactivator gene which is encoded by the virus. For instance, for HIV, TAT acts on target sequences present in the viral long terminal repeat (LTR). HIV-1 recombinant retrovirus (RRV), where the gag, pol and part of env genes have been exchanged for the reporter nlsLacZ gene, expresses the reporter gene only in presence of TAT. When the RRV is tat defective, this activity can be complemented by tat present on a second molecule. The expression of nlsLacZ can then be detected by a simple histochemical staining. If this complementation can also be provided by a wild type virus, then their detection and titration would be greatly simplified.

  6. Polymorphisms of homologous recombination RAD51, RAD51B, XRCC2, and XRCC3 genes and the risk of prostate cancer.

    PubMed

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland. PMID:26339569

  7. Differential activation of transcription versus recombination of transgenic T cell receptor beta variable region gene segments in B and T lineage cells

    PubMed Central

    1994-01-01

    We have tested the ability of the T cell receptor beta (TCR-beta) transcriptional enhancer (E beta) to confer transcriptional activation and tissue-specific V(D)J recombination of TCR-beta V, D, and J segments in a transgenic minilocus recombination substrate. We find that the minimal E beta element, as previously shown for a DNA segment that contained the E mu element, promotes a high level of substrate D to J beta rearrangement in both B and T cells, but only promotes V beta to DJ beta rearrangement in T cells. Thus, both the E mu and E beta elements similarly direct V(D)J recombination of this substrate in vivo, supporting a general role for transcriptional enhancers in the normal regulation of this rearrangement process. Surprisingly, however, we found that both the V beta and DJ beta portion of the constructs were transcribed in an enhancer-dependent fashion (conferred by either E mu or E beta) in both B and T lineage cells, including normal precursor B cells propagated in culture. These findings indicate that, at least in some contexts, transcriptional activation, per se, is not sufficient to confer V(D)J recombinational accessibility to a substrate V gene segment. PMID:8006587

  8. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris.

    PubMed Central

    Lee, Guan-Chiun; Lee, Li-Chiun; Sava, Vasyl; Shaw, Jei-Fu

    2002-01-01

    The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2. PMID:12020350

  9. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    PubMed

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  10. Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

    PubMed Central

    Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone. PMID:23300841

  11. Evolution of Smooth Tubercle Bacilli PE and PE_PGRS Genes: Evidence for a Prominent Role of Recombination and Imprint of Positive Selection

    PubMed Central

    Fabre, Michel; Gutierrez, Maria Cristina; Mardassi, Helmi

    2013-01-01

    Background PE and PE_PGRS are two mycobateria-restricted multigene families encoding membrane associated and secreted proteins that have expanded mainly in the pathogenic species, notably the Mycobacterium tuberculosis complex (MTBC). Several lines of evidence attribute to PE and PE_PGRS genes critical roles in mycobacterial pathogenicity. To get more insight into the nature of these genes, we sought to address their evolutionary trajectories in the group of smooth tubercle bacilli (STB), the putative ancestor of the clonal MTBC. Methodology/Principal Findings By focussing on six polymorphic STB PE/PE_PGRS genes, we demonstrate significant incongruence among single gene genealogies and detect strong signals of recombination using various approaches. Coalescent-based estimation of population recombination and mutation rates (ρ and θ, respectively) indicates that the two mechanisms are of roughly equal importance in generating diversity (ρ/θ = 1.457), a finding in a marked contrast to house keeping genes (HKG) whose evolution is chiefly brought about by mutation (ρ/θ = 0.012). In comparison to HKG, we found 15 times higher mean rate of nonsynonymous substitutions, with strong evidence of positive selection acting on PE_PGRS62 (dN/dS = 1.42), a gene that has previously been shown to be essential for mycobacterial survival in macrophages and granulomas. Imprint of positive selection operating on specific amino acid residues or along branches of PE_PGRS62 phylogenetic tree was further demonstrated using maximum likelihood- and covarion-based approaches, respectively. Strikingly, PE_PGR62 proved highly conserved in present-day MTBC strains. Conclusions/Significance Overall the data indicate that, in STB, PE/PE_PGRS genes have undergone a strong diversification process that is speeded up by recombination, with evidence of positive selection. The finding that positive selection involved an essential PE_PGRS gene whose sequence appears to be driven to

  12. Correction of the nonlinear dose response improves the viability of adenoviral vectors for gene therapy of Fabry disease.

    PubMed

    Ziegler, Robin J; Li, Chester; Cherry, Maribeth; Zhu, Yunxiang; Hempel, Donna; van Rooijen, Nico; Ioannou, Yiannis A; Desnick, Robert J; Goldberg, Mark A; Yew, Nelson S; Cheng, Seng H

    2002-05-20

    Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the

  13. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    SciTech Connect

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  14. Generation of a recombinant rabies Flury LEP virus carrying an additional G gene creates an improved seed virus for inactivated vaccine production.

    PubMed

    Tao, Lihong; Ge, Jinying; Wang, Xijun; Wen, Zhiyuan; Zhai, Hongyue; Hua, Tao; Zhao, Bolin; Kong, Dongni; Yang, Chinglai; Bu, Zhigao

    2011-01-01

    The rabies Flury Low Egg Passage virus (LEP) has been widely used as a seed virus to generate inactive vaccine. Here, we established a reverse genetic system for LEP and generated a recombinant LEP virus (rLEP-G) that carries two identical G genes. This recombinant virus showed similar properties to those of LEP with respect to in vitro growth, neurotropism index, and virulence in mice. rLEP-G produced 4.3-fold more G protein than did LEP in BHK-21 cells. The inactivated vaccine generated from rLEP-G induced significantly higher virus neutralization titers in mice and dogs than those produced in response to LEP-derived vaccine. Our results suggest that rLEP-G is an improved seed virus candidate for inactivated rabies virus vaccine manufacture.

  15. Construction and Characterization of an Infectious Vaccinia Virus Recombinant That Expresses the Influenza Hemagglutinin Gene and Induces Resistance to Influenza Virus Infection in Hamsters

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Murphy, Brian R.; Moss, Bernard

    1983-12-01

    A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.

  16. Gene-by-Diet Interactions Affect Serum 1,25-Dihydroxyvitamin D Levels in Male BXD Recombinant Inbred Mice.

    PubMed

    Fleet, James C; Replogle, Rebecca A; Reyes-Fernandez, Perla; Wang, Libo; Zhang, Min; Clinkenbeard, Erica L; White, Kenneth E

    2016-02-01

    1,25-Dihydroxyvitamin D (1,25[OH]2D) regulates calcium (Ca), phosphate, and bone metabolism. Serum 1,25(OH)2D levels are reduced by low vitamin D status and high fibroblast growth factor 23 (FGF23) levels and increased by low Ca intake and high PTH levels. Natural genetic variation controls serum 25-hydroxyvitamin D (25[OH]D) levels, but it is unclear how it controls serum 1,25(OH)2D or the response of serum 1,25(OH)2D levels to dietary Ca restriction (RCR). Male mice from 11 inbred lines and from 51 BXD recombinant inbred lines were fed diets with either 0.5% (basal) or 0.25% Ca from 4 to 12 weeks of age (n = 8 per line per diet). Significant variation among the lines was found in basal serum 1,25(OH)2D and in the RCR as well as basal serum 25(OH)D and FGF23 levels. 1,25(OH)2D was not correlated to 25(OH)D but was negatively correlated to FGF23 (r = -0.5). Narrow sense heritability of 1,25(OH)2D was 0.67 on the 0.5% Ca diet, 0.66 on the 0.25% Ca diet, and 0.59 for the RCR, indicating a strong genetic control of serum 1,25(OH)2D. Genetic mapping revealed many loci controlling 1,25(OH)2D (seven loci) and the RCR (three loci) as well as 25(OH)D (four loci) and FGF23 (two loci); a locus on chromosome 18 controlled both 1,25(OH)2D and FGF23. Candidate genes underlying loci include the following: Ets1 (1,25[OH]2D), Elac1 (FGF23 and 1,25[OH]2D), Tbc1d15 (RCR), Plekha8 and Lyplal1 (25[OH]D), and Trim35 (FGF23). This report is the first to reveal that serum 1,25(OH)2D levels are controlled by multiple genetic factors and that some of these genetic loci interact with the dietary environment. PMID:26587785

  17. Gene-by-Diet Interactions Affect Serum 1,25-Dihydroxyvitamin D Levels in Male BXD Recombinant Inbred Mice.

    PubMed

    Fleet, James C; Replogle, Rebecca A; Reyes-Fernandez, Perla; Wang, Libo; Zhang, Min; Clinkenbeard, Erica L; White, Kenneth E

    2016-02-01

    1,25-Dihydroxyvitamin D (1,25[OH]2D) regulates calcium (Ca), phosphate, and bone metabolism. Serum 1,25(OH)2D levels are reduced by low vitamin D status and high fibroblast growth factor 23 (FGF23) levels and increased by low Ca intake and high PTH levels. Natural genetic variation controls serum 25-hydroxyvitamin D (25[OH]D) levels, but it is unclear how it controls serum 1,25(OH)2D or the response of serum 1,25(OH)2D levels to dietary Ca restriction (RCR). Male mice from 11 inbred lines and from 51 BXD recombinant inbred lines were fed diets with either 0.5% (basal) or 0.25% Ca from 4 to 12 weeks of age (n = 8 per line per diet). Significant variation among the lines was found in basal serum 1,25(OH)2D and in the RCR as well as basal serum 25(OH)D and FGF23 levels. 1,25(OH)2D was not correlated to 25(OH)D but was negatively correlated to FGF23 (r = -0.5). Narrow sense heritability of 1,25(OH)2D was 0.67 on the 0.5% Ca diet, 0.66 on the 0.25% Ca diet, and 0.59 for the RCR, indicating a strong genetic control of serum 1,25(OH)2D. Genetic mapping revealed many loci controlling 1,25(OH)2D (seven loci) and the RCR (three loci) as well as 25(OH)D (four loci) and FGF23 (two loci); a locus on chromosome 18 controlled both 1,25(OH)2D and FGF23. Candidate genes underlying loci include the following: Ets1 (1,25[OH]2D), Elac1 (FGF23 and 1,25[OH]2D), Tbc1d15 (RCR), Plekha8 and Lyplal1 (25[OH]D), and Trim35 (FGF23). This report is the first to reveal that serum 1,25(OH)2D levels are controlled by multiple genetic factors and that some of these genetic loci interact with the dietary environment.

  18. Efficacy of recombinant adenoviral human p53 gene in the treatment of lung cancer-mediated pleural effusion

    PubMed Central

    LI, KUN-LIN; KANG, JUN; ZHANG, PENG; LI, LI; WANG, YU-BO; CHEN, HENG-YI; HE, YONG

    2015-01-01

    Pleural effusion induced by lung cancer exerts a negative impact on quality of life and prognosis. The aim of the present study was to evaluate the value of the recombinant adenoviral human p53 gene (rAd-p53) in the local treatment of lung cancer and its synergistic effect with chemotherapy. The present study retrospectively recruited 210 patients with lung cancer-mediated pleural effusion who had adopted a treatment strategy of platinum chemotherapy. Pleurodesis was performed via the injection of cisplatin or rAd-p53. Long-term follow-up was conducted to investigate the therapeutic effects of cisplatin and rAd-p53 administration on pleural effusion and other relevant clinical indicators. The short-term effect of pleurodesis was as follows: The efficacy rate of rAd-p53 therapy was significantly higher compared with cisplatin therapy (71.26 vs. 54.47%), and the efficacy of treatment with ≥2×1012 viral particles of rAd-p53 for pleurodesis was significantly greater than treatment with 40 mg cisplatin (P<0.05). Furthermore, efficacy analysis performed 6 and 12 months after pleurodesis indicated that the efficacy rate of rAd-p53 was significantly greater than that of cisplatin (P<0.05). A comparison of median progression-free survival (PFS) time identified a significant difference (P<0.05) between rAd-p53 and cisplatin therapy (3.3 vs. 2.7 months); however, a comparison of median overall survival time identified no significant difference (P>0.05) between rAd-p53 and cisplatin therapy (9.6 vs. 8.7 months). In addition, Cox regression analysis indicated that PFS was not affected by clinical indicators such as age, gender, prognostic staging and smoking status; however, PFS was affected by pathological subtype (adenocarcinoma or squamous carcinoma) in the rAd-p53 group. rAd-p53 administration for pleurodesis exerts long-term therapeutic effects on the local treatment of lung cancer. Thus, a combination of rAd-p53 and chemotherapy may exert a synergistic effect and

  19. Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes.

    PubMed

    Gallardo, H F; Tan, C; Ory, D; Sadelain, M

    1997-08-01

    It is essential for the study of T-cell function and the improvement of adoptive cell therapies to efficiently generate large populations of human primary T cells that reliably express foreign genes. This goal is achieved by using recombinant retroviruses pseudotyped with either the gibbon ape leukemia virus (GaLV) envelope or the vesicular stomatitis virus G (VSV-G) glycoprotein. We show here that both retroviral particles mediate stable gene transfer in CD4+ and in CD8+ peripheral blood lymphocytes cultured under optimized conditions. However, VSV-G-pseudotyped virions may cause transduction artifacts that must be carefully excluded. The VSV-G virions require 10- to 100-fold higher concentrations of infectious particles to achieve levels of gene transfer comparable to GaLV-virions. Nonetheless, the physical stability of VSV-G-coated particles enables the concentration of viral stocks to 10(9) infectious particles per milliliter or more.

  20. Recombinant mouse prion protein alone or in combination with lipopolysaccharide alters expression of innate immunity genes in the colon of mice.

    PubMed

    Dervishi, Elda; Lam, Tran H; Dunn, Suzana M; Zwierzchowski, Grzegorz; Saleem, Fozia; Wishart, David S; Ametaj, Burim N

    2015-01-01

    The objectives of this study were to test whether recombinant mouse (mo)PrP alone or in combination with LPS or under simulated endotoxemia would affect expression of genes related to host inflammatory and antimicrobial responses. To test our hypotheses colon tissues were collected from 16 male mice (FVB/N strain) and mounted in an Ussing chamber. Application of moPrP to the mucosal side of the colon affected genes related to TLR- and NLR- signaling and antimicrobial responses. When LPS was added on the mucosal side of the colon, genes related to TLR, Nlrp3 inflammasome, and iron transport proteins were over-expressed. Addition of LPS to the serosal side of the colon up-regulated genes related to TLR- and NLR-signaling, Nlrp3 inflammasome, and a chemokine. Treatment with both moPrP and LPS to the mucosal side of the colon upregulated genes associated with TLR, downstream signal transduction (DST), inflammatory response, attraction of dendritic cells to the site of inflammation, and the JNK-apoptosis pathway. Administration of moPrP to the mucosal side and LPS to the serosal side of the colon affected genes related to TLR- and NLR-signaling, DST, apoptosis, inflammatory response, cytokines, chemokines, and antimicrobial peptides. Overall this study suggests a potential role for moPrP as an endogenous 'danger signal' associated with activation of colon genes related to innate immunity and antibacterial responses.

  1. Recombinant mouse prion protein alone or in combination with lipopolysaccharide alters expression of innate immunity genes in the colon of mice

    PubMed Central

    Dervishi, Elda; Lam, Tran H; Dunn, Suzana M; Zwierzchowski, Grzegorz; Saleem, Fozia; Wishart, David S; Ametaj, Burim N

    2015-01-01

    ABSTRACT The objectives of this study were to test whether recombinant mouse (mo)PrP alone or in combination with LPS or under simulated endotoxemia would affect expression of genes related to host inflammatory and antimicrobial responses. To test our hypotheses colon tissues were collected from 16 male mice (FVB/N strain) and mounted in an Ussing chamber. Application of moPrP to the mucosal side of the colon affected genes related to TLR- and NLR- signaling and antimicrobial responses. When LPS was added on the mucosal side of the colon, genes related to TLR, Nlrp3 inflammasome, and iron transport proteins were over-expressed. Addition of LPS to the serosal side of the colon up-regulated genes related to TLR- and NLR-signaling, Nlrp3 inflammasome, and a chemokine. Treatment with both moPrP and LPS to the mucosal side of the colon upregulated genes associated with TLR, downstream signal transduction (DST), inflammatory response, attraction of dendritic cells to the site of inflammation, and the JNK-apoptosis pathway. Administration of moPrP to the mucosal side and LPS to the serosal side of the colon affected genes related to TLR- and NLR-signaling, DST, apoptosis, inflammatory response, cytokines, chemokines, and antimicrobial peptides. Overall this study suggests a potential role for moPrP as an endogenous ‘danger signal’ associated with activation of colon genes related to innate immunity and antibacterial responses. PMID:25695140

  2. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

    PubMed

    Xiao, Fei; Deng, Jiali; Yu, Junjie; Guo, Yajie; Chen, Shanghai; Guo, Feifan

    2016-01-01

    Insulin resistance is one of the major factors contributing to metabolic diseases, but the underlying mechanisms are still poorly understood. As an important cofactor, B-cell translocation gene 1 (BTG1) is involved in many physiologic processes; however, the direct effect of BTG1 on insulin sensitivity has not been described. In our study, BTG1 overexpression or knockdown improved or impaired insulin signaling in vitro, respectively. In addition, adenovirus-mediated BTG1 overexpression improved insulin sensitivity in wild-type (WT) and insulin-resistant leptin-receptor mutated (db/db) mice. In addition, transgenic BTG1-overexpressing mice were resistant to high-carbohydrate diet-induced insulin resistance. Adenovirus-mediated BTG1 knockdown consistently impaired insulin sensitivity in WT and insulin-sensitive leucine-deprived mice. Moreover, hepatic BTG1 expression was increased by leucine deprivation via the mammalian target of rapamycin/ribosomal protein S6 kinase 1 pathway. Furthermore, c-Jun expression was up-regulated by BTG1, and adenovirus-mediated c-Jun knockdown blocked BTG1-improved insulin signaling and insulin sensitivity in vitro and in vivo. Finally, BTG1 promoted c-Jun expression via stimulating c-Jun and retinoic acid receptor activities. Taken together, these results identify a novel function for BTG1 in the regulation of hepatic insulin sensitivity and provide important insights into the nutritional regulation of BTG1 expression.- Xiao, F., Deng, J., Yu, J., Guo, Y., Chen, S., Guo, F. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

  3. Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger

    SciTech Connect

    Dai, Ziyu; Aryal, Uma K.; Shukla, Anil; Qian, Wei-Jun; Smith, Richard D.; Magnuson, Jon K.; Adney, William S.; Beckham, Gregg T.; Brunecky, Roman; Himmel, Michael E.; Decker, Stephen R.; Ju, Xiaohui; Zhang, Xiao; Baker, Scott E.

    2013-12-01

    ALG3 is a Family 58 glycosyltransferase enzyme involved in early N-linked glycan synthesis. Here, we investigated the effect of the alg3 gene disruption on growth, development, metabolism, and protein secretion in Aspergillus niger. The alg3 gene deletion resulted in a significant reduction of growth on complete (CM) and potato dextrose agar (PDA) media and a substantial reduction of spore production on CM. It also delayed spore germination in the liquid cultures of both CM and PDA media, but led to a significant accumulation of red pigment on both CM and liquid modified minimal medium (MM) supplemented with yeast extract. The relative abundance of 55 proteins of the total 190 proteins identified in the secretome was significantly different as a result of alg3 gene deletion. Comparison of a Trichoderma reesei cellobiohydrolase (Cel7A) heterologously expressed in A. niger parental and Δalg3 strains showed that the recombinant Cel7A expressed in the mutant background was smaller in size than that from the parental strains. This study suggests that ALG3 is critical for growth and development, pigment production, and protein secretion in A. niger. Functional analysis of recombinant Cel7A with aberrant glycosylation demonstrates the feasibility of this alternative approach to evaluate the role of N-linked glycosylation in glycoprotein secretion and function.

  4. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-10-11

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.

  5. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed Central

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-01-01

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content. Images PMID:7524085

  6. Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris

    PubMed Central

    Jiménez, Juan J.; Gútiez, Loreto; Cintas, Luis M.; Herranz, Carmen; Hernández, Pablo E.

    2015-01-01

    We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. PMID:25821820

  7. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  8. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  9. Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion

    SciTech Connect

    Gangnonngiw, Warachin; Anantasomboon, Gun; Sang-oum, Wiwat; Sriurairatana, Siriporn; Sritunyalucksana, Kallaya; Flegel, Timothy W.

    2009-03-01

    RT-PCR using a commercial kit for yellow head virus (YHV) detection in growth-retarded shrimp yielded an unusual 777 bp amplicon instead of expected amplicons of 277 bp for YHV type-1 (YHV-1) or 406 bp for YHV type-2 (YHV-2). Cloning and sequencing (GenBank (EU170438)) revealed approximately 80% identity to non-structural (NS) ORF1b sequences of both YHV-1 (GenBank (AA083987)) and YHV-2 (GenBank (AF227196)), indicating an atypical YHV type (A-YHV) phylogenetically equidistant from both types. An RT-PCR test specifically designed for A-YHV revealed that it was uncommon and that its occurrence in shrimp culture ponds did not correlate with growth retardation or mortality. By immunohistochemistry with YHV-specific monoclonal antibodies, the A-YHV gave positive reactions for envelope protein gp64 and capsid protein p20, but not for envelope protein gp116, even though gp116 and gp64 originate from a polyprotein of ORF3. Lack of gp116 immunoreactivity correlated with a large ORF3 deletion (GenBank (EU123854)) in the region of the protein targeted by an MAb against gp116. Transmission electron microscopy of A-YHV-infected shrimp revealed only unenveloped pre-virions. During manuscript revision, information received revealed that typing of YHV isolates based on sequences of ORF1b and ORF3 had yielded several geographical types, including one virulent type (YHV-1b) with an ORF3 deletion sequence that matched the sequence of A-YHV. Using these sequences and an additional A-YHV sequence ( (EU853170)) from the ORF1b typing region, A-YHV potentially represents a recombinant between type 1b and type 5. SDS-PAGE and Western blot analysis revealed that type 1b produced a gp116 deletion protein that did not bind with the MAb or polyclonal Ab to normal gp116. Overall, the information suggested that lack of A-YHV virulence was associated with the NS gene sequence linked to ORF1b rather than the deletion in ORF3.

  10. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. PMID

  11. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

  12. C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion.

    PubMed

    Sabouri, Somayeh; Kobayashi, Maki; Begum, Nasim A; Xu, Jianliang; Hirota, Kouji; Honjo, Tasuku

    2014-02-11

    Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.

  13. Sequence and expression of a xylanase gene from the hyperthermophile Thermotoga sp. strain FjSS3-B.1 and characterization of the recombinant enzyme and its activity on kraft pulp.

    PubMed Central

    Saul, D J; Williams, L C; Reeves, R A; Gibbs, M D; Bergquist, P L

    1995-01-01

    A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp. PMID:8526526

  14. Site-specific recombination of nitrogen-fixation genes in cyanobacteria by XisF-XisH-XisI complex: structures and models

    PubMed Central

    Golden, James W.; Pascual, Jaime; Xu, Dong; Cheltsov, Anton

    2014-01-01

    Nitrogen fixation is an important process that converts atmospheric gaseous nitrogen, a form plants cannot utilize, into ammonia that can be easily assimilated. Large serine recombinase XisF (fdxN element site-specific recombinase), together with controlling factors XisH and XisI, plays a critical role in the expression of nitrogen fixation genes of certain Anabaena and Nostoc species of cyanobacteria. All three proteins are required to excise the fdxN DNA element from the chromosome in differentiating heterocysts for the expression of nitrogen fixation related genes. We report the first crystal structures of XisH and XisI proteins, both adopting novel protein folds. Based on the analysis of their sequences and structures, we propose that XisH and XisI proteins function as endonucleases and recombination directionality factors (RDFs), respectively. PMID:25179344

  15. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors.

    PubMed

    Spirin, P V; Baskaran, D; Rubtsov, P M; Zenkova, M A; Vlassov, V V; Chernolovskaya, E L; Prassolov, V S

    2009-07-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

  16. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors

    PubMed Central

    Spirin, P.V.; Baskaran, D.; Rubtsov, P.M.; Zenkova, M.A.; Vlassov, V.V.; Chernolovskaya, E.L.

    2009-01-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented. PMID:22649608

  17. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus.

    PubMed

    Guthrie, Alan J; Quan, Melvyn; Lourens, Carina W; Audonnet, Jean-Christophe; Minke, Jules M; Yao, Jiansheng; He, Ling; Nordgren, Robert; Gardner, Ian A; Maclachlan, N James

    2009-07-16

    We describe the development and preliminary characterization of a recombinant canarypox virus vectored (ALVAC) vaccine for protective immunization of equids against African horse sickness virus (AHSV) infection. Horses (n=8) immunized with either of two concentrations of recombinant canarypox virus vector (ALVAC-AHSV) co-expressing synthetic genes encoding the outer capsid proteins (VP2 and VP5) of AHSV serotype 4 (AHSV-4) developed variable titres (<10-80) of virus-specific neutralizing antibodies and were completely resistant to challenge infection with a virulent strain of AHSV-4. In contrast, a horse immunized with a commercial recombinant canarypox virus vectored vaccine expressing the haemagglutinin genes of two equine influenza H3N8 viruses was seronegative to AHSV and following infection with virulent AHSV-4 developed pyrexia, thrombocytopenia and marked oedema of the supraorbital fossae typical of the "dikkop" or cardiac form of African horse sickness. AHSV was detected by virus isolation and quantitative reverse transcriptase polymerase chain reaction in the blood of the control horse from 8 days onwards after challenge infection whereas AHSV was not detected at any time in the blood of the ALVAC-AHSV vaccinated horses. The control horse seroconverted to AHSV by 2 weeks after challenge infection as determined by both virus neutralization and ELISA assays, whereas six of eight of the ALVAC-AHSV vaccinated horses did not seroconvert by either assay following challenge infection with virulent AHSV-4. These data confirm that the ALVAC-AHSV vaccine will be useful for the protective immunization of equids against African horse sickness, and avoids many of the problems inherent to live-attenuated AHSV vaccines.

  18. ABCG-like transporter of Trypanosoma cruzi involved in benznidazole resistance: gene polymorphisms disclose inter-strain intragenic recombination in hybrid isolates.

    PubMed

    Franco, Jaques; Ferreira, Renata C; Ienne, Susan; Zingales, Bianca

    2015-04-01

    Benznidazole (BZ) is one of the two drugs for Chagas disease treatment. In a previous study we showed that the Trypanosoma cruzi ABCG-like transporter gene, named TcABCG1, is over-expressed in parasite strains naturally resistant to BZ and that the gene of TcI BZ-resistant strains exhibited several single nucleotide polymorphisms (SNPs) as compared to the gene of CL Brener BZ-susceptible strain. Here we report the sequence of TcABCG1 gene of fourteen T. cruzi strains, with diverse degrees of BZ sensitivity and belonging to different discrete typing units (DTUs) and Tcbat group. Although DTU-specific SNPs and amino acid changes were identified, no direct correlation with BZ-resistance phenotype was found. Thus, it is plausible that the transporter abundance is a determinant factor for drug resistance, as pointed out above. Sequence data were used for Bayesian phylogenies and network genealogy analysis. The network showed a high degree of reticulation suggesting genetic exchange between the parasites. TcI and TcII clades were clearly separated. Tcbat sequences were close to TcI. A fourth clade clustered TcABCG1 haplotypes of TcV, TcVI and TcIII strains, with closer proximity to TcI. Analysis of the recombination patterns indicated that hybrid strains contain haplotypes that are mosaics most likely derived by intragenic recombination of parental sequences. The data confirm that TcII and TcIII as the parentals of TcV and TcVI DTUs. Since genetic fingerprint of TcI was found in TcIII, we sustain the previously proposed "Two Hybridization model" for the origin of hybrid strains. Among the twenty best BLASTP hits in databases, orthologues of TcABCG1 transporter were found in Leishmania spp. and African trypanosomes, though their function remains undescribed. PMID:25660041

  19. A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

    PubMed

    Gomes, Michelle M; Wang, Lingyan; Jiang, Han; Kahl, Christoph A; Brigande, John V

    2016-01-01

    There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders. PMID:27259920

  20. Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters.

    PubMed

    Zhang, Zhenjie; Chen, Wenqing; Ma, Chengtai; Zhao, Peng; Duan, Luntao; Zhang, Fushou; Sun, Aijun; Li, Yanpeng; Su, Hongqin; Li, Sifei; Cui, He; Cui, Zhizhong

    2014-07-10

    To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

  1. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  2. Improvement of cloned [alpha]-amylase gene expression in fed-batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

    SciTech Connect

    Shiba, Sumihisa; Nishida, Yoshio; Park, Y.S.; Iijima, Shinji; Kobayashi, Takeshi . Dept. of Biotechnology)

    1994-11-05

    The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the [alpha]-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. To increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy controller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of [alpha]-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory [alpha]-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 392 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled.

  3. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    PubMed

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-10-09

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  4. Deletion of C7L and K1L genes leads to significantly decreased virulence of recombinant vaccinia virus TianTan.

    PubMed

    Liu, Zheng; Wang, Shuhui; Zhang, Qicheng; Tian, Meijuan; Hou, Jue; Wang, Rongmin; Liu, Chang; Ji, Xu; Liu, Ying; Shao, Yiming

    2013-01-01

    The vaccinia virus TianTan (VTT) has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity of these recombinants were evaluated in mouse and rabbit models. Comparing to parental VTT, VTT△C7L and VTT△K1L showed significantly decreased replication capability in CEF, Vero, BHK-21 and HeLa cell lines. In particular, replication of VTT△C7LK1L decreased more than 10-fold in all four cell lines. The virulence of all these mutants were decreased in BALB/c mouse and rabbit models; VTT△C7LK1L once again showed the greatest attenuation, having resulted in no evident damage in mice and erythema of only 0.4 cm diameter in rabbits, compared to 1.48 cm for VTT. VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag elicited as strong cellular and humoral responses against HIV genes as did VTKgpe, while humoral immune response against the vaccinia itself was reduced by 4-8-fold. These data show that deletion of C7L and K1L genes leads to significantly decreased virulence without compromising animal host immunogenicity, and may thus be key to creating a more safe and effective HIV vaccine vector.

  5. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  6. Climate-Driven Reshuffling of Species and Genes: Potential Conservation Roles for Species Translocations and Recombinant Hybrid Genotypes

    PubMed Central

    Scriber, Jon Mark

    2013-01-01

    Comprising 50%–75% of the world’s fauna, insects are a prominent part of biodiversity in communities and ecosystems globally. Biodiversity across all levels of biological classifications is fundamentally based on genetic diversity. However, the integration of genomics and phylogenetics into conservation management may not be as rapid as climate change. The genetics of hybrid introgression as a source of novel variation for ecological divergence and evolutionary speciation (and resilience) may generate adaptive potential and diversity fast enough to respond to locally-altered environmental conditions. Major plant and herbivore hybrid zones with associated communities deserve conservation consideration. This review addresses functional genetics across multi-trophic-level interactions including “invasive species” in various ecosystems as they may become disrupted in different ways by rapid climate change. “Invasive genes” (into new species and populations) need to be recognized for their positive creative potential and addressed in conservation programs. “Genetic rescue” via hybrid translocations may provide needed adaptive flexibility for rapid adaptation to environmental change. While concerns persist for some conservationists, this review emphasizes the positive aspects of hybrids and hybridization. Specific implications of natural genetic introgression are addressed with a few examples from butterflies, including transgressive phenotypes and climate-driven homoploid recombinant hybrid speciation. Some specific examples illustrate these points using the swallowtail butterflies (Papilionidae) with their long-term historical data base (phylogeographical diversity changes) and recent (3-decade) climate-driven temporal and genetic divergence in recombinant homoploid hybrids and relatively recent hybrid speciation of Papilio appalachiensis in North America. Climate-induced “reshuffling” (recombinations) of species composition, genotypes, and genomes

  7. Expression of the phycoerythrin gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and evaluation of the bioactivity of recombinant PE

    NASA Astrophysics Data System (ADS)

    Wen, Ruobing; Sui, Zhenghong; Zhang, Xuecheng; Zhang, Shuang; Qin, Song

    2007-10-01

    Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) ( P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL-1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still

  8. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

    PubMed

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

    2015-04-01

    The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus.

  9. Construction of expression systems for flaA and flaB genes of Helicobacter pylori and determination of immunoreactivity and antigenicity of recombinant proteins

    PubMed Central

    Yan, Jie; Liang, Shao-Hui; Mao, Ya-Fei; Li, Li-Wei; Li, Shu-Ping

    2003-01-01

    AIM: To clone flagellin genes A (flaA) and B (flaB) from a clinical strain of Helicobacter pylori (H pylori) and to construct prokaryotic expression systems of the genes and identify immunity of the fusion proteins. METHODS: The flaA and flaB genes from a clinical H pylori isolate Y06 were amplified by high fidelity PCR. The nucleotide sequences of target DNA amplification fragments from the two genes were sequenced after T-A cloning. The recombinant expression vector pET32a inserted with flaA and flaB genes was constructed, respectively. The expressions of FlaA and FlaB fusion proteins in E. coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations were examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and immunodiffusion assay using self-prepared rabbit antiserum against FlaA (rFlaA) or FlaB (rFlaB) recombinant proteins were applied to the determination of the fusion proteins immunity. ELISA was used to detect the antibodies against rFlaA and rFlaB in sera of 125 H pylori infected patients and to examine rFlaA and rFlaB expression in 98 clinical isolates of H pylori, respectively. RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homologies of the cloned flaA and flaB genes were from 96.28%-97.13% and 96.31%-97.73%, and their putative amino acid sequence homologies were 99.61%-99.80% and 99.41%-100% for the two genes, respectively. The output of rFlaA and rFlaB expressed by pET32a-flaA-BL21DE3 and pET32a-flaB-BL21DE3 systems was as high as 40%-50% of the total bacterial proteins. Both rFlaA and rFlaB were able to combine with the commercial antibodies against whole cell of H pylori and to induce rabbits to produce specific antibodies with the same 1:2 immunodiffusion titers after the animals were immunized with the two recombinant proteins. Ninety-eight and zero point 4 and 92.80% of the serum samples from 125 patients infected with H pylori were

  10. Identification of a novel gene, H34, in wheat using recombinant inbred lines and single nucleotide polymorphism markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hessian fly (HF), Mayetiola destructor, is an important pest of wheat (Triticum aestivum L.) worldwide. Because it has multiple biotypes that are virulent to different wheat HF resistance genes, pyramiding multiple resistance genes in a cultivar can improve resistance durability, and finding DNA mar...

  11. Batch production of coenzyme Q10 by recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis.

    PubMed

    Martínez, Irene; Méndez, Claudia; Berríos, Julio; Altamirano, Claudia; Díaz-Barrera, Alvaro

    2015-09-01

    Coenzyme Q10 (CoQ10) is an important antioxidant used in medicine, dietary supplements, and cosmetic applications. In the present work, the production of CoQ10 using a recombinant Escherichia coli strain containing the decaprenyl diphosphate synthase from Sphingomonas baekryungensis was investigated, wherein the effects of culture medium, temperature, and agitation rate on the production process were assessed. It was found that Luria-Bertani (LB) medium was superior to M9 with glucose medium. Higher temperature (37 °C) and higher agitation rate (900 rpm) improved the specific CoQ10 content significantly in LB medium; on the contrary, the use of M9 medium with glucose showed similar values. Specifically, in LB medium, an increase from 300 to 900 rpm in the agitation rate resulted in increases of 55 and 197 % in the specific CoQ10 content and COQ10 productivity, respectively. Therefore, the results obtained in the present work are a valuable contribution for the optimization of CoQ10 production processes using recombinant E. coli strains.

  12. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  13. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis

    PubMed Central

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. PMID:26197085

  14. Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

    PubMed

    Li, Jing; Zhang, Huayuan; Liu, Jing; Xu, Kangsen

    2006-09-01

    Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity. PMID:16689684

  15. Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

    PubMed

    Li, Jing; Zhang, Huayuan; Liu, Jing; Xu, Kangsen

    2006-09-01

    Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.

  16. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    PubMed

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. PMID:26197085

  17. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    PubMed

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

  18. Bioproduction of 4-vinylphenol from corn cob alkaline hydrolyzate in two-phase extractive fermentation using free or immobilized recombinant E. coli expressing pad gene.

    PubMed

    Salgado, José Manuel; Rodríguez-Solana, Raquel; Curiel, José Antonio; de Las Rivas, Blanca; Muñoz, Rosario; Domínguez, José Manuel

    2014-05-10

    In situ extractive fermentation was used to produce 4-vinyl derivatives from hydroxycinnamic acids extracted from corn cobs by recombinant Escherichia coli cells expressing Lactobacillus plantarum phenolic acid descarboxylase (PAD) gene. This microorganism mainly produced 4-vinylphenol (4VP) from p-coumaric acid (p-CA). In the first study , we observed that the concentrations of 4VP are higher than 1g/L which had a negative impact on decarboxylation of p-CA to 4VP by recombinant E. coli cells. Because of this, and in order to improve the downstream process, a two-phase aqueous-organic solvent system was developed. The results of the extractive fermentation indicated that it was possible to use hydrolyzates as aqueous phase to bioproduce 4VP, and recover simultaneously the product in the organic phase containing hexane. The detoxification of pre-treated corn cob alkaline hydrolyzate improved 4VP production up to 1003.5mg/L after 24h fermentation (QP=41.813mg/Lh). Additionally, preliminary experiments using cells immobilized in calcium alginate showed to be a good system for the biotransform of p-CA to 4VP in extractive fermentation, although the process hindered partially the recovery of 4VP in the organic phase. PMID:24731821

  19. Characterizing the Roles of Cryphonectria parasitica RNA-Dependent RNA Polymerase-Like Genes in Antiviral Defense, Viral Recombination and Transposon Transcript Accumulation

    PubMed Central

    Zhang, Dong-Xiu; Spiering, Martin J.; Nuss, Donald L.

    2014-01-01

    An inducible RNA-silencing pathway, involving a single Dicer protein, DCL2, and a single Argonaute protein, AGL2, was recently shown to serve as an effective antiviral defense response in the chestnut blight fungus Cryphonectria parasitica. Eukaryotic RNA-dependent RNA polymerases (RdRPs) are frequently involved in transcriptional and posttranscriptional gene silencing and antiviral defense. We report here the identification and characterization of four RdRP genes (rdr1–4) in the C. parasitica genome. Sequence relationships with other eukaryotic RdRPs indicated that RDR1 and RDR2 were closely related to QDE-1, an RdRP involved in RNA silencing (“quelling”) in Neurospora crassa, whereas RDR3 was more closely related to the meiotic silencing gene SAD-1 in N. crassa. The RdRP domain of RDR4, related to N. crassa RRP-3 of unknown function, was truncated and showed evidence of alternative splicing. Similar to reports for dcl2 and agl2, the expression levels for rdr3 and rdr4 increased after hypovirus CHV-1/EP713 infection, while expression levels of rdr1 and rdr2 were unchanged. The virus-responsive induction patterns for rdr3 and rdr4 were altered in the Δdcl2 and Δagl2 strains, suggesting some level of interaction between rdr3 and rdr4 and the dcl2/agl2 silencing pathway. Single rdr gene knockouts Δrdr1–4, double knockouts Δrdr1/2, Δrdr2/3, Δrdr1/3, and a triple knockout, Δrdr1/2/3, were generated and evaluated for effects on fungal phenotype, the antiviral defense response, viral RNA recombination activity and transposon expression. None of the single or multiple rdr knockout strains displayed any phenotypic differences from the parental strains with or without viral infection or any significant changes in viral RNA accumulation or recombination activity or transposon RNA accumulation, indicating no detectable contribution by the C. parasitica rdr genes to these processes. PMID:25268858

  20. Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

    PubMed Central

    Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

    2009-01-01

    Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

  1. OBTAINING OF THE TRANSGENIC HELIANTHUS TUBEROSUS L. PLANTS, CALLUS AND "HAIRY" ROOT CULTURES ABLE TO EXPRESS THE RECOMBINANT HUMAN INTERFERON ALPHA-2b GENE.

    PubMed

    Maistrenko, O M; Luchakivska, Yu S; Zholobak, N M; Spivak, M Ya; Kuchuk, M V

    2015-01-01

    This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein. PMID:26638495

  2. OBTAINING OF THE TRANSGENIC HELIANTHUS TUBEROSUS L. PLANTS, CALLUS AND "HAIRY" ROOT CULTURES ABLE TO EXPRESS THE RECOMBINANT HUMAN INTERFERON ALPHA-2b GENE.

    PubMed

    Maistrenko, O M; Luchakivska, Yu S; Zholobak, N M; Spivak, M Ya; Kuchuk, M V

    2015-01-01

    This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein.

  3. A recombinant fowlpox virus vaccine expressing glycoprotein B gene from CVI988/Rispens strain of MDV: protection studies in different chickens.

    PubMed

    Liu, X; Peng, D; Wu, X; Xing, L; Zhang, R

    1999-01-01

    Recombinant fowlpox virus (rFPV) was constructed to express glycoprotein B (gB) gene from CVI988/Rispens strain of Marek's disease virus (MDV). The rFPV-gB/R alone and in combination with herpesvirus of turkey (HVT) preparations were evaluated for their protective efficacy against challenge with very virulent MDV strains Md5 and RB1B in different chickens. The rFPV-gB/R alone induced protection comparable to that by HVT vaccines in both Ab- SPF chickens and Ab+ production chickens. Significant protective synergism was observed in one of these two types of commercial production chickens when rFPV-gB/R was combined with HVT of either cell-associated or cell-free preparations. Immunogenesis studies showed that rFPV-gB/R, just like conventional vaccines, significantly reduced the level of viremia, splenocytes infection and feather follicle shedding of challenge virus in vaccinated chickens.

  4. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  5. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    PubMed

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  6. Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.

    PubMed

    Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

    2015-05-21

    β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine.

  7. Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering

    PubMed Central

    Liu, Qingshu; Shen, Qiyao; Bian, Xiaoying; Chen, Hanna; Fu, Jun; Wang, Hailong; Lei, Ping; Guo, Zhaohui; Chen, Wu; Li, Dingjun; Zhang, Youming

    2016-01-01

    Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera. PMID:27687863

  8. Recombinant porcine reproductive and respiratory syndrome virus expressing luciferase genes provide a new indication of viral propagation in both permissive and target cells.

    PubMed

    Gao, Fei; Qu, Zehui; Li, Liwei; Yu, Lingxue; Jiang, Yifeng; Zhou, Yanjun; Yang, Shen; Zheng, Hao; Huang, Qinfeng; Tong, Wu; Tong, Guangzhi

    2016-08-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has a condensed single-stranded positive-sense RNA genome that contains several overlapping regions. The transcription regulatory sequence (TRS) is the important cis-acting element participating in PRRSV discontinuous transcription process. Based on reverse genetic system of type 2 highly pathogenic PRRSV cell-passage attenuated strain pHuN4-F112, firefly luciferase or Renilla luciferase genes were inserted between ORF1b and ORF2. An extra TRS6 was embedded behind the foreign luciferase genes. pA-Fluc and pA-Rluc were constructed and successfully rescued in MARC-145 cells. The phenotypical characteristics of the progeny virus were indistinguishable from those of vHuN4-F112 and were genetically stable for at least 25 cell passages. Mutant virus-infected cells were lysed at different time points to assess luciferase activities and measure foreign gene expression levels. The results showed identical variations in the luciferase activities of the recombinants in MARC-145 cells, indicating that they were suitable for monitoring viral propagation in PRRSV-permissive cell cultures. They were also used to infect pulmonary alveolar macrophages, which yielded similar variations in luciferase activities. Therefore, vA-Fluc and vA-Rluc present powerful new tools to monitor PRRSV propagation in both passaged and target cells. PMID:27473986

  9. Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.

    PubMed

    Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

    2015-01-01

    β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine. PMID:25994151

  10. Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

    PubMed

    Hermens, W T; ter Brake, O; Dijkhuizen, P A; Sonnemans, M A; Grimm, D; Kleinschmidt, J A; Verhaagen, J

    1999-07-20

    Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

  11. Gene therapy for human colorectal cancer cell lines with recombinant adenovirus 5 based on loss of the insulin-like growth factor 2 imprinting.

    PubMed

    Sun, Huiling; Pan, Yuqin; He, Bangshun; Deng, Qiwen; Li, Rui; Xu, Yeqiong; Chen, Jie; Gao, Tianyi; Ying, Houqun; Wang, Feng; Liu, Xian; Wang, Shukui

    2015-04-01

    The recombinant oncolytic adenovirus is a novel anticancer agent to replicate selectively in colon cancer cell lines. Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon. We utilized the IGF2 LOI in gene therapy for the malignant tumor cell lines. We investigated the tumoricidal effects of IGF2 LOI on four cell lines by oncolytic adenovirus, and constructed novel adenovirus vectors Ad312-E1A and Ad312-EGFP. The expression of E1A was monitored by real-time PCR and western blot analysis. The viability and apoptosis of colorectal cells infected with Ad312-E1A were tested by CCK-8 and flow cytometry. In addition, we established a colorectal cancer model in nude mice. The results showed that HCT-8 and HT-29 with IGF2 LOI were infected with Ad312-EGFP and then produced the EGFP. Nevertheless, SW480 and GES-1, which were IGF2 MOI, did not produce the EGFP. The Ad312-E1A obviously reduced the cell viability and induced apoptosis in HCT-8 and HT-29 in vitro, and successfully suppressed tumor growth in HT-29 xenografts in nude mice. In summary, the conditionally replicative adenovirus with loss of IGF2 imprinting system has a positive effect on gene therapy.

  12. Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

    PubMed

    Wei, Wang; Yushuang, Wang; Lanlan, Huang; Zijian, Jian; Xinhua, Wang; Shouren, Liu; Wenhui, Pi

    2016-09-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts. PMID:27644744

  13. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  14. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  15. The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression

    PubMed Central

    Kelloniemi, Annina; Szabo, Zoltan; Serpi, Raisa; Näpänkangas, Juha; Ohukainen, Pauli; Tenhunen, Olli; Kaikkonen, Leena; Koivisto, Elina; Bagyura, Zsolt; Kerkelä, Risto; Leosdottir, Margret; Hedner, Thomas; Melander, Olle

    2015-01-01

    The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio. PMID:26098115

  16. Selection against recombinant hybrids maintains reproductive isolation in hybridizing Populus species despite F1 fertility and recurrent gene flow.

    PubMed

    Christe, Camille; Stölting, Kai N; Bresadola, Luisa; Fussi, Barbara; Heinze, Berthold; Wegmann, Daniel; Lexer, Christian

    2016-06-01

    Natural hybrid zones have proven to be precious tools for understanding the origin and maintenance of reproductive isolation (RI) and therefore species. Most available genomic studies of hybrid zones using whole- or partial-genome resequencing approaches have focused on comparisons of the parental source populations involved in genome admixture, rather than exploring fine-scale patterns of chromosomal ancestry across the full admixture gradient present between hybridizing species. We have studied three well-known European 'replicate' hybrid zones of Populus alba and P. tremula, two widespread, ecologically divergent forest trees, using up to 432 505 single-nucleotide polymorphisms (SNPs) from restriction site-associated DNA (RAD) sequencing. Estimates of fine-scale chromosomal ancestry, genomic divergence and differentiation across all 19 poplar chromosomes revealed strikingly contrasting results, including an unexpected preponderance of F1 hybrids in the centre of genomic clines on the one hand, and genomically localized, spatially variable shared variants consistent with ancient introgression between the parental species on the other. Genetic ancestry had a significant effect on survivorship of hybrid seedlings in a common garden trial, pointing to selection against early-generation recombinants. Our results indicate a role for selection against recombinant genotypes in maintaining RI in the face of apparent F1 fertility, consistent with the intragenomic 'coadaptation' model of barriers to introgression upon secondary contact. Whole-genome resequencing of hybridizing populations will clarify the roles of specific genetic pathways in RI between these model forest trees and may reveal which loci are affected most strongly by its cyclic breakdown. PMID:26880192

  17. Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone.

    PubMed

    Jonsson, Nina; Sävneby, Anna; Gullberg, Maria; Evertsson, Kim; Klingel, Karin; Lindberg, A Michael

    2015-06-01

    Recombination is an important feature in the evolution of the Enterovirus genus. Phylogenetic studies of enteroviruses have revealed that the capsid genomic region (P1) is type specific, while the parts of the genome coding for the non-structural proteins (P2-P3) are species specific. Hence, the genome may be regarded as consisting of two modules that evolve independently. In this study, it was investigated whether the non-structural coding part of the genome in one type could support replication of a virus with a P1 region from another type of the same species. A cassette vector (pCas) containing a full-length cDNA copy of coxsackievirus B5 (CVB5) was used as a replicative backbone. The P1 region of pCas was replaced with the corresponding part from coxsackievirus B3 Nancy (CVB3N), coxsackievirus B6 Schmitt (CVB6S), and echovirus 7 Wallace (E7W), all members of the Enterovirus B species. The replication efficiency after transfection with clone-derived in vitro transcribed RNA was studied and compared with that of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values, tissue culture infectivity dose 50 %, and plaque-forming unit titers comparable to viruses generated from the pCas construct. In addition to this, a clone without the P1 region was also constructed, and Western Blot and immunofluorescence staining analysis showed that the viral genome could be translated and replicated despite the lack of the structural protein-coding region. To conclude, the replicative backbone of the CVB5 cassette vector supports replication of intraspecies constructs with P1 regions derived from other members of the Enterovirus B species. In addition to this, the replicative backbone can be both translated and replicated without the presence of a P1 region.

  18. Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

    PubMed Central

    Saljoughian, Noushin; Taheri, Tahereh; Zahedifard, Farnaz; Taslimi, Yasaman; Doustdari, Fatemeh; Bolhassani, Azam; Doroud, Delaram; Azizi, Hiva; Heidari, Kazem; Vasei, Mohammad; Namvar Asl, Nabiollah; Papadopoulou, Barbara; Rafati, Sima

    2013-01-01

    Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. PMID:23638195

  19. Construction, expression and immunoassay detection of recombinant plasmid encoding fusion protein of Roman chicken complement C3d and Newcastle disease virus F gene.

    PubMed

    Liu, D; Niu, Z-X

    2008-12-01

    The terminal degradation product (C3d) of mammalian complement component C3 plays an important role in modulation of the adaptive immune response through the interaction with complement receptor type 2 (CR2) on B cells. In this study, the gene fragment coding for the complement protein C3d (chC3d) from Roman chicken was cloned and expressed as a fusion protein for its application in the vaccine study of chicken, and for in vitro experiments. The chC3d fragment strengthened B-cell responses when complexed with antigen. Three potential vaccine construct units were engineered to contain two, four and six copies of chC3d coding gene linked to the F gene of Newcastle disease virus (NDV), an economically important pathogen of chicken that is classified as a list A contagious disease of poultry by the Office International des Epizooties. The cloned chC3d protein and different repeats of C3d proteins in addition to the F gene of NDV were generated separately in Escherichia coli and chicken embryo fibroblast cells with the help of expression vectors. All recombinant proteins were analysed by SDS-PAGE and Western blotting. Analysis of the immunogenicity of different repeats of C3d revealed that chC3d had an enhancing effect on the immunogenicity of antigens, and that six or more repeats of C3d may be necessary for efficient enhancement of antigen-specific immune responses. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice, rabbits and cattle. The adjuvant properties of C3d have not been assessed in poultry using homologous C3d in association with antigens relevant to the target species. The Roman chicken C3d fusion proteins described in this study is the first report and will provide a basis for immunization trials in chicken, studies of receptor binding and cell activation of chicken lymphocytes, and investigations of new types of vaccines, including recombinant vaccines and DNA vaccines for future use against other

  20. Duplicated Clock genes with unique polyglutamine domains provide evidence for nonhomologous recombination in Chinook salmon (Oncorhynchus tshawytscha).

    PubMed

    O'Malley, K G; Banks, M A

    2008-01-01

    Circadian rhythms underlie diverse life functions ranging from cellular activities to behavior. Multiple clock genes play a central role in the generation of these rhythms. We partially characterized two copies of the Clock gene from Chinook salmon (Oncorhynchus tshawytscha), OtsClock1a and OtsClock1b. The 6,460 bp OtsClock1a sequence contains 16 exons, 15 introns and encompasses three highly conserved domains indicating it is a novel member of the bHLH-PAS superfamily of transcription factors. The second copy, OtsClock1b, consists of five exons and five introns spanning 1,945 bp. A polyglutamine repeat motif (PolyQ), characteristic of a majority of CLOCK proteins, is present in both OTSCLOCK1a and OTSCLOCK1b. However, the Chinook PolyQ domains are uniquely positioned inside the gene. Interestingly, a 1,200 bp non-coding segment located downstream of the OtsClock1a PolyQ domain is absent from OtsClock1b. This insertion/deletion is 91% similar to the Salmo salar Transferrin gene. A phylogenetic analysis of 11 CLOCK proteins shows that OtsClock1a and OtsClock1b are paralogs which likely arose subsequent to the salmonid genome-wide duplication event. Ultimately, the Chinook salmon Clock genes are key components to our understanding the genetic mechanisms underlying temporally regulated life history traits in Pacific salmonids. PMID:17503191

  1. Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer

    PubMed Central

    Wong, Carmen M.; Poulin, Kathy L.; Tong, Grace; Christou, Carin; Kennedy, Michael A.; Falls, Theresa; Bell, John C.; Parks, Robin J.

    2016-01-01

    Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo. PMID:26986751

  2. Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer.

    PubMed

    Wong, Carmen M; Poulin, Kathy L; Tong, Grace; Christou, Carin; Kennedy, Michael A; Falls, Theresa; Bell, John C; Parks, Robin J

    2016-01-01

    Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo. PMID:26986751

  3. Determination of resistance spectra of the Pi-ta and Pi-k genes to US races of Magnaporthe oryzae causing rice blast in a recombinant inbred line population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resistance (R) genes to ten common races of Magnaporthe oryzae were mapped using an F10 recombinant inbred line population of a cross of a tropical japonica cultivar Katy with a breeding line RU9101001. Katy was found to confer resistance to all common races IA-45, IB-1, IB-45, IB-49, IB-54, IC-17,...

  4. Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

    PubMed Central

    Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

    2014-01-01

    Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

  5. Prolonged expression of the BX1 signature enzyme is associated with a recombination hotspot in the benzoxazinoid gene cluster in Zea mays

    PubMed Central

    Zheng, Linlin; McMullen, Michael D.; Bauer, Eva; Schön, Chris-Carolin; Gierl, Alfons; Frey, Monika

    2015-01-01

    Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription. PMID:25969552

  6. Evidence that intragenic recombination contributes to allelic diversity of the S-RNase gene at the self-incompatibility (S) locus in Petunia inflata.

    PubMed

    Wang, X; Hughes, A L; Tsukamoto, T; Ando, T; Kao, T

    2001-02-01

    For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population of Petunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5' region of S(19) allele was derived from an ancestor allele closely related to S(2), whereas its 3' region was derived from an ancestor allele closely related to S(8). A similar evolutionary relationship was found among S(3), S(12), and S(15) alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of the S(1) allele identified in this work is completely identical to that of the previously identified S(1) allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity, S(6) and S(9) alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed. PMID:11161057

  7. Prolonged expression of the BX1 signature enzyme is associated with a recombination hotspot in the benzoxazinoid gene cluster in Zea mays.

    PubMed

    Zheng, Linlin; McMullen, Michael D; Bauer, Eva; Schön, Chris-Carolin; Gierl, Alfons; Frey, Monika

    2015-07-01

    Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140 kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription.

  8. Engineering a Recombinant Baculovirus with a Peptide Hormone Gene and its Effect on the Corn Earworm, Helicoverpa zea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The helicokinins are peptides identified from Helicoverpa zea that when injected into the larvae were found to cause excessive diuresis and loss of feeding activity. Of the three peptides, helicokinin II (HezK-II) was found to be most potent. A synthetic gene encoding HezK-II was constructed based o...

  9. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants.

    PubMed

    Nada, Reham M

    2016-04-01

    Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6-9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16-17 and 14-15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley. PMID:27436915

  10. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants.

    PubMed

    Nada, Reham M

    2016-04-01

    Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6-9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16-17 and 14-15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.

  11. Recombinant plasmids carrying promoters, genes and the origin of DNA replication of the early region of bacteriophage T7.

    PubMed Central

    Scherzinger, E; Lauppe, H F; Voll, N; Wanke, M

    1980-01-01

    Two full-length contiguous HpaI fragments of the 0 to 18.2% region of T7 H DNA (HpF-H and HpG) were inserted into plasmids pHV14 or pC194 using oligo(dG . dC) connectors or synthetic HindIII adaptors. Amplification of the two early T7 fragments was achieved by transforming lysostaphin-treated S. aureus W57 with the hybrid plasmids. Experimental evidence is presented suggesting that neither of these T7 segments can be cloned in an intact form in E. coli. One of the hybrids, pHV14-HpF-H, proved to be unstable even in B. subtilis 168. The supercoiled recombinant plasmids were tested for their capacity to support RNA synthesis by purified E. coli or T7 RNA polymerases and to serve as templates in a cell-free T7 DNA replication system. The results of these in vitro studies indicate the presence of active "early" promoters in the cloned fragment HpF-H and active "late" promoters, as well as a functional origin of replication in the cloned fragment HpG. Images PMID:7433121

  12. Immunoglobulin genes and the acquisition of HIV infection in a randomized trial of recombinant adenovirus HIV vaccine.

    PubMed

    Pandey, Janardan P; Namboodiri, Aryan M; Bu, Shizhong; De Dieu Tapsoba, Jean; Sato, Alicia; Dai, James Y

    2013-06-20

    Our knowledge of the host genetic factors that contribute to the acquisition of HIV infection is limited. To identify the host genetic correlates of HIV1 acquisition, we genotyped 777 participants of a randomized trial of recombinant adenovirus HIV1 vaccine for Fcγ receptor IIa (FcγRIIa), FcγRIIIa, and several GM and KM alleles-genetic markers of immunoglobulin γ and κ chains, respectively. None of the genotypes by itself was significantly associated with the acquisition of HIV1 infection. However, particular combinations of GM and KM as well as those of GM and FcγRIIIa loci were significantly associated with the acquisition of HIV1 infection epistatically: KM1/3-GM3/17 (interaction p=0.0246; FDR=0.2952), KM1/3-GM5/21 (interaction p=0.0016; FDR=0.0960), and GM23+/-FcγRIIIa (interaction p=0.0060; FDR=0.1200). These results suggest the involvement of GM, KM, and FcγRIIIa loci in the acquisition of HIV infection. Additional studies are warranted.

  13. Recombinant Leishmania tarentolae expressing the A2 virulence gene as a novel candidate vaccine against visceral leishmaniasis.

    PubMed

    Mizbani, Amir; Taheri, Tahereh; Zahedifard, Farnaz; Taslimi, Yasaman; Azizi, Hiva; Azadmanesh, Kayhan; Papadopoulou, Barbara; Rafati, Sima

    2009-12-10

    Visceral leishmaniasis is the most severe form of leishmaniasis. To date, there is no effective vaccine against this disease. Many antigens have been examined so far as protein- or DNA-based vaccines, but none of them conferred complete long-term protection. The use of live attenuated vaccines has recently emerged as a promising vaccination strategy. In this study, we stably expressed the Leishmania donovani A2 antigen in Leishmania tarentolae, a non-pathogenic member of the genus Leishmania, and evaluated its protective efficacy as a live vaccine against L. infantum challenge. Our results show that a single intraperitoneal administration of the A2-recombinant L. tarentolae strain protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-gamma production prior and after challenge. This is accompanied by reduced levels of IL-5 production after challenge, leading to a potent Th1 immune response. In contrast, intravenous injection elicited a Th2 type response, characterized by higher levels of IL-5 and high humoral immune response, resulting in a less efficient protection. All together, these results indicate the promise of A2-expressing L. tarentolae as a safe live vaccine against visceral leishmaniasis.

  14. Contribution of RecFOR machinery of homologous recombination to cell survival after loss of a restriction-modification gene complex.

    PubMed

    Handa, Naofumi; Ichige, Asao; Kobayashi, Ichizo

    2009-07-01

    Loss of a type II restriction-modification (RM) gene complex, such as EcoRI, from a bacterial cell leads to death of its descendent cells through attack by residual restriction enzymes on undermethylated target sites of newly synthesized chromosomes. Through such post-segregational host killing, these gene complexes impose their maintenance on their host cells. This finding led to the rediscovery of type II RM systems as selfish mobile elements. The host prokaryote cells were found to cope with such attacks through a variety of means. The RecBCD pathway of homologous recombination in Escherichia coli repairs the lethal lesions on the chromosome, whilst it destroys restricted non-self DNA. recBCD homologues, however, appear very limited in distribution among bacterial genomes, whereas homologues of the RecFOR proteins, responsible for another pathway, are widespread in eubacteria, just like the RM systems. In the present work, therefore, we examined the possible contribution of the RecFOR pathway to cell survival after loss of an RM gene complex. A recF mutation reduced survival in an otherwise rec-positive background and, more severely, in a recBC sbcBC background. We also found that its effect is prominent in the presence of specific non-null mutant forms of the RecBCD enzyme: the resistance to killing seen with recC1002, recC1004, recC2145 and recB2154 is severely reduced to the level of a null recBC allele when combined with a recF, recO or recR mutant allele. Such resistance was also dependent on RecJ and RecQ functions. UV resistance of these non-null recBCD mutants is also reduced by recF, recJ or recQ mutation. These results demonstrate that the RecFOR pathway of recombination can contribute greatly to resistance to RM-mediated host killing, depending on the genetic background. PMID:19389761

  15. Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

    PubMed

    Shigeto, Hajime; Nakatsuka, Keisuke; Ikeda, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Funabashi, Hisakage

    2016-08-16

    This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner. PMID:27458920

  16. Molecular characterization of infectious bronchitis virus isolates from Russia and neighbouring countries: identification of intertypic recombination in the S1 gene.

    PubMed

    Ovchinnikova, Evgeniya V; Bochkov, Yury A; Shcherbakova, Lidiya O; Nikonova, Zoya B; Zinyakov, Nikolay G; Elatkin, Nikolay P; Mudrak, Nataliya S; Borisov, Alexander V; Drygin, Vladimir V

    2011-10-01

    Infectious bronchitis virus (IBV) isolates recovered in Russia, Ukraine, and Kazakhstan between 2007 and 2010 were subjected to molecular characterization and compared with those isolated a decade ago. The IBV genome was detected in 202 out of 605 field samples from chickens with various clinical signs. Partial sequencing of the S1 gene revealed 153 vaccine strains and 49 field isolates of several genetic groups. Massachusetts, 793/B and D274 remained the predominant IBV genotypes along with QX, whereas B1648, Italy-02, Arkansas and variants accounted for about 12% of the total number. Three IBVs contained recombinant S1 gene sequences comprising genome fragments of QX-type field isolates and vaccine strains H120 (UKR/02/2009) or 4/91 (RF/03/2010), and vaccine strains H120 and D274 (RF/01/2010). The results of the present study showed a significant decline in prevalence of variant IBVs and a further spread of QX-type isolates in commercial chicken flocks in Russia as compared with the 1998 to 2002 data.

  17. Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.

    PubMed Central

    Berka, R M; Schneider, P; Golightly, E J; Brown, S H; Madden, M; Brown, K M; Halkier, T; Mondorf, K; Xu, F

    1997-01-01

    A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase. PMID:9251203

  18. High-level expression of a novel α-galactosidase gene from Rhizomucor miehei in Pichia pastoris and characterization of the recombinant enyzme.

    PubMed

    Chen, Zhou; Yan, Qiaojuan; Jiang, Zhengqiang; Liu, Yu; Li, Yuchen

    2015-06-01

    The second α-galactosidase gene (designated as RmgalB) was cloned from the thermophilic fungus Rhizomucor miehei and expressed in Pichia pastoris. The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241bp encoding 746 amino acids with two introns. The recombinant α-galactosidase (RmgalB) was secreted at high levels of 1953.9Uml(-1) in high cell density fermentor, which is the highest yield obtained for a α-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1kDa in SDS-PAGE. The enzyme exhibited a very high specific activity of 505.5Umg(-1). The optimum temperature and pH of RmgalB were determined to be 55°C and pH 5.5, respectively. It was stable within pH 5.5-9.5 and up to 55°C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries.

  19. Treatment of Parkinson disease with C17.2 neural stem cells overexpressing NURR1 with a recombined republic-deficit adenovirus containing the NURR1 gene.

    PubMed

    Li, Qing-Jun; Tang, Ya-Mei; Liu, Jun; Zhou, Dao-You; Li, Xiang-Pen; Xiao, Song-Hua; Jian, Dong-Xing; Xing, Yi-Gang

    2007-12-01

    To study the potential benefit of the NURR1 gene in Parkinson's disease (PD), we constructed a recombinant republic-deficit adenovirus containing the NURR1 gene (Ad-NURR1) and expressed it in transplanted neural stem cells (NSC). Ad-NURR1 was constructed, and NURR1 mRNA and protein expression were identified by in situ hybridization and western blot analysis, respectively. The identified NURR1 protein could directly or indirectly induce NSC differentiation into neurons. To identify a potential therapeutic use for the transfected NSCs, cells were transplanted into 6-hydroxydopamine lesioned rats. Histopathological and behavioral alterations were evaluated via immunohistochemistry and the ration test, respectively, in rats transplanted with NSCs with or without the Ad-NURR1 adenovirus. The Ad-NURR1 construct effectively expressed the NURR1 protein, which could directly or indirectly induce NSC differentiation into neurons. Both histopathological and behavioral alterations were seen in rats treated with NSCs with or without the Ad-NURR1 construct, although in the case of the latter, the benefits were more robust. These results suggest a potential therapeutic benefit for Ad-NURR1-expressing cells in the treatment of PD. The Ad-NURR1 modification induced NSC differentiation and therefore represents a potential therapy for PD.

  20. A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D-- phenotype.

    PubMed Central

    Kemp, T. J.; Poulter, M.; Carritt, B.

    1996-01-01

    We have studied the arrangement of Rh (rhesus) genes in donors who are completely null for the products of one of them, RHCE. We show that five of six homozygous individuals with the so-called Rh D-- phenotype, who express no red-cell antigens of the C/c and E/e series, have rearranged RHCE genes in which internal sequences have been replaced by the corresponding sequences from RHD. Moreover, although there is heterogeneity at the 3' end, the 5' boundary of this chimerism is within the same small interval around exon 2. This interval is characterized by an exceptionally high degree of sequence homology between RHCE and RHD, a high density of dispersed repetitive elements, and the presence of an alternating purine-pyrimidine copolymer tract. We suggest that these features may explain the mechanistic basis for the origin of the rearrangement. Images Figure 2 Figure 3 Figure 4 PMID:8900235