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T. F. Meyer - One of the best experts on this subject based on the ideXlab platform.
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Construction of Hermes shuttle vectors: a versatile system useful for Genetic Complementation of transformable and non-transformable
Molecular & general genetics : MGG, 1996Co-Authors: Eva-maria Kupsch, Dominique Aubel, Carol P. Gibbs, Andreas F. Kahrs, Thomas Rudel, T. F. MeyerAbstract:A versatile shuttle system has been developed for Genetic Complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, ororifd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the Genetic manipulation of pathogenicNeisseriae are described.
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construction of hermes shuttle vectors a versatile system useful for Genetic Complementation of transformable and non transformableneisseria mutants
Molecular Genetics and Genomics, 1996Co-Authors: Eva-maria Kupsch, Dominique Aubel, Carol P. Gibbs, Andreas F. Kahrs, Thomas Rudel, T. F. MeyerAbstract:A versatile shuttle system has been developed for Genetic Complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, ororifd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the Genetic manipulation of pathogenicNeisseriae are described.
Alastair G. Mcewan - One of the best experts on this subject based on the ideXlab platform.
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Phenotypic characterisation and Genetic Complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus
FEMS microbiology letters, 1995Co-Authors: Tracey C. Bonnett, Paul A. Cobine, R. Elizabeth Sockett, Alastair G. McewanAbstract:Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further Genetic Complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides.
Helen M. Blau - One of the best experts on this subject based on the ideXlab platform.
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Genetic Complementation reveals a novel regulatory role for 3′ untranslated regions in growth and differentiation
Cell, 1993Co-Authors: Farzan Rastinejad, Helen M. BlauAbstract:Abstract Differentiated skeletal muscle cells cease dividing and sustain expression of a battery of tissue-specific genes. To identify regulators of growth and differentiation, we used a Genetic Complementation approach. Following introduction of a cDNA expression library into a differentiation-defective myoblast mutant (NMU2), cDNAs were isolated that activated muscle-specific promoters. The complementing cDNAs were identified as muscle structural genes, troponin I, tropomyosin, and α-cardiac actin, and their activity was mapped to the 3′ untranslated region (3′UTR). The 3′UTRs augmented the differentiation of wild-type muscle cells. Upon expression in 10T12 fibroblasts, proliferation was suppressed, indicating that the effects of the 3′UTRs are not limited to myogenic cells. These data suggest that 3′UTRs of certain differentiation-specific RNAs are trans-acting regulators in a feedback loop that inhibits cell division and promotes differentiation.
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Genetic Complementation reveals a novel regulatory role for 3 untranslated regions in growth and differentiation
Cell, 1993Co-Authors: Farzan Rastinejad, Helen M. BlauAbstract:Abstract Differentiated skeletal muscle cells cease dividing and sustain expression of a battery of tissue-specific genes. To identify regulators of growth and differentiation, we used a Genetic Complementation approach. Following introduction of a cDNA expression library into a differentiation-defective myoblast mutant (NMU2), cDNAs were isolated that activated muscle-specific promoters. The complementing cDNAs were identified as muscle structural genes, troponin I, tropomyosin, and α-cardiac actin, and their activity was mapped to the 3′ untranslated region (3′UTR). The 3′UTRs augmented the differentiation of wild-type muscle cells. Upon expression in 10T12 fibroblasts, proliferation was suppressed, indicating that the effects of the 3′UTRs are not limited to myogenic cells. These data suggest that 3′UTRs of certain differentiation-specific RNAs are trans-acting regulators in a feedback loop that inhibits cell division and promotes differentiation.
Tahir A. Rizvi - One of the best experts on this subject based on the ideXlab platform.
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Role of a heterologous retroviral transport element in the development of Genetic Complementation assay for mouse mammary tumor virus (MMTV) replication.
Virology, 2009Co-Authors: Tahir A. Rizvi, Jahabar Ali, Pretty Susan Phillip, Akela Ghazawi, Preethi Jayanth, Farah MustafaAbstract:The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo Genetic Complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.
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Role of Mason-Pfizer Monkey Virus (MPMV) Constitutive Transport Element (CTE) in the Propagation of MPMV Vectors by Genetic Complementation Using Homologous/HeterologousenvGenes
Virology, 1996Co-Authors: Tahir A. Rizvi, Kathy A. Lew, Edwin C. Murphy, Russell D. SchmidtAbstract:Abstract To study Mason-Pfizer monkey virus (MPMV) replication over a single round, virus particles were generated that contain a replication-defective vector encoding a dominant selectable marker, the hygromycin B phosphotransferase (hyg r ) gene. Genetic Complementation with a homologous MPMV envelope glycoprotein (Env-gp) or pseudotyping by several heterologous Env-gps from a variety of viruses resulted in infectious MPMV particles containing the replication-defective RNA. Recently, it has been shown that human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) Rev and Rev-responsive element (RRE) functions can be substituted in vitro by a cis -acting sequence, the constitutive transport element (CTE), from simian type D retroviruses like MPMV and simian retrovirus type 1 (SRV-1). To determine whether CTE of MPMV is necessary for MPMV nucleic acid propagation, an MPMV vector that lacked the terminally located CTE was generated. Propagation of this vector was completely abrogated in the absence of CTE, showing the importance of CTE in MPMV replication. Insertion of CTE back into the MPMV genome in the sense orientation rescued replication to wild-type levels. Slot-blot analysis of nuclear versus cytoplasmic RNA fractions revealed that most of the messages were sequestered in the nucleus of cells transfected with the CTE(−) vectors and very little was transported to the cytoplasm. To test whether HIV-1 or SIV RREs could complement CTE function, the HIV-1 or SIV RREs were inserted in the CTE(−) vectors. trans Complementation of CTE(−)RRE(+) vectors with Env- and Rev- expression plasmids rescued propagation of the CTE(−) vectors. Computer analysis predicted an RNA secondary structure in MPMV CTE analogous to the HIV-1 and SIV RREs that could form three stable stem loops, the first of which contains a site similar to the Rev-binding domain in the HIV-1 RRE. The presence of a higher-order CTE structure was analyzed by mutational analysis. We conclude that CTE is important in the replication of MPMV and affects the nucleocytoplasmic transport and/or stability of viral messages similar to the Rev/RRE regulatory system of HIV-1 and SIV.
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Propagation of SIV vectors by Genetic Complementation with a heterologous env gene.
AIDS research and human retroviruses, 1992Co-Authors: Tahir A. Rizvi, Antonito T PanganibanAbstract:In order to study SIV replication over a single round of replication virus particles were generated that contain a replication-defective vector containing a selectable marker. Genetic Complementation between an env-deficient SIV variant and plasmid that expresses the env gene of an amphotropic murine retrovirus resulted in infectious SIV particles containing the vector. These pseudotyped particles exhibited an expanded host range through the use of an alternative receptor. This system should be useful in the Genetic analysis of SIV nucleic acid replication. To determine whether the terminal cis acting components of the SIV genome might be sufficient for viral nucleic acid propagation a vector was generated which lack the internally located rev-responsive element. Propagation of this vector was reduced by at least 100-fold.
Eva-maria Kupsch - One of the best experts on this subject based on the ideXlab platform.
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Construction of Hermes shuttle vectors: a versatile system useful for Genetic Complementation of transformable and non-transformable
Molecular & general genetics : MGG, 1996Co-Authors: Eva-maria Kupsch, Dominique Aubel, Carol P. Gibbs, Andreas F. Kahrs, Thomas Rudel, T. F. MeyerAbstract:A versatile shuttle system has been developed for Genetic Complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, ororifd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the Genetic manipulation of pathogenicNeisseriae are described.
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construction of hermes shuttle vectors a versatile system useful for Genetic Complementation of transformable and non transformableneisseria mutants
Molecular Genetics and Genomics, 1996Co-Authors: Eva-maria Kupsch, Dominique Aubel, Carol P. Gibbs, Andreas F. Kahrs, Thomas Rudel, T. F. MeyerAbstract:A versatile shuttle system has been developed for Genetic Complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, ororifd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the Genetic manipulation of pathogenicNeisseriae are described.