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Results: 201 to 206 of 206

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Vaccinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters.
Blakely RD, Clark JA, Rudnick G, Amara SG
(1991) Anal Biochem 194: 302-8
MeSH Terms: Animals, Base Sequence, Carrier Proteins, Cell Membrane, Cloning, Molecular, DNA, DNA-Directed RNA Polymerases, Genetic Vectors, HeLa Cells, Humans, Kinetics, Molecular Sequence Data, Monosaccharide Transport Proteins, Rabbits, Transfection, Vaccinia virus, Viral Proteins
Show Abstract · Added July 10, 2013
The vaccinia/T7 transient expression system, which results in rapid, high-level expression of proteins encoded by plasmids bearing T7 promoters, provides a powerful strategy for the expression cloning of membrane transporters. To test the feasibility of this approach, we introduced the rabbit Na+/glucose transporter by liposome-mediated transfection into vaccinia infected HeLa cells and determined the characteristics and sensitivity of induced [14C]alpha-methyl D-glucopyranoside uptake. We observed a rapid (4-12 h) expression of saturable (Kt = 342 microM) [14C]alpha-methyl D-glucopyranoside uptake following transfection, with substrate and inhibitor sensitivities of the native carrier, including Na+ and temperature dependence and appropriate phloridzin sensitivity (KI = 9.1 microM). The time-dependent increase in alpha-methyl D-glucopyranoside uptake coincided with a decline in endogenous Na+/D-aspartate transport. Maximal levels of expression achieved were nearly 10-fold higher than that reported for transient expression of Na+/glucose transporters in the COS cell system. Rate and dilution estimates demonstrates a sensitivity of detection of single clones diluted several thousand fold by nonspecific plasmid DNA. A further 3-fold increase in transport sensitivity was achieved after transfection of plasmid constructs bearing additional 5'-T7 stem-loop and 3'-T7 termination signals. When cell lines with low endogenous transport were coupled with substrates of high specific activity, as with measurements of induced [3H]gamma-aminobutyric acid uptake, we were able to detect expression from transporter bearing plasmids diluted as much as 10,000-fold by non-specific plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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17 MeSH Terms
Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells.
Reddy S, DeGregori JV, von Melchner H, Ruley HE
(1991) J Virol 65: 1507-15
MeSH Terms: Animals, Blotting, Northern, Blotting, Southern, Cell Line, Clone Cells, Escherichia coli, Genetic Vectors, Methionine, Mice, Moloney murine leukemia virus, Poly A, Promoter Regions, Genetic, RNA, RNA, Messenger, Recombinant Fusion Proteins, Repetitive Sequences, Nucleic Acid, Restriction Mapping, Transcription, Genetic, Transfection, beta-Galactosidase
Show Abstract · Added May 29, 2014
A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus. The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter. Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting. In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA. Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth-arrested and log phase cells, respectively.
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20 MeSH Terms
Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding.
Wada A, Waterman MR
(1992) J Biol Chem 267: 22877-82
MeSH Terms: Adrenodoxin, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cattle, Cholesterol Side-Chain Cleavage Enzyme, Escherichia coli, Genetic Vectors, Kinetics, Lysine, Molecular Sequence Data, Molecular Weight, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Plasmids, Rabbits, Rats, Recombinant Proteins, Restriction Mapping, Sequence Homology, Amino Acid, Spectrophotometry
Show Abstract · Added February 12, 2015
Utilizing site-directed mutagenesis and an Escherichia coli expression system for bovine cholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are found to play crucial roles in binding bovine adrenodoxin, required for transfer of electrons to mitochondrial P450s. These lysine residues are conserved among mitochondrial P450s and have been implicated previously by chemical modification studies as being important for adrenodoxin binding. In the present study, site-directed mutagenesis producing either neutral or positive amino acids at 377 or 381 has no effect on the structure of side chain cleavage cytochrome P450 as determined spectrally or on the enzymatic conversion of cholesterol to pregnenolone. However, the estimated Ks of adrenodoxin binding is increased approximately 150-600-fold depending on the particular mutation. Therefore these conserved positively charged residues in mitochondrial P450s are the key sites for adrenodoxin binding which is electrostatic in nature.
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22 MeSH Terms
Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector.
Benamira M, Singh U, Marnett LJ
(1992) J Biol Chem 267: 22392-400
MeSH Terms: Bacteriophage M13, Base Sequence, Chromatography, High Pressure Liquid, DNA Damage, DNA, Bacterial, Deoxyguanosine, Frameshift Mutation, Genetic Vectors, Histidine, Magnetic Resonance Spectroscopy, Malondialdehyde, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Proprotein Convertase 2, Salmonella typhimurium, Serine Endopeptidases
Show Abstract · Added March 5, 2014
Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.
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17 MeSH Terms
Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1.
Sorrentino BP, Brandt SJ, Bodine D, Gottesman M, Pastan I, Cline A, Nienhuis AW
(1992) Science 257: 99-103
MeSH Terms: Alkaloids, Antineoplastic Agents, Phytogenic, Base Sequence, Bone Marrow, Bone Marrow Transplantation, DNA, Drug Resistance, Erythrocytes, Genetic Vectors, Hematopoietic Stem Cells, Humans, Molecular Sequence Data, Oligodeoxyribonucleotides, Paclitaxel, Polymerase Chain Reaction, Proviruses, Retroviridae, Transfection
Show Abstract · Added March 5, 2014
Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo. When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed. This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy.
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18 MeSH Terms
Selective disruption of genes expressed in totipotent embryonal stem cells.
von Melchner H, DeGregori JV, Rayburn H, Reddy S, Friedel C, Ruley HE
(1992) Genes Dev 6: 919-27
MeSH Terms: Alcohol Oxidoreductases, Animals, Base Sequence, Blotting, Northern, Cell Line, Chimera, Female, Genetic Vectors, Kanamycin Kinase, Male, Mice, Molecular Sequence Data, Mutagenesis, Insertional, Phosphotransferases, Promoter Regions, Genetic, Recombinant Fusion Proteins, Stem Cells
Show Abstract · Added May 29, 2014
Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.
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17 MeSH Terms