Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple selection system has been developed for the cloning and expression of open reading frames in vaccinia virus. The selection system is based on a conditional lethal (host range) mutant of vaccinia virus. A deletion mutant of the vaccinia virus WR strain was generated by insertion of the neomycin resistance gene from transposon Tn5 and selection with the antibiotic G418. This deletion recombinant, vP293, lacked approximately 21.7 kilobases of DNA beginning 3.8 kilobases from the left end of the genome, vP293, was capable of plaquing on primary chicken embryo fibroblasts and two monkey cell lines (BSC-40 and Vero) but was defective in replication in the human cell line MRC-5. Insertion of the host range gene K1L into vP293 restored the ability to grow on MRC-5 cells. A series of plasmids were constructed which in addition to the K1L gene contained a vaccinia virus early-late promoter, H6, followed by a unique polylinker sequence, translational initiation and termination signals, and an early transcription termination signal. These plasmids, pHES1 through 4, allowed for rapid single-step cloning and expression of any open reading frame when recombined in vivo with vP293 and scored for growth on MRC-5 cells.
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PMID:Cloning and expression of foreign genes in vaccinia virus, using a host range selection system. 254 99

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
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PMID:Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants. 301 37

Recombinant vaccinia virus is a useful and powerful tool for the expression and study of foreign genes. Methods that are currently available for the selection of vaccinia virus recombinants include the restoration of viral plaque-forming phenotype, the replication of viral DNA in the presence of BUdR or mycophenolic acid, and the maturation and propagation of virus under antibiotic selection. Though effective, each of these methods requires several weeks of concerted effort to isolate, purify, and amplify a potential recombinant virus. Here we report the development of a bifunctional enzyme (BiZyme) to simplify and expedite the isolation and purification of vaccinia virus recombinants. This novel selection marker is composed of an in-frame fusion between the genes encoding gfp and the neomycin phosphotransferase enzyme (neo). Remarkably, expression of the chimeric gfp-neo cassette in the presence of G418 confers both viability and fluorescence to transfected or recombinant virus-infected cells, indicating that both activities are retained within the fusion protein. Therfore, BiZyme was incorporated into a recombination plasmid (pGNR) to enable the concomitant insertion of a foreign gene of interest. Here we demonstrate that this selection/amplification process requires a minimum of 11 days to produce the desired vaccinia virus recombinants. Furthermore, recombinants produced in this fashion have been shown to express both biologically active enzymes and antigenically authenticforeign antigens. In addition to its use in the vaccinia virus vector system, the BiZyme bifunctional selection scheme should be applicable to other eukaryotic and prokaryotic expression systems, simply by coupling it to the appropriate host-specific transcription regulatory signals.
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PMID:BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance. 1201 92