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)

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.
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PMID:Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. 230 58

In this study, we compared the relative abilities of human thymidylate synthase (hTS) and Escherichia coli thymidylate synthase (eTS) expression to confer resistance to the cytotoxic effects of treatment with the TS inhibitor 5-fluorodeoxyuridine (FdUrd). G418-selected clones expressing either form of the protein were significantly more resistant than the lacZ-expressing clone, VALZ2, to FdUrd-induced cytotoxicity. Although eTS-expressing clones expressed 2- to 3-fold more TS protein than hTS-overexpressing clones, the representative eTS-expressing clone, VAEG8, and hTS-overexpressing clone, VAHGC, were equally sensitive to an FdUrd-induced loss of clonogenicity; in addition, a large fraction of either form of exogenously expressed TS appeared to be inactive in the intact cell. The clones differed, however, in their responses to leucovorin (LV). Although LV significantly enhanced FdUrd-induced TS inhibition, growth inhibition, and cytotoxicity in VAHGC cells, it had no effect on these parameters in VAEG8 cells. These results suggest that eTS may more efficiently confer resistance to FdUrd plus LV when expressed for the purposes of a "host protection" strategy in vivo.
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PMID:Escherichia coli thymidylate synthase expression protects human cells from the cytotoxic effects of 5-fluorodeoxyuridine more effectively than human thymidylate synthase overexpression. 1097 79