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)

Diphtheria toxin (DT), a bacterial protein exotoxin, inactivates mammalian cell elongation factor 2 after toxin internalization by receptor-mediated endocytosis. To isolate the DT receptor, we cotransfected DT-resistant wild-type mouse L-M cells with a cDNA library constructed from RNA of highly toxin-sensitive monkey Vero cells and with a neomycin-resistance gene. Stably transfected G418-resistant L-M colonies were screened for DT sensitivity in a replica plate assay. After screening of 8000 colonies, one DT-sensitive (DTS) colony was isolated. The purified DTS mouse cells are highly toxin-sensitive; they are at least 1000-fold more sensitive than wild-type L-M cells and only approximately 10-fold less sensitive than Vero cells. Incubation of the DTS mouse cells with CRM 197, a nontoxic form of DT that competitively inhibits the binding of native DT to the toxin receptor, protected them from DT-mediated toxicity. More important, these DTS mouse cells express receptors on their cell surface that bind radioiodinated DT in a specific fashion, a property hitherto readily demonstrable only with highly toxin-sensitive cells of monkey origin. Furthermore, HA6DT, a DT fragment comprising the Mr 6000 carboxyl-terminal receptor-binding domain, inhibited the binding of radioiodinated toxin to these DTS mouse cells to the same extent as unlabeled DT. With these DTS mouse cells as a source of monkey cDNA, it should be possible to clone the gene encoding the DT receptor.
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PMID:Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin. 154 77

In attempting to produce a mutant mouse with embryonic stem cells, the critical step is the efficient isolation of homologous recombinants; the frequency of the homologous recombination is usually low and the potency of the cells to differentiate into germ cells is unstable in culture. Here, we report an efficacious method for such isolation in which the diphtheria toxin A-fragment gene is used to negatively select nonhomologous recombinants. In contrast to the use of the herpes simplex virus thymidine kinase gene, the selection can be made singly by the neomycin analog G418 without using a drug such as ganciclovir, a nucleoside analog. At the c-fyn locus, the diphtheria-toxin negative selection enriched the recombinants about 10-fold, and half of the cells integrating with the neomycin phosphotransferase gene were homologous recombinants.
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PMID:Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection. 226 43

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neor-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.
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PMID:Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids. 343 21

As an alternative to directing plant or bacterial toxins to surface receptors, we are investigating the possibility of killing tumor cells by the expression of an exogenously introduced toxin gene (i.e., cell suicide). Tissue-specific gene regulatory elements might thus be exploited to achieve selective killing. To assess the feasibility of such an approach, we have transfected human cells (HeLa, B-lymphoblastoid, and 293 cells) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. The presence of the DT-A sequence lowered the level of transient expression of chloramphenicol acetyltransferase from a cotransfected plasmid, pSV2cat. This expression level in B-cells was further diminished by the inclusion of an immunoglobulin enhancer in the DT-A plasmid. In cotransfection experiments with a DT-A plasmid lacking an enhancer, chloramphenicol acetyltransferase expression was much more strongly inhibited in 293 cells (which express adenovirus E1A and E1B products) than in the other cell types; furthermore, the presence of the DT-A sequence eliminated recovery of G418-resistant 293 cell transformants after transfection with a plasmid containing the neo selectable marker. These results suggest that cell-specific regulatory mechanisms can be exploited to achieve selective cell killing by expression of an introduced toxin gene.
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PMID:Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. 346 Jun 97

In producing mutant mice by gene targeting in embryonic stem (ES) cells, the efficient isolation of the homologous recombinants is still a critical step. We previously reported on a negative selection using the diphtheria toxin A (DT-A) fragment gene for homologous recombinants (1). It was efficient but limited to gene loci expressed in ES cells. For wider applicability of this negative selection to many gene loci not expressed or expressed at low levels in ES cells, we exploited a novel targeting vector composed of a polyA-less neo gene, a mRNA destabilizing signal, a pausing signal for RNA polymerase II from the minute virus of mice, and the DT-A gene. There was about a 30-fold decrease in frequency of G418-resistant colonies with this strategy against that using only the neo gene in the vector, and homologous recombinants were obtained at frequencies of more than 1/50 among G418 resistant cells at fyn, csk, c-mos, and insulin receptor substrate-1 gene loci.
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PMID:A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. 825 Feb 58