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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Mechanism of action of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. II. A resistant human cell mutant with an altered transcriptional machinery.

To determine the role of DRB in transcription, we isolated a resistant (DRBR) HeLa cell mutant. After mutagenesis with N-methyl-N'-nitro-nitrosoguanidine, cell colonies able to grow at 20 micrograms DRB/ml (63 microM) were selected. One of these colonies, DRBR-1, was stable and able to grow at concentrations of DRB three to five times higher than tolerated by normal HeLa cells. The DNA of DRBR-1 was able to confer resistance to DRB to other HeLa cells by transfection. Uridine uptake was reduced by DRB to a similar extent in both wild-type and mutant cells. In contrast, transcription in the mutant cells, as measured by [3H]uridine incorporation into RNA in short pulses, was resistant to DRB. Cell-free extracts prepared from DRBR-1 cells are able to transcribe the epsilon-globin or the adenovirus 2 major late promoter genes at DRB concentrations that eliminate the transcriptional activity of HeLa cell extracts. Thus the transcriptional machinery of the mutant is altered. The presence of both DRB-resistant and DRB-sensitive transcriptional activities in extracts from DRBR-1 cells, grown in the presence of the drug, suggests constitutive expression of this cellular component. Efficient somatic cell hybridization with an alpha-amanitin-resistant RNA polymerase II mouse mutant indicates cross-complementation in vivo. This DRBR mutant provides a useful tool for the biochemical analysis of the mechanism of action of DRB on transcription. It also serves as a genetic handle for selection of the gene responsible for DRB resistance.[1]


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