Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II.
Cultures of the rat skeletal muscle myoblast cell line, L6, were treated with the mutagen ethylmethanesulfonate and grown in the presence of alpha-amanitin, an inhibitor of RNA polymerase II in vitro. One clonal cell line, Ama102, resistant tc the cytotoxic action of 2 mu-g/ml of alpha-amanitin was isolated and extensively characterized. Ama102 cells were about 30-fold more resistant to alpha-amanitin than their Ama+ parent cells based on a comparison of the concentration of alpha-amanitin required to reduce their plating efficiencies to similar extents. The RNA polymerase activities from Ama+ and Ama102 cells were solubilized and separated by DEAE-Sephadex chromatography. Whereas all of the Ama+ RNA polymerase II activity was inhibited by 0.1 mu-g/ml of alpha-amanitin, about 30% of the activity in the Ama102 RNA polymerase II peak was resistant to this concentration of alpha-amanitin and was inhibited only by much higher concentrations (25 mu-g/ml) of alpha-amanitin. This alpha-amanitin-resistant activity in Ama102 cells was identified as a bona fide RNA polymerase II by its chromatographic behavior on DEAE-Sephadex, salt optimum, preference for denatured DNA as template, insensitivity to inhibition by potassium phosphate, thermal inactivation kinetics, and inactivation by anti-RNA polymerase II antiserum. Both RNA polymerase IIa and IIb from Ama102 cells exhibited the partial alpha-amanitin resistance, as did this activity when purified further on phosphocellusose. Unlike the parental Ama+ cells, Ama102 cells neither fused at confluence nor showed an increase in the specific activity of creatine kinase. The altered sensitivity of the Ama102 RNA polymerase II to alpha-amanitin appears to account for the drug-resistant phenotype of these cells.
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004351:Drug Resistance; D007106:Immune Sera; D007328:Insulin / Q000494:pharmacology; D009154:Mutation; D009379:Neoplasms, Muscle Tissue / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D010710:Phosphates / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D013696:Temperature; D013950:Thymus Gland / Q000201:enzymology; D014500:Uracil Nucleotides / Q000378:metabolism
D000546:Amanitins; D007106:Immune Sera; D007328:Insulin; D010710:Phosphates; D014500:Uracil Nucleotides; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases