RNA polymerase II from mutant and wild type strains of Caenorhabditis elegans
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Studies of transcription in Caenorhabditis elegans, a model organism for the study of developmental genetics, should contribute to an understanding of the developmental process in metazoans. This research describes preliminary characterization of RNA polymerase II, the enzyme that transcribes messenger RNA. RNA polymerase I, II, and III from C. elegans were isolated, and their sensitivities to the fungal toxin O-amanitin measured. Sensitivities of these enzymes to amanitin were similar to those of the coresponding RNA polymerases from vertebrates. RNA polymerase II from the nematode was 50% inhibited by 7 [mu]g/ml of the amatoxin and RNA polymerase III by 80 [mu]g/ml, whereas RNA polymerase I was insensitive to 500 [mu]g/ml of the toxin. Mutants of C. elegans were isolated which can grow and reproduce in concentrations of amanitin which arrest development of wild type worms. One of these mutant strains (DR432) was shown to contain an altered RNA polymerase II which when purified was 150 times less sensitive to the amatoxin than wild type enzyme. The mutation in DR432, ama-l(ml30), is dominant and located on linkage group IV. RNA polymerase II isolated from ama-l/+ heterozygotes contains equal proportions of two components, corresponding in amanitin sensitivity to the enzymes from DR432 and wild type. Thus, ama-1 appears to affect a subunit of RNA polymerase II. A procedure was desinged for obtaining highly purified RNA polymerase II from C. elegans. The structure of the enzyme was examined by denaturing gel electrophoresis and found to consist of two large subunits ([greater than] 100 kd), and eight smaller subunits ([less than] 50 kd). The structure of the nematode RNA polymerase II closely resembles that of the corresponding enzyme from other animals. Polyclonal antibodies against C, elegans and Drosophila RNA polymerase II were shown to bind to several subunits of the C. elegans RNA polymerase II in protein blots.
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