RNA polymerase III (Rpo m) transcribes genes encoding SS and transfer RNA's. This enzyme and its associated factors have been studied extensively in other systems, but Rpo m function has not been studied genetically in a metazoan. Developmental regulation of Rpo m activity in C. elegans has been suggested by the molecular genetic analysis of nonsense suppressors (Kondo et al., J. Mol. Biol. 215:7-19, 1990). The gene for the largest subunit of Rpo m, rpc-l, has been cloned (Bird and Riddle, Mol. Cell. Biol. 9:4119-4130, 1989) and positioned on the physical map between unc44 and unc-24 on chromosome IV. The availability of cloned genes and an in vitro transcription system (Honda et al., Nucl. Acid Res. 14B:869-881, 1986) provides potential tools for a detailed analysis of Rpo III structure and function using mutants, if appropriate mutants can be identified. Two approaches to genetic analysis of rpc-l have been employed. The first approach utilized resistance to the fungal toxin, ct-amanitin. A unique strain with an extremely amanitin-resistant Rpo II (Rogalski et al., Genetics 126:889-898, 1990) was used as a parent to select even higher levels of resistance. One such mutation was mapped on chromosome IV, and preliminary nuclear run-on assays indicate that Rpo III is abnormally resistant to amanitin, in appropriate recombinants derived from this mutant strain. The mutant Rpo III transcribes SS RNA in the presence of 200 g/ml amanitin, whereas wild-type Rpo m is inhibited by 25 g/ml. These results suggest that the resistance mutation affects Rpo III, and it may be within the rpc-l gene. A second approach to rpc-l genetics utilized a collection of EMSinduced lethal mutations generated by Denise Clark. We mapped fourteen of her lethal mutations to the interval between unc44 and unc-24, a region estimated to contain approximately 30 essential genes. These lethal mutations are now being assigned to complementation groups. Lethal derivatives of the putative amanitin-resistant rpc-l allele will also be sought, and selected lethal strains will be injected with the cloned rpc-l gene in an effort to identify the genetic correlate to rpc-l by DNA transformation. Genetic, phenotypic, and biochemical analysis of these mutants may provide a useful entree into structure- function relationships in this important enzyme.