The nematode, Caenorhabditis elegans, is currently utilized as a laboratory model for a variety of biomedical research applications, including the study of metazoan development. The enzyme RNA polymerase II, which is responsible for the synthesis of messenger RNA, plays such a central role in cellular activity that its expression, control and activity must be examined if the genetic and cellular mechanisms that underlie development and aging are to be understood. The goal of the genetic work is to characterize genes encoding subunits of the enzyme, select lethal and conditional-lethal alleles of these genes, and determine the effect of such mutations on normal development and on the expression of selected mutant phenotypes. Two genes already have been identified by selection of mutants resistant to the RNA polymerase II inhibitor, alpha-amanitin(amanitin). One dominant mutant, ama-1(m118)IV, produces an RNA polymerase II that is 100-fold more resistant to the toxin in vitro than is the wild-type enzyme. Six lethal alleles of ama-1 have been characterized. Additional lethal alleles will be isolated and a fine-structure genetic map of the ama-1 gene will be generated. Revertants of lethal alleles will be selected as a means to obtain additional variants with altered activity, and as a means to identify suppressor mutations that may define structural genes for other RNA polymerase subunits, transcription factors, or for regulatory elements controlling the level of RNA polymerase synthesis. The in vitro activity and amanitin sensitivity of RNA polymerase isolated from strains carrying mutations in ama-2 and let-276 (two genes that influence sensitivity to amanitin) will be determined. Also, the properties of RNA polymerase extracted from dauer larvae (a non-feeding, developmentally arrested dispersal stage formed in response to starvation or overcrowding) will be examined, since this unique larval stage does not appear to contain significant amounts of mRNA that can be translated in vitro. The DNA sequence of the entire ama-1 gene will be determined. Coding regions and introns will be defined, and promoter structure determined. Mutational alterations in certain ama-1 mutants will be characterized to correlate the DNA sequence with the fine-structure genetic map. The size and amount of mRNA complementary to the ama-1 gene will be determined. ama-1 mRNA from revertants of ama-1 lethal and conditional-lethal alleles will be quantitated to detect any promoter mutants with abnormally high levels of ama-1 mRNA.
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