Worm Breeder's Gazette 10(3): 84
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The ama-1 gene encodes the largest subunit of RNA polymerase II, a protein of approximately 200,000 MW. Rare, dominant mutations in the ama-1 gene result in an RNA polymerase II that is more resistant to alpha-amanitin than the wild-type enzyme (Sanford, Golomb and Riddle, J. Biol. Chem. 258:12804-12809,1983; Rogalski, Bullerjahn and Riddle, Genetics 120:409-422, 1988). Although these mutations decrease the amanitin sensitivity of RNA polymerase II, presumably by affecting the site where amanitin binds, they do not eliminate it completely. For example, RNA polymerase II from the resistant ama-1( m118) strain requires 1.0 g/ml amanitin for 50% inhibition in vitro compared to 7 ng/ml for the wild-type enzyme (Sanford, Golomb and Riddle, op. cit.). To determine whether it was possible to obtain a completely resistant enzyme, we devised a selection for a super-resistant derivative of the resistant ama-1(m118) parent. Resistant hermaphrodites are unaffected in vivo by amanitin concentrations up to and including 800 g/ml. However, the addition of 0.5% Triton X-100 to the growth medium increases the sensitivity of the ama-1(m118) strain, such that these animals are unable to grow at a concentration of about 80 g/ml amanitin. This has allowed us to select for mutants that will grow under conditions where ama-1(m118) hermaphrodites will not. One mutant has been found among 4x10+E6 F1 progeny of EMS- mutagenized worms. In contrast to the resistant parent, which exhibits near normal growth and fertility, the super-resistant mutant develops slowly, has a much reduced brood size at 20 C, and is sterile at 25 C. The initial genetic analysis indicated that this new mutation, m526, was closely linked to ama-1, and further 3-factor mapping placed it at the ama-1 locus. Strains carrying various combinations of the wild-type (+), resistance (R) and super-resistance (SR) ama-1 alleles have been tested for their level of amanitin resistance, and the genotypes are listed below in order of increasing resistance. +/+ < +/SR < +/R < R/R < R/SR < SR/SR These data show that resistance is dominant to wild-type as in other organisms. Surprisingly, the +/SR worms are much less resistant than the +/R worms. In fact, +/SR hermaphrodites are only marginally more resistant than +/+ animals, suggesting that the super-resistant subunit may not function well in the presence of the wild-type subunit. Preliminary results indicate that RNA polymerase II from the ama-1( m118m526) strain is more resistant to amanitin in vitro than RNA polymerase III, which is 10,000-fold more resistant than the wild-type polII and 100-fold more resistant than polII from ama-1(m118) homozygotes (M. Golomb, this issue). Thus, in the super-resistant strain amanitin sensitivity is presumably due to inhibition of RNA polymerase III. We plan to select for amanitin-resistant polIII mutants in the super-resistant polII genetic background. To aid in finding the m526 site in the 8.7 kb ama-1 gene, we have attempted to position m526 relative to m118 on the ama-1 fine- structure map (Bullerjahn and Riddle, Genetics 120:423-434, 1988). One putative recombinant has been obtained and is currently being analyzed. The data thus far suggest that m526 is very close to m118, so we are cloning and sequencing this region of the super-resistant allele. [See Figure 1]