Worm Breeder's Gazette 7(1): 77
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 fungal toxin, alpha-amanitin is an effective inhibitor of C. elegans RNA polymerase II. Larvae hatched from eggs into medium containing 20 g/ml amanitin do not grow. Larvae placed in fresh medium after 4 days incubation in toxin are unable to resume development and eventually die. In vitro, partially purified RNA polymerase II is 50% inhibited by 10 Ng/ml amanitin. We have isolated seven EMS-induced mutants resistant to amanitin. Preliminary genetic crosses indicate that all the mutations are closely linked to dpy-13 IV, and at least some mutants are semi-dominant. Partial purification of RNA polymerase involves sonication of the worms, ammonium sulfate fractionation of the extract, and heparin sepharose and DEAE-cellulose chromatography. Assays consist of 20- minute DNA-dependent incorporations of [3H]-UTP into acid-precipitable material. The amount of in vitro activity is comparable to that found in Drosophila or in mammalian cells. The figure below shows the results of assays from fractions of a DEAE-cellulose column run on a wild-type extract. Polymerase activity is eluted in the salt gradient in two peaks. Open circles show the results of assays performed in the presence of amanitin, while the filled circles represent assays done without added toxin. The first peak contains amanitin-resistant activity, presumably representing polymerases I and III. The smaller peak represents amanitin-sensitive activity, and is therefore polymerase II. The chromatographic behavior and stability of the enzyme seem quite similar to the mammalian enzyme in all respects. Assays on extracts from amanitin-resistant mutants are in progress. Should a polymerase gene be identified, conditional-lethal alleles will be sought, and biochemical studies will be pursued so that functional comparisons can be made between normal and mutant enzymes. [See Figure 1]