Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtii

The inhibitory activities of amatoxins on the growth of Chlamydomonas reinhardtii have been determined using a convenient assay based upon incubation in multiwell tissue culture plates followed by turbidimetric estimates of growth on a multiwell plate reader. Values for the inhibitory dosage at which growth is 50% of untreated culture (ID(50)) of 5.4, 6.6, and 5.6 micromolar were obtained for alpha-amanitin, O-methyl-alpha-amanitin, and amaninamide, respectively. Treatment of liquid cultures with 1 microgram per milliliter N-methyl-N' -nitro-N-nitrosoguanidine followed by growth in agar pour tubes containing 25 micromolar alpha-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID(50) values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar alpha-amanitin provided partially purified RNA polymerase activities that were 160-fold and 5600-fold more resistant to inhibition than the analogous enzyme activity from the wild-type strain. These studies provide evidence that Chlamydomonas reinhardtii does not contain significant activity capable of inactivating alpha-amanitin and that this amatoxin may be used to select for RNA polymerase mutants.