Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtii

Abstract

The inhibitory activities of amatoxins on the growth of Chiamydomonas 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.) of 5.4, 6.6, and 5.6 micromolar were obtained for ci-amanitin, O-methyl-a-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 a-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID.0 values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar a-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 a-amanitin and that this amatoxin may be used to select for RNA polymerase mutants.Significant progress has recently been made with respect to our understanding of the structures and functions of eucaryotic DNA-dependent RNA polymerases (1, 4). Three classes of enzyme, i.e. I, II, and III, have been defined for the respective transcription of genes coding for ribosomal, messenger, and tRNA. While a number of properties have served to distinguish each class, the most definitive now being subunit structures and amino acid and/or DNA sequences, the unique sensitivity of the RNA polymerase II to inhibition by the fungal peptide AMA2 has been an important factor assisting in the purification and characterization of this enzyme from a number of sources. The relative specificity of AMA for the inhibition of transcription of mRNA in animals and plants appears to 2 Abbreviations: AMA, a-amanitin; meAMA, 6'0-methyl-a-amanitin; TAP, tris-acetate-phosphate medium; PMSF, phenylmethylsulfonyl fluoride; BME, 8-mercaptoethanol; MNNG, N-methyl-N'-ditro-N-nitrosoguanidine; deAMA, 6'-deoxy-a-amanitin or amaninamide; ID50, inhibitory dosage at which growth is 50% of untreated culture; IC-50, inhibitory concentration at which 50% of the enzyme activity remains;WT, wild type.RNA polymerase II (KD of 10-9-10-8M). This specificity has been of particular value in the selection and isolation of RNA polymerase II mutants as amanitin-resistant mutants and has permitted the identification and/or cloning and sequencing of RNA polymerase II genes from several animal sources (1).Plant systems have not been amenable to the use of amatoxins for the selection of RNA polymerase II mutants, although all of the RNA polymerase II activities which have been purified from plants show a sensitivity to inhibition by a-amanitin similar to the enzyme from animal sources (3, 6, 12). Both carrot (15,19...