the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID.0 ..... acterization of carrot cell mutants resistant to alpha-amanitin.
Plant Physiol. (1988) 87, 286-290
0032-0889/88/87/0286/05/$Ol .00/0
Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtiil EVIDENCE FOR RNA POLYMERASE MUTANTS Received for publication September 9, 1987 and in revised form January 18, 1988
DAVID M. DUSEK AND JAMES F. PRESTON 11I* Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611 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 derive from its high affinity for I Supported by SAES Project on Genetic Mechanisms in Plant Improvement (Project No. FLA-MCS-02183-BI) from the University of Florida Experiment Station of the Institute of Food and Agricultural Science. Journal Series No. 8379. 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) and tobacco (19) cultures are relatively resistant to inhibition by AMA as a result of amanitin-inactivating activities, and meAMA was required to select for amatoxin resistant lines. A careful examination of several amatoxin-resistant lines of Daucus carota has indicated the basis for their resistance to be other than the presence of amatoxin resistant RNA polymerase activities (16). A report conflicting with this (27) indicated AMA-resistant clones of D. carota have been isolated. With the objective of further investigating the interaction of amatoxins with plant RNA polymerases, we have initiated studies on DNA-dependent RNA polymerases from the green alga Chlamydomonas reinhardtii. A number of features recommend this organism as a model for photosynthetic eucaryotes, including (a) growth as single cells in liquid or solid media of defined composition, (b) vegetative growth as a haploid with the potential for meiosis following zygote formation, (c) extensive genetic characterization and the existence of numerous markers to facilitate gene mapping (7). Transformation systems have been developed for this organism based upon selection of resistance to aminoglycoside antibiotic inhibition (9) and arginine prototrophy (21). In this paper, we have evaluated the sensitivity of cultures of C. reinhardtii to inhibition by different amatoxins, determined the sensitivities of partially purified RNA polymerase activities to inhibition by AMA, and selected AMA-resistant lines. Two of these lines have been shown to contain AMAresistant RNA polymerase activities.
MATERIAL AND METHODS Culture Conditions. Chlamydomonas reinhardtii strain CC125, WT, mating type plus, maintained in the Duke University culture collection, was a gift from Dr. Elizabeth Harris of Duke University. All media were sterilized by autoclaving for 20 min at 1.05 kg/cm2. Incubations of all cultures were at 25°C under continuous light of approximately 3000 lux generated by 20 W softwhite fluorescent bulbs. Cells were grown on TAP medium prepared according to protocols provided by E. Harris. Plating media consisted of the above TAP medium containing 1.5% Difco bacto agar supplemented with AMA where appropriate. Liquid cultures were shaken on a New Brunswick G2 gyrotory shaker 286
AMANITIN RESISTANT LINES OF CHLAMYDOMONAS REINHARDTII
(New Brunswick Scientific Co., Inc., Edison, NJ) at 150 rpm. Growth curves were determined by measuring the absorbance in a Klett-Summerson colorimeter using filter No. 66 or on a Dynatech Minireader II plate reader (Dynatech Laboratories, Inc., Alexandria, VA) at 630 nm. Cells to be fractionated for RNA polymerase isolation were collected as a pellet in a Delaval cream separator at 4°C, washed one time with distilled water followed by centrifugation, and stored at - 70°C. Chemicals and Reagents. All reagents were ACS reagent grade. Glass distilled deionized water was used for preparation of solutions. RNase-free sucrose and [5-3H]UTP (21 Ci/mmol) were from Schwarz/Mann (now with ICN Biomedical, Inc., Costa Mesa, CA). Calf thymus native DNA, BSA recrystallized two times, PMSF, and MNNG were purchased from Sigma Chemical Co., St. Louis, MO. Resins for chromatography were from Pharmacia (DEAE Sephadex A25, Pharmacia, Inc., Piscataway, NJ) and Whatman (DE52, Whatman Ltd, Kent, England). Polymin P was a gift from BASF (Rhein, Germany). Buffers for isolation of RNA were: buffer A containing (0.05 M Tris-HCl [pH 7.9], 4°C; 0.25 M sucrose; 25 mM BME; 1.0 mM PMSF; 1.0 mM EDTA; 0.5 mM MgCl2); buffer B (0.05 M Tris-HCl [pH 7.9], 1.0 mM EDTA, 5.0 mM BME, 1.0 mM PMSF); buffer C (0.05 M TrisHCl [pH 7.9], 1.0 mm EDTA, 15 mm BME, 25% v/v glycerol). BME was added just before use to minimize oxidation of the thiol by the DMSO used as the solvent in the stock 0.1 M PMSF solutions. Polymin P was prepared as a 10% solution as described (10). Inhibitors. AMA was a preparation from our laboratory and was purified as previously described (14). meAMA was synthesized according to Little et al. (17). deAMA was purified from methanolic extracts of Amanita virosa as described (15). Toxins were quantified by measuring absorbance at 304 nm for AMA and meAMA, using a molar absorptivity of 15,400. deAMA was measured at 278 nm using a molar absorptivity of 12,500 (28). Cycloheximide was obtained from Sigma. Aqueous solutions of inhibitors were sterilized by filtration through MILLEX-GV 0.2 ,.m filters (Millipore Corp., Bedford, MA). Quantification of Growth and Inhibition. Log phase cultures were diluted in sterile TAP medium to approximately 104 to 105 cells/ml, and 190 ,ul aliquots were dispensed aseptically into each of 96 wells in sterile, flat-bottomed Corning tissue culture plates (Coming Glassworks, Corning, NY). To each well was added 10 ,1 of the inhibitor dissolved in TAP. Controls of TAP medium alone and sterile TAP medium without cells were included in each assay. Determinations at each inhibitor concentration were made in triplicate and the wild-type culture was included as a positive control with each analysis of an amatoxin-resistant line. Turbidity (A) was measured on a Dynatech multiplate reader with a 630 filter and was essentially 0.0 for all cultures at the beginning of each analysis. Measurements of A630 for estimating extent of growth were usually taken at 24 to 72 h following incubation at 25°C and 3000 lux with shaking. The tissue culture plates with plastic covers were wrapped with Parafilm (American Can Co., Greenwich, CT) to reduce evaporation. Since each culture acted as its own control, any differences in growth rate among the various strains did not affect a comparison of strains with respect to inhibition. Percent of control (Figs. 1 and 2) was determined from the mean of triplicate samples divided by the mean of uninhibited culture multiplied by 100. Mutagenesis of Chiamydomonas reinhardtii. Log phase cells of TAP-grown cultures were mutagenized with 1 ,ug/ml MNNG in 20 mm citrate buffer (pH 5.0), for 30 min in the dark, as described by Harris et al. (8). Following washing of the cells via low speed centrifugation and resuspension in TAP, the culture was incubated at 25°C, 3000 lux overnight. Selection of AMAresistant lines was carried out in pour tubes prepared by mixing 1.0 ml of culture with 1.0 ml 2.0% molten (50°C) agar TAP
287
containing 50 uM AMA. Incubation at 25°C under constant illumination followed. From 248 resistant clones, 62 were selected from nonsurface colonies and subcultured onto TAP agar plates containing 0, 50, and 100 p.M AMA. Of the 62, 28 were found to be resistant to 100 uM AMA under these conditions. These were retested and found to retain the AMA-resistant phenotype after passage two or more times on AMA-free media. Cultures were maintained on TAP agar media with and without 100 AM AMA. Long-term maintenance of cultures was carried out vegetatively on TAP agar media under reduced light and in liquid nitrogen as described by Hwang and Hudock (11). Storage in liquid nitrogen although potentially variable was viewed as a supplement to vegetative maintenance. Cultures of the two AMAresistant clones were deposited with Dr. Elizabeth Harris at the Duke University, Chlamydomonas Genetics Cenlter, Department of Botany. Isolation, Purification, and Assay of RNA Polymerase. All procedures were performed at 0 to 4°C. Cell disruption was carried out in a French pressure cell at 840 kg/cm2 + 300 kg/cm2 (American Instrument Co., Silver Spring, MD) of a slurry prepared using 4.1 ml of 2 x buffer A per gram, wet weight, of cells. One pas§ was sufficient to produce 99% breakage as determined microscopically. Cells were centrifuged 15 min at 15,000g. The supematant was treated with 0.4% (w/v) Polymin P (10) by slow addition and mixing for 10 min. After centrifugation for 15 min at 15,000g, the pellet was weighed and resuspended in buffer B containing 0.4 M (NH4)2SO4, 1.5 ml/g in a Potter-Elvehjem homogenizer. Centrifugation at 15,000g for 15 min was followed by adjusting the supernatant solution to 60% saturated (NH4)2S04 with slow addition of saturated ammonium sulfate and stirring for 10 min. The pellet obtained by centrifugation as above was dissolved in buffer C containing 0.05 M (NH4)2SO4. This solution was dialyzed against two changes of the same buffer for 10 to 18 h or desalted on a Pharmacia PD-10 column. This preparation, designiated FR5, was applied to a DEAE-cellulose column (Whatman DE52) or DEAE Sephadex column, 2.5 x 20 cm, washed with several bed volumes of buffer C containing 0.05 M (NH4)2SO4 and then eluted with a linear gradient of 0.05 to 0.5 M (NH4)2SO4 in buffer C or step-eluted progressively with 0.05 M, 0.1 M, 0.275 M, and 0.5 M (NH4)2SO4 in buffer C. Step-gradient-eluted fractions from mutant and WT cultures were carried out concurrently on similar columns of DE52 cellulose having bed volumes of approximately 5 ml. Protein was measured via the microtiter plate method of Redinbaugh and Turley (20) modified to remove interfering substances via precipitation of protein with 6% TCA in the presence of 0.0125% sodium deoxycholate. Crystalline BSA was used as a standard. Assay for DNA-Dependent RNA Polymerase Inhibition by aAmanitin. The RNA polymerase activity was measured at 30°C under conditions similar to those used in previous studies (2, 18). The transcription mixture contained in a final volume of 100 p.1:0.1 M Tris-HCl (pH 7.8), 0.1 mM EDTA, 0.1 mm DTT, 4.0 mM thioglycerol, 3.0 mM MnCl2, 100 mm (NH4)2SO4, 12% v/v glycerol, 200 ,ug/ml BSA, 120 p.g/ml calf thymus heat-denatured DNA, 1.0 mm each CTP, GTP, and ATP, and 16 pM [3H]UTP (2 Ci/mmol). After 10 min of incubation, the reaction was stopped with the addition of excess UTP and carrier macromolecules, treated with cold 10% TCA, and further processed as previously described (18). Enzyme was preincubated for 10 min with the inhibitor in 50 ,ul volumes at 25°C, at which time 50 ,ul of the above transcription mix was added and incubated for 10 min. One unit of enzyme activity is defined as the incorporation of 1 pmol of UMP into TCA precipitable material in 10 min at 30°C. Plots of the percentage of control activity versus log of the aamanitin concentration were generated using a least squares lin-
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ear regression of data. IC50 values were then interpolated from a curve representing the best fit for those data.
RESULTS Inhibition of Cell Cultures. Figure 1 compares three amatoxins with respect to their inhibitory effect on cultures of C. reinhardtii. The three amatoxins, AMA, meAMA, and deAMA, are nearly equal with regard to the concentrations required for IDs. Growth inhibition is linearly related to amatoxin concentration over the range 0.5 to 10 pM producing an ID50 between 5.3 and 6.6 p.M. Cycloheximide, a protein synthesis inhibitor, was included as a positive control. Isolation of AMA Resistant Clones. A total of 62 clones of C.
-J
0
z
IL
0 I-
0
100
200
a- AMANITIN (juM)
reinhardtii resistant to 25 AM AMA were initially isolated following MNNG mutagenesis. Retesting on 100 p.M AMA-TAP FIG. 2. Growth response of AMA-resistant clones obtained following plates produced 28 clones demonstrating resistance to 100 p.M MNNG mutagenesis and retesting on TAP plates containing 100 /LM AMA. These 28 clones were derived from 2.3 x 108 cells, giving AMA. (U), (0), 28B; (F7), 23A; (O), 25A; (A), WT. Results a frequency for the appearance of AMA resistance of 1 x 10-7. expressed as 28C; in Figure 1 except after 71 h incubation. Control A30, was Control cultures plated in parallel did not demonstrate spontaneously resistant colonies. The resistance of the clones to AMA 0.70. was titered using the described tissue culture plate system. Titers for four AMA-resistant strains, 23A, 25A, 28B, 28C, and the WT are shown in Figure 2. Both 23A and 28B show ID50 values in excess of 200 uM, while the ID50 for 28C, 25A, and the WT strains are 200, 36, and 8 /.M, respectively. Based on ID_o values, three classes of resistance to amatoxin have been tentatively assigned: high level, greater than 200 AM; intermediate level, 200 gM; and low level, 35 pM. Fractionation of WT Cell Extracts and Isolation of RNA Polymerase Activity. Fraction 5 from WT cells was applied to either a DEAE-Sephadex A-25 column or a DE52 column and fractionated via gradient or step elution. Column fractions were assessed for RNA polymerase activity and sensitivity of this activity to 0.5 and 50 F.M AMA. DEAE-Sephadex chromatography produced two peaks of activity (not shown) eluting at 0.1 and 0.16 M (NH4)2SO4, both of which were sensitive to inhibition by 0.5 p.M AMA. The major peak, eluting at 0.1 M (NH4)2SO4, was 50% inhibited by 0.02 p.M AMA (data not shown). Later at20 40 60 tempts using DEAE-Sephadex were frustrated by low recoveries FRACTION NUMBER of polymerase activity prompting the use of DEAE-cellulose FIG. 3. Amanitin inhibition of WT RNA polymerase activities. FRchromatography. As shown in Figure 3, step elution with 5 from WT cell extracts was eluted from a DE-52 column with a step (NH4)2SO4 from DE52 produced an activity (peak I) that was gradient of (NH4)2S04 in buffer C. RNA polymerase activity (0) without insensitive to 0.5 p.M AMA but sensitive to 50 p.M. A second AMA, (A) with 0.5 AM AMA, and (LI) 50 AM AMA were assessed for activity (peak II) was sensitive to 0.5 AM AMA, with an IC50 for the eluted fraction. Protein (0) was determined using BCA reagent. AMA of 0.04 FM (Fig. 5).
0 I-
z
0 C.)
50
LIL 0
0
2.0
4.0
6.0
8.0
10.0
INHIBITOR CONCENTRATION (AM) FIG. 1. Growth response of cell suspensions to AMA, meAMA, deAMA, and cycloheximide. Results expressed as percent of control culture A630 after 27 h of incubation at 25°C, 3000 lux constant. (LO, AMA; (0), meAMA; (A), deAMA; (0), cycloheximide. Control A630 was
0.37.
Amanitin Sensitivity of Partily Purified RNA Polymerase from Amanitin Resistant Strains. Partial purification of RNA polymerase activities from AMA-resistant lines and WT was carried out simultaneously on identical minicolumns of DE-52 cellulose (Fig. 4). Both lines produce activity profiles similar to the WT. Peak I eluting with 0.1 M (NH4)2SO4 was similar in its sensitivity to AMA for all three strains. However, for the two AMA resistant lines, peak II eluting with 0.275 M (NH4)2SO4 was very resistant to both 0.5 and 50 uM AMA. In contrast the WT peak II was very sensitive to both concentrations. The lower activities for peaks I and II in Figure 4C (WT) compared to Figure SA (resistant line 23A) and Figure 4B (resistant line 28B) is due in part to the use of one-half as much starting material for the preparation of fraction S from the WIT line. It was noted that the peak I activity from the WT was significantly more labile upon storage at 0°C than either the analogous activity peak the peak from AMA-resistant lines or the peak II activities from all three. The activity of WT peak I was preserved if the preparation was frozen in liquid nitrogen and stored at - 70°C. The observed loss of activity for the preparation shown in Figure 4C may be
AMANITIN RESISTANT LINES OF CHLAMYDOMONAS REINHARDTII
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-J
0 I-z 0
3C)
U.
o 50-
6-.
0.
o
20 lo 30 VOLUME (ml) FIG. 4. Comparison of amanitin sensitivities of partially purified RNA polymerase activities from WT and amanitin-resistant cells on DE52. (NH4)2SO4 pellets of 23A, 28B, and WT cell extracts treated as described in "Materials and Methods" were applied to DE52 columns (bed volume = 5.0 ml) and eluted with a step gradient of (NH4)2SO4 in buffer C as indicated. A, 23A; B, 28B; C, WT. RNA polymerase activity (0) without AMA, (A) with 0.5 ,/M AMA, and (LI) 50 ,UM AMA were assessed for the eluted fraction. The (NH4)2SO4 concentration of each fraction was adjusted to between 25 and 70 ,LM for optimum polymerase activity (data not shown). Peak I elutes with 0.1 M (NH4)2SO4 and peak II elutes with 0.275 M (NH4)2S04.
a result of the lower protein concentration. As noted in the legend to Figure 5, the specific enzyme activities of peak I and peak II were comparable for preparations from all three lines. The data from Figure 5 provides IC_o values of 6.5 and 225 ,UM for peak II from 23A and 28B, respectively. The corresponding value for WT as stated earlier was 0.04 /lM. Peak I IC50 for 23A, 28B, and WT were 76, 35.5, and 30 l.M, respectively. The IC50 value for WT peak I was determined on a separate preparation which was assayed before significant loss of this relatively unstable activity.
DISCUSSION Cultures of C. reinhardtii have been shown in these studies to exhibit a sensitivity to amatoxins similar to that found with mammalian cells (2, 24, 26), insects (5), and nematodes (23). These results contrast with those obtained with carrot and tobacco cell
cultures, both of which were significantly resistant to inhibition by AMA and required derivatives of amatoxins lacking a free 6'hydroxyl group on the indole moiety to obtain significant inhibition (15, 19). AMA, meAMA, and deAMA are equally effective in inhibiting cells of C. reinhardtii (Fig. 1), indicating this alga lacks an activity which destroys the toxin in culture or at least the inactivating potential is much less than in higher plants (15) or fungi (22). IC50 values for the AMA inhibition of RNA polymerase activities partially purified by DE52 anion exchange chromatography indicate at least two different RNA polymerases are present. One is less sensitive to AMA and elutes from DE52 at a concentration of (NH4)2SO4 (0.10 M) expected for RNA polymerase I and/or III (25). A second activity which elutes at
cx-AMANITIN (,uM) FIG. 5. Log-linear plot of AMA concentration versus RNA polymerase activity for peak I (solid symbols) and peak II (open symbols) from WT (A, A), 23A (U, [), and 28B (0, 0). The straight line was derived by least-squares linear regression analysis and produced an rvalue of: A = 0.991, A = 0.88, * = 0.96, 2 = 0.95, 0 = 0.991, O = 0.91. Control cpm values, less background were A = 501, A = 901, * = 590, a = 2394, 0 = 2356, and 0 = 4450. Specific activities (activity units/mg protein) were A = 619, A = 53, * = 608, 0I = 67, * = 413, and 0 = 147.
a higher (NH4)2SO4 concentration is very sensitive to AMA. The sensitivity to AMA and position of elution on DE52 indicate this activity is RNA polymerase II. Keller et al. (13) working with nuclei from C. reinhardtii showed that 1 /iM AMA inhibited tubulin mRNA synthesis but only 60% of the total RNA syntheses, supporting the interpretation that AMA is a relatively specific inhibitor of RNA polymerase II in this organism as in most other
eucaryotes. The marked sensitivity of the C. reinhardtii to AMA provided a strong selective pressure for isolating amanitin-resistant cell lines and potential RNA polymerase II mutants. That this has been accomplished is evidenced by the dramatic differences in IC50 values for AMA inhibition of Peak II fractions from the WT and lines 23A and 28B. Since the RNA polymerase activities eluting from DE52 cellulose as peak II are 160- and 5600-fold more resistant to AMA inhibition for lines 23A and 28B, respectively, compared to the WT, a mutation in a structural gene coding for a subunit of this activity is a plausible basis for the amanitin resistance displayed by cultures of these lines. Final proof of this will require purification and characterization of this activity with respect to structure and amanitin interaction, as well as genetic studies on linkage of the amatoxin resistance phenotype. The mutagenesis procedure described produced several variants with different levels of resistance to AMA. These different classes of resistance are clearly demonstrated using the multiwell tissue culture plate method described in the text. The convenience of this system enabled titration and screening of all the cultures at several concentrations of AMA with minimal use of reagents and recording time when compared to the more conventional tube assay method. Several hundred cultures could be conveniently evaluated using this system. This approach resulted in the identification of three classes of variants having ID_,0 s of approximately 35, 200, and greater than 200 ,UM AMA. Previous studies in our laboratory established that D. carota and Nicotiana tabacum cultures have the capacity to oxidatively inactivate a-amanitin (15, 16, 19). Only amatoxin derivatives in which the 6'-hydroxyl group of the indole moiety was either absent, i.e. deAMA, or methylated, i.e. meAMA, proved to be
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effective inhibitors of suspension cultures of these species. Two cell lines of D. carota were selected on the basis of resistance to inhibition by meAMA; a careful evaluation of the amanitin sensitivities of the RNA polymerase activities purified from these lines indicated that the basis for their resistance to growth inhibition by meAMA was not due to resistance of RNA polymerase II to inhibition by amanitin (16). Hence, the use of these protected amatoxins does not guarantee a resistant cell line will be an RNA polymerase mutant. Vergara et al. (27) have reported the isolation of carrot cell lines resistant to AMA based on selection in the presence of unmodified a-amanitin and on the basis of crude extract sensitivity have claimed two of these to be RNA polymerase mutants. Further characterization of the lines has not been reported. These studies clearly demonstrate that AMA resistant lines of C. reinhardtii have been isolated which have resistant RNA polymerase activities. Based upon preliminary characterization of these activities, it is probable that the amatoxin-resistant phenotype is the result of a mutation in a structural gene coding for a polypeptide component of the DNA-dependent RNA polymerase. Such a development strengthens the support for use of C. reinhardtii as a model system for studying transcription and RNA polymerase in plants.
9. 10. 11. 12.
13. 14. 15.
16. 17. 18.
Acknowledgments-We thank Dr. Michael C. Little of BioRad Laboratories for the meAMA and Dr. E. H. Harris for the culture of wild-type Chlamydomonas reinhardtii. We thank Ms. Martina Champion for her patience during the typing of this manuscript and Ms. Donna Huseman for preparing the figures.
20.
LITERATURE CITED
21.
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