Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines.

A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the cytotoxic action of alpha-amanitin have been isolated. The alpha-amanitin sensitivity of the different mutant cell lines varied widely, but correlated well with the alpha-amanitin sensitivity of the RNA polymerase II activity in each of these mutant cell lines. In comparison with the RNA polymerase II of wild-type cells, three mutants, Ama39, Ama6, and Amal, required respectively 2- to 3-fold, 8- to 10-fold, and about 800-fold higher concentrations of alpha-amanitin for inhibition of their polymerase II activity. Determination of the equilibrium dissociation constants (KD) for complexes between 0-[3H]methyl-demethyl-gamma-amanitin and RNA polymearse II indicated that differences in alpha-amanitin sensitivity were reflected in differences in the ability of the enzymes to bind amanitin. Hybrids formed by fusion of mutants with cells of wild-type sensitivity contained both mutant and wild-type polymerase II activities. Thus, each of the different alpha-amanitin resistance mutations was expressed co-dominantly. A test for complementation between two of these mutations by measurement of both the alpha-amanitin sensitivity and the [3H]amanitin binding by RNA polymerase II in Ama6 X Amal hybrid cells did not reveal any wild-type RNA polymerase II activity. These data provide evidence that the mutation to alpha-amanitin resistance involves structural changes in the gene coding for the alpha-amanitin binding subunit of RNA polymerase II. These changes appear to account for the alpha-amanitin-resistant phenotypes of these mutant cells.     

Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II.

Cultures of the rat skeletal muscle myoblast cell line, L6, were treated with the mutagen ethylmethanesulfonate and grown in the presence of alpha-amanitin, an inhibitor of RNA polymerase II in vitro. One clonal cell line, Ama102, resistant tc the cytotoxic action of 2 mu-g/ml of alpha-amanitin was isolated and extensively characterized. Ama102 cells were about 30-fold more resistant to alpha-amanitin than their Ama+ parent cells based on a comparison of the concentration of alpha-amanitin required to reduce their plating efficiencies to similar extents. The RNA polymerase activities from Ama+ and Ama102 cells were solubilized and separated by DEAE-Sephadex chromatography. Whereas all of the Ama+ RNA polymerase II activity was inhibited by 0.1 mu-g/ml of alpha-amanitin, about 30% of the activity in the Ama102 RNA polymerase II peak was resistant to this concentration of alpha-amanitin and was inhibited only by much higher concentrations (25 mu-g/ml) of alpha-amanitin. This alpha-amanitin-resistant activity in Ama102 cells was identified as a bona fide RNA polymerase II by its chromatographic behavior on DEAE-Sephadex, salt optimum, preference for denatured DNA as template, insensitivity to inhibition by potassium phosphate, thermal inactivation kinetics, and inactivation by anti-RNA polymerase II antiserum. Both RNA polymerase IIa and IIb from Ama102 cells exhibited the partial alpha-amanitin resistance, as did this activity when purified further on phosphocellusose. Unlike the parental Ama+ cells, Ama102 cells neither fused at confluence nor showed an increase in the specific activity of creatine kinase. The altered sensitivity of the Ama102 RNA polymerase II to alpha-amanitin appears to account for the drug-resistant phenotype of these cells.     

Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells.

In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D.     

Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II.

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.     

Human diploid fibroblast mutants with altered RNA polymerase II.

Clones resistant to the cytotoxic action of alpha-amanitin have been isolated from a strain of fetal human lung diploid fibroblasts. Resistant clones were recovered at a frequencey of 5 X 10(-8) after single-step selections following mutagenesis with the mutagen ethyl methane sulfonate. Following propagation in drug-free medium, the clones retained the selected phenotype and in both growth and plating experiments showed a 10-50-fold higher resistance than wild-type cells to the cytotoxicity of 0.25 microgram/ml alpha-amanitin. The alpha-amanitin sensitivity of RNA polymerase II purified from the mutant cells suggests the presence of two forms of the enzyme, one similar to that found in wild-type cells and a second form with increased resistance to alpha-amanitin inhibition. These results are consistent with previous evidence that alpha-amanitin resistance behaves as a codominant marker in mammalian cells.     

alpha-Amanithin-resistant mutants of Chinese hamster ovary (CHO) cells

Spontaneous and EMS-induced alpha-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent RNA polymerase II in cell-free extracts from a mutant (ARM-1) was partially resistant to alpha-amanitin. Growing mutants for several generations in the presence or absence of alpha-amanitin did not change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without alpha-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.     

Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.     

Purification and characterization of RNA polymerase II resistant to alpha-amanitin from the mushroom Agaricus bisporus

The DNA-dependent RNA polymerases II or B (ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from the mushroom Agaricus bisporus has been purified to apparent homogeneity. The purification procedures involve precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-cellulose chromatography, and exclusion chromatography on Bio-Gel A-1.5M. With this procedure 11 mg of RNA polymerase II is recovered from 1.5 kg of mushroom tissue. RNA polymerase II from Agaricus bisporus has 12 subunits with the following molecular weights: 182,000, 140,000, 89,000, 69,000, 53,000, 41,000, 37,000, 31,000, 29,000, 25,000, 19,000, and 16,500. Purified RNA polymerase II from Agaricus bisporous was half-maximally inhibited by the mushroom toxin alpha-amanitin at a concentration of 6.5 microgram/mL (7 X 10(-6) M), which is 650-fold more resistant than mammalian RNA polymerases II. The apparent Ki for the alpha-amanitin-RNA polymerase complex was estimated to be 12 X 10(-6) M. The activity of purified RNA polymerase II from the mushroom was quite typical of other eukaryotic RNA polymerase II with regard to template preference, salt optima, and divalent metal cation optima.     

Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II.

Following EMS mutagenesis we recovered a mutant of D. melanogaster that grows at concentrations of alpha-amanitin lethal to wild-type. To our knowledge this mutant represents the first example of an amanitin-resistant eucaryotic organism. The amanitin resistance of the mutant (AmaC4) is due to an alteration in its DNA-dependent RNA polymerase II, which is approximately 250 times less sensitive to inhibition by amanitin than the wild-type polymerase II whether tested in nuclei, in partially-fractionated extracts or as a highly purified enzyme. While the wild-type enzyme activity is inhibited 50% by 2.1 x 10(-8) M alpha-amanitin, inhibition of 50% of the AmaC4 RNA polymerase II activity requires a toxin concentration of 5.6 x 10(-6) M. The mutation responsible for the amanitin resistance of AmaC4 is on the X chromosome near the vermillion locus.     

DNA-mediated transfer of an RNA polymerase II gene: reversion of the temperature-sensitive hamster cell cycle mutant TsAF8 by mammalian DNA

Treatment of the TsAF8 temperature-sensitive (TS) mutant of Syrian hamster BHK-21 cells, with calcium phosphate precipitates of genomic TS+ DNAs from a variety of mammalian cell lines permitted the selection of TS+ colonies at 40 degrees C. TS+ transformation events were distinguished from spontaneous TS+ reversions in experiments in which alpha-amanitin-sensitive (Amas) TS+ DNA was used to transform an AmaR derivative of TsAF8 cells and AmaR TS+ DNA was used to transform Amas TsAF8 cells. In each case it was possible to demonstrate the unselected acquisition of the appropriate Amas or AmaR phenotype with the selected TS+ allele. Each of these TS+ transformed cell lines when grown at 40 degrees C contained an RNA polymerase II activity with a sensitivity to inhibition by alpha-amanitin characteristic of the particular DNA used to transform the TS cells, whereas at 34 degrees C the same cells contained a mixture of AmaR and Amas polymerase II activities. Together, these data provide convincing evidence that the RNA polymerase II gene determining sensitivity to inhibition by alpha-amanitin can be transferred to TsAF8 cells and that the TS defect in TsAF8 is a polymerase II mutation.     

Alpha-amanitin blocks translocation by human RNA polymerase II

Our laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using alpha-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with alpha-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, alpha-amanitin inhibits elongation at each translocation step.     

Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines.

RNA polymerase II polypeptides present in [35S]methionine-labeled Chinese hamster ovary (CHO) cell extracts have been quantitatively immunoprecipitated with an anti-calf thymus RNA polymerase II serum. Analyses of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels indicated that the immunoprecipitated polymerase II of both wild type CHO cells and the alpha-amanitin-resistant mutant Ama1 had polypeptides of molecular weight 214,000, 140,000, 34,000, 25,000, 23,000, 20,500, and 16,500. In heterozygous alpha-amanitin-resistant/alpha-amanitin-sensitive hybrid CHO cells, growth in the presence of alpha-amanitin results in the inactivation of the alpha-amanitin-sensitive RNA polymerase II activity and a compensating increase in the activity of the alpha-amanitin-resistant enzyme. Determination of the rates of synthesis and degradation of RNA polymerase II polypeptides using [35S]methionine labeling and polymerase II immunoprecipitation demonstrated that this increase in activity of alpha-amanitin-resistant polymerase II resulted from a co-ordinate increase in the rate of synthesis of at least three polypeptides of RNA polymerase II. At the same time, there was an enhanced rate of degradation of the alpha-amanitin-inactivated RNA polymerase II polypeptides.     

Alpha-amanitin resistance: a dominant mutation in CHO cells.

Hybrids of CHO cells were constructed consisting of either a 1:1 or 1:2 ratio of alpha-amanitin-resistant and sensitive cells, respectively. The resistance of such hybrids to killing by the drug was similar but slightly less than that of the resistant parent. The hybrids contained both resistant and wild-type RNA polymerase II, in amounts related to the expected gene dosage. The alpha-amanitin marker therefore is expressed codominantly.     

Partial purification and characterization of DNA-dependent RNA polymerases I and II from cherry salmon (Oncorhynchus masou)

1. DNA-dependent RNA polymerases I and II were purified approx 3900- and 13,000-fold, respectively, from sonicated nuclear extract of cherry salmon (Oncorhynchus masou) liver by DEAE-Sephadex, heparin-Sepharose and DNA-cellulose column chromatography. 2. The purified RNA polymerases exhibited a requirement for four kinds of ribonucleoside 5'-triphosphates, an exogeneous template and divalent cation. 3. The activities of RNA polymerases I and II were inhibited by Actinomycin D (24 micrograms/ml) but not by Rifampicin (200 micrograms/ml). 4. RNA polymerase I preferred native DNA as template, while polymerase II preferred single-stranded DNA. 5. RNA polymerase II was inhibited by a low concentration of alpha-amanitin (0.02 micrograms/ml). RNA polymerase I was also inhibited by the relatively high concentration of alpha-amanitin (IC50 = 100 micrograms/ml and IC70 = 750 micrograms/ml). 6. RNA polymerases from cherry salmon exhibited a higher activity at low temperature than from rat liver.