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.     

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.     

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.     

alpha-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to alpha-amanitin

Spontaneous and EMS-induced alpha-amanitin-resistant Aedes albopictus cells have been isolated and characterized. Two mutant sublines, one of intermediate resistance (alpha A2) and the other highly resistant (Ama18) contained RNA polymerase II activity, the resistance of which in vitro to alpha-amanitin correlated well with the resistance of these cells in vivo. The resistance of these cells to alpha-amanitin can likely be attributed to the presence of an altered RNA polymerase II.     

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.     

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.     

Properties of mutationally altered RNA polymerases II of Drosophila

We tested and compared several in vitro properties of wild type and mutant RNA polymerases II from Drosophila melanogaster, using several different mutants of a single X-linked genetic locus, RpIIC4 (Greenleaf, A. L., Weeks, J. R., Voelker, R. A., Ohnishi, S., and Dickson, B. (1980) Cell 21, 785-792); the mutants tested included the original amanitin-resistant mutant, C4, which is nonconditional, plus the temperature-sensitive mutants A9, C20, E28, and 1Fb40. Using a tritium-labeled amanitin derivative, we demonstrated that C4 polymerase has a reduced binding affinity for amanitin. The C4 polymerase was as stable to thermal denaturation as the wild type enzyme, and the two enzymes had similar specific activities, ionic strength and Mn2+ requirements, and apparent Km values for UTP and GTP when assayed in the presence of Mn2+. However, with Mg2+ as the divalent cation, C4 polymerase was less active than wild type and had 2-fold higher apparent Km values for UTP and GTP. Three of the temperature-sensitive mutants, A9, C20, and E28, were derived from the amanitin-resistant mutant C4; the polymerase II activities from these mutants displayed resistance to alpha-amanitin in vitro identical with that of the C4 enzyme. C20, E28, and 1Fb40 polymerases were markedly less stable to thermal denaturation in vitro than wild type polymerase. The results presented indicate that the mutations at the RNA polymerase locus (RpIIC4-) directly alter the structure of the enzyme, providing conclusive evidence that the locus is a structural gene for a polymerase II subunit.     

Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.     

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.     

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.     

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.     

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.     

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.