Amanitin binding to RNA polymerase II in α-amanitin-resistant rat myoblast mutants

Crerar, Michael M.; Andrews, S.; David, Emina S.; Somers, Donald G.; Mandel, Jean‐Louis; Pearson, Mark

doi:10.1016/s0022-2836(77)80147-2

Cited by 38 publications

(11 citation statements)

References 19 publications

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“…2A) and fused into myotubes within a few days (Fig. 2B) Cells were harvested, frozen, and thawed four times as described for (A) and then sonicated, diluted, and centrifuged as described previously (8,39). S-100 supernatants (-0.5 x 107 to -1.0 x 107 cell equivalents per ml) were assayed for RNA polymerase activity in 0.1 M ammonium sulfate as previously described (8.…”

Section: Myogenic Phenotypes Of Amar Mutants (I)mentioning

confidence: 99%

“…Estimates of the inhibition constant for amanitin, K,1ama), were Of the remaining three AmaR mutants that had IIs/IIR ratios greater than 1, Amal7-1 had a ratio of -4:1, and the other two mutants, Ama21-1 and Ama28-1, yielded ratios of 2.5:1. Karyotype analysis of Amal7-1 showed that it contained a pseudotetraploid complement of chromosomes (75 ± 1) in accord with the higher ratio of the two forms of the enzyme (8). However, both Ama21-1 and Ama28-1 had pseudodiploid karyotypes (chromosome numbers of 39 ± 1 and 38 ± 1, respectively).…”

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confidence: 96%

“…We (11,41,42) or bacteriophage growth (13,14,27). L6 mutant cell lines containing altered forms of RNA polymerase II can be obtained by selecting mutants able to grow in the presence of the mushroom toxin a-amanitin (8,39), since this drug is a specific inhibitor of RNA polymerase 11 (25,28). Most of these L6 myoblast a-amanitin-resistant (AmaR) mutants, like those isolated in other mammalian cell lines (1, 5-7, 21-23, 44), contain altered RNA polymerase II activities.…”

mentioning

confidence: 99%

“…Most of these L6 myoblast a-amanitin-resistant (AmaR) mutants, like those isolated in other mammalian cell lines (1, 5-7, 21-23, 44), contain altered RNA polymerase II activities. In the myoblast AmaR mutants isolated from diploid and tetraploid parent lines, both wild-type and mutant forms of the enzyme are expressed, indicating that the ama R mutation is dominant in L6 cells (8,40), as it is in CHO cell hybrids (29). Consequently, growth of such mutant cells in amanitin is not significantly impaired, although the sensitive wild-type activity of the enzyme is lost.…”

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confidence: 99%

“…The first explanation is based on the known pleiotropic effects of RNA polymerase mutations on bacterial sporulation or phage development (cited above). The latter explanation is based on the observation that acquisition of the neoplastic "transformed" phenotype results in a loss of the myogenicpositive phenotype in the rat myoblasts (24 (8,9,40). Under these conditions, RNA polymerase activity is dependent on the endogenous DNA template (9).…”

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confidence: 99%

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Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

Crerar¹,

Leather²,

David³

et al. 1983

Mol. Cell. Biol.

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To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent cx-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two-to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo-phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo-phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation.The L6 rat myoblast cell line isolated by Yaffe (45) forms multinucleate myotubes and synthesizes elevated levels of several muscle-characteristic proteins when grown to confluence in physiological levels of calcium (10,19,30,31,(35)(36)(37)43

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Section: Myogenic Phenotypes Of Amar Mutants (I)mentioning

confidence: 99%

mentioning

confidence: 96%

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confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

Crerar¹,

Leather²,

David³

et al. 1983

Mol. Cell. Biol.

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α‐Amanitin and 5‐fluorouridine inhibition of serumstimulated DNA synthesis in quiescent AKR‐2B mouse embryo cells

Wells

Stoddard

Getz

et al. 1979

Journal Cellular Physiology

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AKR-2B mouse embryo cells undergoing the serum-stimulated transition from a quiescent to a proliferating state exhibit an increase in the rate of hnRNA synthesis which appears to be mediated through an increase in the actual number of RNA polymerase II molecules. alpha-Amanitin, administered early in the prereplication interval following stimulation, effectively inhibits hnRNA synthesis, polysomal mRNA accumulation, polyribosome formation, and subsequent DNA synthesis, and cell division. Unexpectedly, alpha-amanitin treatment also produces almost complete inhibition of the synthesis of 45S rRNA precursor and the increase in accumulation of cytoplasmic rRNA following serum stimulation. In order to determine whether the inhibition of new ribosomal synthesis might in itself be sufficient to prevent serum-stimulated DNA synthesis, the effects of 5-fluorouridine (5-FU), a specific inhibitor of 45S rRNA processing, were investigated. If added within eight hours following serum stimulation, 5-FU was found to completely inhibit subsequent DNA synthesis. These results suggest that quiescent AKR-2B cells do not contain a sufficient excess of ribosomes to support the synthesis of proteins which are required for DNA synthesis in response to serum growth factors. Furthermore, an early polymerase II mediated synthesis of mRNA(s) coding for some factor(s) necessary for ribosomal gene transcription may be an essential step in the serum-stimulated synthesis of new ribosomes.

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Changes in RNA polymerase II in a cell cycle‐specific temperature‐sensitive mutant of hamster cells

Rossini

Baserga

Huang

et al. 1980

Journal Cellular Physiology

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tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bine [3H]-gamma-amanitin decrease in tsAF8 cells at 40.6 degrees, with a half-life of approximately 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34 degrees or in BHK cells at either 34 degrees or 40.6 degrees. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6 degrees. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.

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Amanitin binding to RNA polymerase II in α-amanitin-resistant rat myoblast mutants

Cited by 38 publications

References 19 publications

Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

α‐Amanitin and 5‐fluorouridine inhibition of serumstimulated DNA synthesis in quiescent AKR‐2B mouse embryo cells

Changes in RNA polymerase II in a cell cycle‐specific temperature‐sensitive mutant of hamster cells

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