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. 1978 Jan;75(1):405–409. doi: 10.1073/pnas.75.1.405

Temperature-sensitive RNA polymerase II mutations in Chinese hamster ovary cells

C James Ingles 1
PMCID: PMC411257  PMID: 272657

Abstract

Mutant Chinese hamster ovary cell lines temperature-sensitive (TS) for growth and containing TS mutations in RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) have been isolated. Wild-type cells were treated with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine and a population of cells possessing mutations in RNA polymerase II was initially selected by isolating α-amanitin-resistant clones at 34°. Of 168 such α-amanitin-resistant isolates screened for temperature sensitivity, nine were TS for growth at 39.5°. By examining the behavior of the α-amanitin resistance of these TS cell lines in somatic cell hybrids, the TS mutation in a number of them was shown to be in RNA polymerase II. Hybrid cells obtained by the fusion of the TS and α-amanitin-resistant cells with cells possessing α-amanitin-sensitive polymerase II grew at both 34° and 39.5°; the TS mutations were recessive. At 34° all the hybrids were α-amanitin-resistant and possessed a mixture of α-amanitin-resistant and sensitive polymerase II. At 39.5° the α-amanitin-resistant polymerase II activities in hybrids of four of the TS cell lines were lost; these four lines were α-amanitin-sensitive and possessed only α-amanitin-sensitive polymerase II. Temperature-insensitive revertants of two of these mutants were isolated. Reversion of the TS phenotype for mutants TsAmaR-1 and TsAmaR-8 was accompanied by an alteration in the level of α-amanitin resistance of the RNA polymerase II activities in the revertant cells. Together these data provide convincing evidence that TS mutations in RNA polymerase II can be coselected with α-amanitin resistance.

Keywords: α-amanitin resistance, nitrosoguanidine mutagenesis, cell hybridization, reversion, RNA nucleotidyltransferase

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Selected References

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  1. Amati P., Blasi F., Di Porzio U., Riccio A., Traboni C. Hamster alpha-amanitine-resistant RNA polymerase II able to transcribe polyoma virus genome in somatic cell hybrids. Proc Natl Acad Sci U S A. 1975 Feb;72(2):753–757. doi: 10.1073/pnas.72.2.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brodner O. G., Wieland T. Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B. Biochemistry. 1976 Aug 10;15(16):3480–3484. doi: 10.1021/bi00661a013. [DOI] [PubMed] [Google Scholar]
  3. Buchwald M., Ingles C. J. Human diploid fibroblast mutants with altered RNA polymerase II. Somatic Cell Genet. 1976 May;2(3):225–233. doi: 10.1007/BF01538961. [DOI] [PubMed] [Google Scholar]
  4. Chan V. L., Whitmore G. F., Siminovitch L. Mammalian cells with altered forms of RNA polymerase II. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3119–3123. doi: 10.1073/pnas.69.11.3119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crerar M. M., Andrews S. J., David E. S., Somers D. G., Mandel J. L., Pearson M. L. Amanitin binding to RNA polymerase II in alpha-amanitin-resistant rat myoblast mutants. J Mol Biol. 1977 May 15;112(2):317–329. doi: 10.1016/s0022-2836(77)80147-2. [DOI] [PubMed] [Google Scholar]
  6. Dawes I. W., Mackinnon D. A., Ball D. E., Hardie I. D., Sweet D. M., Ross F. M., Macdonald F. Identifying sites of simultaneous DNA replication in eukaryotes by N-methyl-N'-nitro-N-nitrosoguanidine multiple mutagenesis. Mol Gen Genet. 1977 Mar 28;152(1):53–57. doi: 10.1007/BF00264939. [DOI] [PubMed] [Google Scholar]
  7. Guerola N., Ingraham J. L., Cerdá-Olmedo E. Induction of closely linked multiple mutations by nitrosoguanidine. Nat New Biol. 1971 Mar 24;230(12):122–125. doi: 10.1038/newbio230122a0. [DOI] [PubMed] [Google Scholar]
  8. Guialis A., Beatty B. G., Ingles C. J., Crerar M. M. Regulation of RNA polymerase II activity in alpha-amanitin-resistant CHO hybrid cells. Cell. 1977 Jan;10(1):53–60. doi: 10.1016/0092-8674(77)90139-8. [DOI] [PubMed] [Google Scholar]
  9. Hong J. S., Ames B. N. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3158–3162. doi: 10.1073/pnas.68.12.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ingles C. J., Guialis A., Lam J., Siminovitch L. Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines. J Biol Chem. 1976 May 10;251(9):2729–2734. [PubMed] [Google Scholar]
  11. Kao F. T., Puck T. T. Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline. Genetics. 1967 Mar;55(3):513–524. doi: 10.1093/genetics/55.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kirschbaum J. B., Claeys I. V., Nasi S., Molholt B., Miller J. H. Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2375–2379. doi: 10.1073/pnas.72.6.2375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lobban P. E., Siminovitch L. Alpha-amanitin resistance: a dominant mutation in CHO cells. Cell. 1975 Feb;4(2):167–172. doi: 10.1016/0092-8674(75)90123-3. [DOI] [PubMed] [Google Scholar]
  14. McBurney M. W., Whitmore G. F. Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell. 1974 Jul;2(3):173–182. doi: 10.1016/0092-8674(74)90091-9. [DOI] [PubMed] [Google Scholar]
  15. Milman G., Lee E., Ghangas G. S., McLaughlin J. R., George M., Jr Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4589–4593. doi: 10.1073/pnas.73.12.4589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Norwood T. H., Zeigler C. J., Martin G. M. Dimethyl sulfoxide enhances polyethylene glycol-mediated somatic cell fusion. Somatic Cell Genet. 1976 May;2(3):263–270. doi: 10.1007/BF01538964. [DOI] [PubMed] [Google Scholar]
  17. Oeschger M. P., Berlyn M. K. A simple procedure for localized mutagenesis using nitrosoguanidine. Mol Gen Genet. 1974;134(1):77–83. doi: 10.1007/BF00332814. [DOI] [PubMed] [Google Scholar]
  18. Oeschger M. P., Berlyn M. K. Regulation of RNA polymerase synthesis in Escherichia coli: a mutant unable to synthesize the enzyme at 43 degrees. Proc Natl Acad Sci U S A. 1975 Mar;72(3):911–915. doi: 10.1073/pnas.72.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Siminovitch L. On the nature of hereditable variation in cultured somatic cells. Cell. 1976 Jan;7(1):1–11. doi: 10.1016/0092-8674(76)90249-x. [DOI] [PubMed] [Google Scholar]
  20. Somers D. G., Pearson M. L., Ingles C. J. Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II. J Biol Chem. 1975 Jul 10;250(13):4825–4831. [PubMed] [Google Scholar]
  21. Somers D. G., Pearson M. L., Ingles C. J. Regulation of RNA polymerase II activity in a mutant rat myoblast cell line resistant to alpha-amanitin. Nature. 1975 Jan 31;253(5490):372–374. doi: 10.1038/253372a0. [DOI] [PubMed] [Google Scholar]
  22. Stanners C. P., Eliceiri G. L., Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52–54. doi: 10.1038/newbio230052a0. [DOI] [PubMed] [Google Scholar]
  23. Steinberg R. A., O'Farrell P. H., Friedrich U., Coffino P. Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells. Cell. 1977 Mar;10(3):381–391. doi: 10.1016/0092-8674(77)90025-3. [DOI] [PubMed] [Google Scholar]
  24. Worton R. G., Ho C. C., Duff C. Chromosome stability in CHO cells. Somatic Cell Genet. 1977 Jan;3(1):27–45. doi: 10.1007/BF01550985. [DOI] [PubMed] [Google Scholar]
  25. Wulf E., Bautz L. RNA polymerase B from an alpha-amanitin resistant mouse myeloma cell line. FEBS Lett. 1976 Oct 15;69(1):6–10. doi: 10.1016/0014-5793(76)80641-2. [DOI] [PubMed] [Google Scholar]

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