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. 1986 Mar;83(6):1554–1558. doi: 10.1073/pnas.83.6.1554

Isolation of structural genes for yeast RNA polymerases by immunological screening.

M Riva, S Memet, J Y Micouin, J Huet, I Treich, J Dassa, R Young, J M Buhler, A Sentenac, P Fromageot
PMCID: PMC323121  PMID: 2937059

Abstract

A lambda gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the lambda A-190 and lambda C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bréant B., Huet J., Sentenac A., Fromageot P. Analysis of yeast RNA polymerases with subunit-specific antibodies. J Biol Chem. 1983 Oct 10;258(19):11968–11973. [PubMed] [Google Scholar]
  2. Buhler J. M., Huet J., Davies K. E., Sentenac A., Fromageot P. Immunological studies of yeast nuclear RNA polymerases at the subunit level. J Biol Chem. 1980 Oct 25;255(20):9949–9954. [PubMed] [Google Scholar]
  3. Buhler J. M., Iborra F., Sentenac A., Fromageot P. Structural studies on yeast RNA polymerases. Existence of common subunits in RNA polymerases A(I) and B(II). J Biol Chem. 1976 Mar 25;251(6):1712–1717. [PubMed] [Google Scholar]
  4. Greenleaf A. L. Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit. J Biol Chem. 1983 Nov 25;258(22):13403–13406. [PubMed] [Google Scholar]
  5. Huet J., Riva M., Sentenac A., Fromageot P. Yeast RNA polymerase C and its subunits. Specific antibodies as structural and functional probes. J Biol Chem. 1985 Dec 5;260(28):15304–15310. [PubMed] [Google Scholar]
  6. Huet J., Schnabel R., Sentenac A., Zillig W. Archaebacteria and eukaryotes possess DNA-dependent RNA polymerases of a common type. EMBO J. 1983;2(8):1291–1294. doi: 10.1002/j.1460-2075.1983.tb01583.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huet J., Sentenac A., Fromageot P. Spot-immunodetection of conserved determinants in eukaryotic RNA polymerases. Study with antibodies to yeast RNA polymerases subunits. J Biol Chem. 1982 Mar 10;257(5):2613–2618. [PubMed] [Google Scholar]
  8. Ingles C. J., Himmelfarb H. J., Shales M., Greenleaf A. L., Friesen J. D. Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2157–2161. doi: 10.1073/pnas.81.7.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Miller J. S., Paterson B. M., Ricciardi R. P., Cohen L., Roberts B. E. Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules. Methods Enzymol. 1983;101:650–674. doi: 10.1016/0076-6879(83)01046-0. [DOI] [PubMed] [Google Scholar]
  10. Riva M., Buhler J. M., Sentenac A., Fromageot P., Hawthorne D. C. Natural variation in yeast RNA polymerase A. Formation of a mosaic RNA polymerase A in a meiotic segregant from an interspecific hybrid. J Biol Chem. 1982 Apr 25;257(8):4570–4577. [PubMed] [Google Scholar]
  11. Ruet A., Sentenac A., Fromageot P., Winsor B., Lacroute F. A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro. J Biol Chem. 1980 Jul 10;255(13):6450–6455. [PubMed] [Google Scholar]
  12. Searles L. L., Jokerst R. S., Bingham P. M., Voelker R. A., Greenleaf A. L. Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging. Cell. 1982 Dec;31(3 Pt 2):585–592. doi: 10.1016/0092-8674(82)90314-2. [DOI] [PubMed] [Google Scholar]
  13. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem. 1985;18(1):31–90. doi: 10.3109/10409238509082539. [DOI] [PubMed] [Google Scholar]
  14. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  15. Winsor B., Lacroute F., Ruet A., Sentenac A. Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity. Mol Gen Genet. 1979 Jun 7;173(2):145–151. doi: 10.1007/BF00330304. [DOI] [PubMed] [Google Scholar]
  16. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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