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. 1986 May;6(5):1633–1639. doi: 10.1128/mcb.6.5.1633

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

J F Jerome, J A Jaehning
PMCID: PMC367690  PMID: 3537708

Abstract

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

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Selected References

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

  1. Bach M. L., Lacroute F., Botstein D. Evidence for transcriptional regulation of orotidine-5'-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):386–390. doi: 10.1073/pnas.76.1.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker S. M., Okkema P. G., Jaehning J. A. Expression of the Saccharomyces cerevisiae GAL7 gene on autonomous plasmids. Mol Cell Biol. 1984 Oct;4(10):2062–2071. doi: 10.1128/mcb.4.10.2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell G. I., DeGennaro L. J., Gelfand D. H., Bishop R. J., Valenzuela P., Rutter W. J. Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs. J Biol Chem. 1977 Nov 25;252(22):8118–8125. [PubMed] [Google Scholar]
  4. Cameron J. R., Loh E. Y., Davis R. W. Evidence for transposition of dispersed repetitive DNA families in yeast. Cell. 1979 Apr;16(4):739–751. doi: 10.1016/0092-8674(79)90090-4. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elder R. T., St John T. P., Stinchcomb D. T., Davis R. W., Scherer S., Davis R. W. Studies on the transposable element Ty1 of yeast. I. RNA homologous to Ty1. II. Recombination and expression of Ty1 and adjacent sequences. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 2):581–591. doi: 10.1101/sqb.1981.045.01.075. [DOI] [PubMed] [Google Scholar]
  7. Ernest M. J., Schutz G., Feigelson P. RNA synthesis in isolated hen oviduct nuclei. Biochemistry. 1976 Feb 24;15(4):824–829. doi: 10.1021/bi00649a015. [DOI] [PubMed] [Google Scholar]
  8. Ide G. J. Nucleoside 5'-[gamma-S]triphosphates will initiate transcription in isolated yeast nuclei. Biochemistry. 1981 Apr 28;20(9):2633–2638. doi: 10.1021/bi00512a042. [DOI] [PubMed] [Google Scholar]
  9. Jost J. P., Geiser M., Seldran M. Specific modulation of the transcription of cloned avian vitellogenin II gene by estradiol-receptor complex in vitro. Proc Natl Acad Sci U S A. 1985 Feb;82(4):988–991. doi: 10.1073/pnas.82.4.988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Laughon A., Gesteland R. F. Isolation and preliminary characterization of the GAL4 gene, a positive regulator of transcription in yeast. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6827–6831. doi: 10.1073/pnas.79.22.6827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  12. Manley J. L., Sharp P. A., Gefter M. L. RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor. Proc Natl Acad Sci U S A. 1979 Jan;76(1):160–164. doi: 10.1073/pnas.76.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schultz L. D. Transcriptional role of yeast deoxyribonucleic acid dependent ribonucleic acid polymerase III. Biochemistry. 1978 Feb 21;17(4):750–758. doi: 10.1021/bi00597a031. [DOI] [PubMed] [Google Scholar]
  14. Shaw K. J., Olson M. V. Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene. Mol Cell Biol. 1984 Apr;4(4):657–665. doi: 10.1128/mcb.4.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
  16. Struhl K., Davis R. W. Transcription of the his3 gene region in Saccharomyces cerevisiae. J Mol Biol. 1981 Nov 5;152(3):535–552. doi: 10.1016/0022-2836(81)90267-9. [DOI] [PubMed] [Google Scholar]
  17. Struhl K. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucleic Acids Res. 1985 Dec 9;13(23):8587–8601. doi: 10.1093/nar/13.23.8587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Treisman R., Maniatis T. Simian virus 40 enhancer increases number of RNA polymerase II molecules on linked DNA. Nature. 1985 May 2;315(6014):73–75. doi: 10.1038/315072a0. [DOI] [PubMed] [Google Scholar]
  19. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  20. Winkley C. S., Keller M. J., Jaehning J. A. A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae. J Biol Chem. 1985 Nov 15;260(26):14214–14223. [PubMed] [Google Scholar]
  21. de Kloet S. R., Beltz W. R. Control of the formation of ribonucleic acid in yeast: synthesis of ribonucleic acid in a nuclear fraction of Saccharomyces carlsbergensis. Arch Biochem Biophys. 1975 Mar;167(1):322–334. doi: 10.1016/0003-9861(75)90468-3. [DOI] [PubMed] [Google Scholar]

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