Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Dec;5(12):3545–3551. doi: 10.1128/mcb.5.12.3545

Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs.

J B McNeil, M Smith
PMCID: PMC369185  PMID: 3915780

Abstract

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

Full text

PDF
3549

Images in this article

Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  3. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  4. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dobson M. J., Tuite M. F., Roberts N. A., Kingsman A. J., Kingsman S. M., Perkins R. E., Conroy S. C., Fothergill L. A. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982 Apr 24;10(8):2625–2637. doi: 10.1093/nar/10.8.2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Faye G., Leung D. W., Tatchell K., Hall B. D., Smith M. Deletion mapping of sequences essential for in vivo transcription of the iso-1-cytochrome c gene. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2258–2262. doi: 10.1073/pnas.78.4.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guarente L., Hoar E. Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box". Proc Natl Acad Sci U S A. 1984 Dec;81(24):7860–7864. doi: 10.1073/pnas.81.24.7860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guarente L., Lalonde B., Gifford P., Alani E. Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell. 1984 Feb;36(2):503–511. doi: 10.1016/0092-8674(84)90243-5. [DOI] [PubMed] [Google Scholar]
  9. Guarente L., Mason T. Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell. 1983 Apr;32(4):1279–1286. doi: 10.1016/0092-8674(83)90309-4. [DOI] [PubMed] [Google Scholar]
  10. Guarente L., Ptashne M. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199–2203. doi: 10.1073/pnas.78.4.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hsu Y. P., Schimmel P. Yeast LEU1. Repression of mRNA levels by leucine and relationship of 5'-noncoding region to that of LEU2. J Biol Chem. 1984 Mar 25;259(6):3714–3719. [PubMed] [Google Scholar]
  13. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lowry C. V., Weiss J. L., Walthall D. A., Zitomer R. S. Modulator sequences mediate oxygen regulation of CYC1 and a neighboring gene in yeast. Proc Natl Acad Sci U S A. 1983 Jan;80(1):151–155. doi: 10.1073/pnas.80.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  16. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Orkin S. H., Sexton J. P., Cheng T. C., Goff S. C., Giardina P. J., Lee J. I., Kazazian H. H., Jr ATA box transcription mutation in beta-thalassemia. Nucleic Acids Res. 1983 Jul 25;11(14):4727–4734. doi: 10.1093/nar/11.14.4727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Osborne T. F., Gaynor R. B., Berk A. J. The TATA homology and the mRNA 5' untranslated sequence are not required for expression of essential adenovirus E1A functions. Cell. 1982 May;29(1):139–148. doi: 10.1016/0092-8674(82)90098-8. [DOI] [PubMed] [Google Scholar]
  19. Piatak M., Ghosh P. K., Norkin L. C., Weissman S. M. Sequences locating the 5' ends of the major simian virus 40 late mRNA forms. J Virol. 1983 Nov;48(2):503–520. doi: 10.1128/jvi.48.2.503-520.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rymond B. C., Zitomer R. S., Schümperli D., Rosenberg M. The expression in yeast of the Escherichia coli galK gene on CYC1::galK fusion plasmids. Gene. 1983 Nov;25(2-3):249–262. doi: 10.1016/0378-1119(83)90229-9. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Siliciano P. G., Tatchell K. Transcription and regulatory signals at the mating type locus in yeast. Cell. 1984 Jul;37(3):969–978. doi: 10.1016/0092-8674(84)90431-8. [DOI] [PubMed] [Google Scholar]
  23. Somasekhar M. B., Mertz J. E. Sequences involved in determining the locations of the 5' ends of the late RNAs of simian virus 40. J Virol. 1985 Dec;56(3):1002–1013. doi: 10.1128/jvi.56.3.1002-1013.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Struhl K. Promoter elements, regulatory elements, and chromatin structure of the yeast his3 gene. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):901–910. doi: 10.1101/sqb.1983.047.01.104. [DOI] [PubMed] [Google Scholar]
  25. Zalkin H., Paluh J. L., van Cleemput M., Moye W. S., Yanofsky C. Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase. J Biol Chem. 1984 Mar 25;259(6):3985–3992. [PubMed] [Google Scholar]
  26. Zarucki-Schulz T., Tsai S. Y., Itakura K., Soberon X., Wallace R. B., Tsai M. J., Woo S. L., O'Malley B. W. Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription. J Biol Chem. 1982 Sep 25;257(18):11070–11077. [PubMed] [Google Scholar]
  27. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES