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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Feb;81(4):1144–1148. doi: 10.1073/pnas.81.4.1144

Molecular cloning of hormone-responsive genes from the yeast Saccharomyces cerevisiae.

G L Stetler, J Thorner
PMCID: PMC344782  PMID: 6366798

Abstract

A method for identifying yeast genes whose transcription is differentially regulated was developed. The technique is based on incorporation of the analog 4-thiouridine into nascent RNAs, which allows their purification. The purified RNAs are used to prepare cDNA copies for screening of genomic DNA libraries by hybridization. Using this procedure, several cloned yeast DNA segments were found whose transcription in MATa haploids in vivo is apparently modulated in dramatic fashion within 10-15 min after exposure to the mating pheromone, alpha factor. Subsequent analysis indicated that these sequences fall into three major classes: (i) genes expressed in vegetatively growing cells that are no longer transcribed after alpha-factor administration ("turn-off" genes); (ii) genes whose expression is increased 10- to 20-fold after exposure of the MATa cells to alpha factor ("turn-up" genes); and (iii) genes that are expressed only after alpha-factor treatment ("turn-on" genes). The first class may encode products required for cell cycle progression; the third class may code for products uniquely involved in the mating process.

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Selected References

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  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brake A. J., Julius D. J., Thorner J. A functional prepro-alpha-factor gene in Saccharomyces yeasts can contain three, four, or five repeats of the mature pheromone sequence. Mol Cell Biol. 1983 Aug;3(8):1440–1450. doi: 10.1128/mcb.3.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chan R. K., Otte C. A. Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):11–20. doi: 10.1128/mcb.2.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chan R. K., Otte C. A. Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):21–29. doi: 10.1128/mcb.2.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ciejek E., Thorner J. Recovery of S. cerevisiae a cells from G1 arrest by alpha factor pheromone requires endopeptidase action. Cell. 1979 Nov;18(3):623–635. doi: 10.1016/0092-8674(79)90117-x. [DOI] [PubMed] [Google Scholar]
  6. Fehrenbacher G., Perry K., Thorner J. Cell-cell recognition in Saccharomyces cerevisiae: regulation of mating-specific adhesion. J Bacteriol. 1978 Jun;134(3):893–901. doi: 10.1128/jb.134.3.893-901.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hartwell L. H. Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J Cell Biol. 1980 Jun;85(3):811–822. doi: 10.1083/jcb.85.3.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hereford L., Bromley S., Osley M. A. Periodic transcription of yeast histone genes. Cell. 1982 Aug;30(1):305–310. doi: 10.1016/0092-8674(82)90036-8. [DOI] [PubMed] [Google Scholar]
  9. Jenness D. D., Burkholder A. C., Hartwell L. H. Binding of alpha-factor pheromone to yeast a cells: chemical and genetic evidence for an alpha-factor receptor. Cell. 1983 Dec;35(2 Pt 1):521–529. doi: 10.1016/0092-8674(83)90186-1. [DOI] [PubMed] [Google Scholar]
  10. Jensen R., Sprague G. F., Jr, Herskowitz I. Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus. Proc Natl Acad Sci U S A. 1983 May;80(10):3035–3039. doi: 10.1073/pnas.80.10.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Karn J., Brenner S., Barnett L., Cesareni G. Novel bacteriophage lambda cloning vector. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5172–5176. doi: 10.1073/pnas.77.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Liao H., Thorner J. Yeast mating pheromone alpha factor inhibits adenylate cyclase. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1898–1902. doi: 10.1073/pnas.77.4.1898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mangiarotti G., Ceccarelli A., Lodish H. F. Cyclic AMP stabilizes a class of developmentally regulated Dictyostelium discoideum mRNAs. Nature. 1983 Feb 17;301(5901):616–618. doi: 10.1038/301616a0. [DOI] [PubMed] [Google Scholar]
  14. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  15. Murdoch G. H., Rosenfeld M. G. Eukaryotic transcriptional regulation and chromatin-associated protein phosphorylation by cyclic AMP. Science. 1982 Dec 24;218(4579):1315–1317. doi: 10.1126/science.6293056. [DOI] [PubMed] [Google Scholar]
  16. Nagamine Y., Sudol M., Reich E. Hormonal regulation of plasminogen activator mRNA production in porcine kidney cells. Cell. 1983 Apr;32(4):1181–1190. doi: 10.1016/0092-8674(83)90301-x. [DOI] [PubMed] [Google Scholar]
  17. Nasmyth K. A. Molecular genetics of yeast mating type. Annu Rev Genet. 1982;16:439–500. doi: 10.1146/annurev.ge.16.120182.002255. [DOI] [PubMed] [Google Scholar]
  18. Nasmyth K. Molecular analysis of a cell lineage. Nature. 1983 Apr 21;302(5910):670–676. doi: 10.1038/302670a0. [DOI] [PubMed] [Google Scholar]
  19. Reed S. I., Ferguson J., Groppe J. C. Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28. Mol Cell Biol. 1982 Apr;2(4):412–425. doi: 10.1128/mcb.2.4.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reed S. I. The selection of S. cerevisiae mutants defective in the start event of cell division. Genetics. 1980 Jul;95(3):561–577. doi: 10.1093/genetics/95.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reeve A. E., Smith M. M., Pigiet V., Huang R. C. Incorporation of purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro. Biochemistry. 1977 Oct 4;16(20):4464–4470. doi: 10.1021/bi00639a021. [DOI] [PubMed] [Google Scholar]
  22. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Schekman R., Brawley V. Localized deposition of chitin on the yeast cell surface in response to mating pheromone. Proc Natl Acad Sci U S A. 1979 Feb;76(2):645–649. doi: 10.1073/pnas.76.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sprague G. F., Jr, Blair L. C., Thorner J. Cell interactions and regulation of cell type in the yeast Saccharomyces cerevisiae. Annu Rev Microbiol. 1983;37:623–660. doi: 10.1146/annurev.mi.37.100183.003203. [DOI] [PubMed] [Google Scholar]
  25. Sprague G. F., Jr, Herskowitz I. Control of yeast cell type by the mating type locus. I. Identification and control of expression of the a-specific gene BAR1. J Mol Biol. 1981 Dec 5;153(2):305–321. doi: 10.1016/0022-2836(81)90280-1. [DOI] [PubMed] [Google Scholar]
  26. St John T. P., Davis R. W. Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell. 1979 Feb;16(2):443–452. doi: 10.1016/0092-8674(79)90020-5. [DOI] [PubMed] [Google Scholar]
  27. Tait R. C., Rodriguez R. L., West R. W., Jr The rapid purification of T4 DNA ligase from a lambda T4 lig lysogen. J Biol Chem. 1980 Feb 10;255(3):813–815. [PubMed] [Google Scholar]
  28. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thorner J. An essential role for cyclic AMP in growth control: the case for yeast. Cell. 1982 Aug;30(1):5–6. doi: 10.1016/0092-8674(82)90004-6. [DOI] [PubMed] [Google Scholar]
  30. Tuite M. F., Plesset J., Moldave K., McLaughlin C. S. Faithful and efficient translation of homologous and heterologous mRNAs in an mRNA-dependent cell-free system from Saccharomyces cerevisiae. J Biol Chem. 1980 Sep 25;255(18):8761–8766. [PubMed] [Google Scholar]
  31. Vande Woude G. F., Oskarsson M., Enquist L. W., Nomura S., Sullivan M., Fischinger P. J. Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4464–4468. doi: 10.1073/pnas.76.9.4464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Williams J. G., Tsang A. S., Mahbubani H. A change in the rate of transcription of a eukaryotic gene in response to cyclic AMP. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7171–7175. doi: 10.1073/pnas.77.12.7171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Woo S. L. A sensitive and rapid method for recombinant phage screening. Methods Enzymol. 1979;68:389–395. doi: 10.1016/0076-6879(79)68028-x. [DOI] [PubMed] [Google Scholar]

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