Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Oct;7(10):3446–3451. doi: 10.1128/mcb.7.10.3446

Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.

N F Lue 1, D I Chasman 1, A R Buchman 1, R D Kornberg 1
PMCID: PMC367995  PMID: 3316976

Abstract

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

Full text

PDF
3447

Images in this article

Selected References

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

  1. Abraham J., Nasmyth K. A., Strathern J. N., Klar A. J., Hicks J. B. Regulation of mating-type information in yeast. Negative control requiring sequences both 5' and 3' to the regulated region. J Mol Biol. 1984 Jul 5;176(3):307–331. doi: 10.1016/0022-2836(84)90492-3. [DOI] [PubMed] [Google Scholar]
  2. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Bram R. J., Kornberg R. D. Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci U S A. 1985 Jan;82(1):43–47. doi: 10.1073/pnas.82.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bram R. J., Lue N. F., Kornberg R. D. A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription. EMBO J. 1986 Mar;5(3):603–608. doi: 10.1002/j.1460-2075.1986.tb04253.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brody R. S., Westheimer F. H. The purification of orotidine-5'-phosphate decarboxylase from yeast by affinity chromatography. J Biol Chem. 1979 May 25;254(10):4238–4244. [PubMed] [Google Scholar]
  7. Emini E. A., Hughes J. V., Perlow D. S., Boger J. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol. 1985 Sep;55(3):836–839. doi: 10.1128/jvi.55.3.836-839.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [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. Hashimoto H., Kikuchi Y., Nogi Y., Fukasawa T. Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4. Mol Gen Genet. 1983;191(1):31–38. doi: 10.1007/BF00330886. [DOI] [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. Johnston S. A., Hopper J. E. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6971–6975. doi: 10.1073/pnas.79.22.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnston S. A., Zavortink M. J., Debouck C., Hopper J. E. Functional domains of the yeast regulatory protein GAL4. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6553–6557. doi: 10.1073/pnas.83.17.6553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keegan L., Gill G., Ptashne M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science. 1986 Feb 14;231(4739):699–704. doi: 10.1126/science.3080805. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Laughon A., Driscoll R., Wills N., Gesteland R. F. Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):268–275. doi: 10.1128/mcb.4.2.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Laughon A., Gesteland R. F. Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):260–267. doi: 10.1128/mcb.4.2.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Levine H. L., Brody R. S., Westheimer F. H. Inhibition of orotidine-5'-phosphate decarboxylase by 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid, 6-azauridine 5'-phosphate, and uridine 5'-phosphate. Biochemistry. 1980 Oct 28;19(22):4993–4999. doi: 10.1021/bi00563a010. [DOI] [PubMed] [Google Scholar]
  23. Lohr D., Hopper J. E. The relationship of regulatory proteins and DNase I hypersensitive sites in the yeast GAL1-10 genes. Nucleic Acids Res. 1985 Dec 9;13(23):8409–8423. doi: 10.1093/nar/13.23.8409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lorch Y., Kornberg R. D. A region flanking the GAL7 gene and a binding site for GAL4 protein as upstream activating sequences in yeast. J Mol Biol. 1985 Dec 20;186(4):821–824. doi: 10.1016/0022-2836(85)90400-0. [DOI] [PubMed] [Google Scholar]
  25. Ma J., Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987 Mar 13;48(5):847–853. doi: 10.1016/0092-8674(87)90081-x. [DOI] [PubMed] [Google Scholar]
  26. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nogi Y., Shimada H., Matsuzaki Y., Hashimoto H., Fukasawa T. Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. II. The isolation and dosage effect of the regulatory gene GAL80. Mol Gen Genet. 1984;195(1-2):29–34. doi: 10.1007/BF00332719. [DOI] [PubMed] [Google Scholar]
  28. Perlman D., Hopper J. E. Constitutive synthesis of the GAL4 protein, a galactose pathway regulator in Saccharomyces cerevisiae. Cell. 1979 Jan;16(1):89–95. doi: 10.1016/0092-8674(79)90190-9. [DOI] [PubMed] [Google Scholar]
  29. Reyes P., Sandquist R. B. Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives. Anal Biochem. 1978 Aug 1;88(2):522–531. doi: 10.1016/0003-2697(78)90452-9. [DOI] [PubMed] [Google Scholar]
  30. Rose M., Grisafi P., Botstein D. Structure and function of the yeast URA3 gene: expression in Escherichia coli. Gene. 1984 Jul-Aug;29(1-2):113–124. doi: 10.1016/0378-1119(84)90172-0. [DOI] [PubMed] [Google Scholar]
  31. Torchia T. E., Hamilton R. W., Cano C. L., Hopper J. E. Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes. Mol Cell Biol. 1984 Aug;4(8):1521–1527. doi: 10.1128/mcb.4.8.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Umezu K., Amaya T., Yoshimoto A., Tomita K. Purification and properties of orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase from baker's yeast. J Biochem. 1971 Aug;70(2):249–262. doi: 10.1093/oxfordjournals.jbchem.a129637. [DOI] [PubMed] [Google Scholar]
  33. West R. W., Jr, Yocum R. R., Ptashne M. Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol Cell Biol. 1984 Nov;4(11):2467–2478. doi: 10.1128/mcb.4.11.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES