Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985

Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

R R Yocum, S Hanley, R West Jr, M Ptashne
PMCID: PMC369015  PMID: 6390181

Abstract

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.

Full text

PDF
1985

Selected References

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

  1. Adams B. G. Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol. 1972 Aug;111(2):308–315. doi: 10.1128/jb.111.2.308-315.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beier D. R., Young E. T. Characterization of a regulatory region upstream of the ADR2 locus of S. cerevisiae. Nature. 1982 Dec 23;300(5894):724–728. doi: 10.1038/300724a0. [DOI] [PubMed] [Google Scholar]
  3. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  4. Broach J. R. Galactose regulation in Saccharomyces cerevisiae. The enzymes encoded by the GAL7, 10, 1 cluster are co-ordinately controlled and separately translated. J Mol Biol. 1979 Jun 15;131(1):41–53. doi: 10.1016/0022-2836(79)90300-0. [DOI] [PubMed] [Google Scholar]
  5. Carlson M., Osmond B. C., Botstein D. Mutants of yeast defective in sucrose utilization. Genetics. 1981 May;98(1):25–40. doi: 10.1093/genetics/98.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cirillo V. P. Galactose transport in Saccharomyces cerevisiae. I. Nonmetabolized sugars as substrates and inducers of the galactose transport system. J Bacteriol. 1968 May;95(5):1727–1731. doi: 10.1128/jb.95.5.1727-1731.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Citron B. A., Donelson J. E. Sequence of the Saccharomyces GAL region and its transcription in vivo. J Bacteriol. 1984 Apr;158(1):269–278. doi: 10.1128/jb.158.1.269-278.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Citron B. A., Feiss M., Donelson J. E. Expression of the yeast galactokinase gene in Escherichia coli. Gene. 1979 Jul;6(3):251–264. doi: 10.1016/0378-1119(79)90061-1. [DOI] [PubMed] [Google Scholar]
  9. DOUGLAS H. C., HAWTHORNE D. C. ENZYMATIC EXPRESSION AND GENETIC LINKAGE OF GENES CONTROLLING GALACTOSE UTILIZATION IN SACCHAROMYCES. Genetics. 1964 May;49:837–844. doi: 10.1093/genetics/49.5.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donahue T. F., Daves R. S., Lucchini G., Fink G. R. A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast. Cell. 1983 Jan;32(1):89–98. doi: 10.1016/0092-8674(83)90499-3. [DOI] [PubMed] [Google Scholar]
  11. Douglas H. C., Hawthorne C. D. Uninducible mutants in the gal i locus of Saccharomyces cerevisiae. J Bacteriol. 1972 Mar;109(3):1139–1143. doi: 10.1128/jb.109.3.1139-1143.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Douglas H. C., Hawthorne D. C. Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics. 1966 Sep;54(3):911–916. doi: 10.1093/genetics/54.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Federoff H. J., Eccleshall T. R., Marmur J. Regulation of maltase synthesis in Saccharomyces carlsbergensis. J Bacteriol. 1983 Jun;154(3):1301–1308. doi: 10.1128/jb.154.3.1301-1308.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fromm M., Berg P. Transcription in vivo from SV40 early promoter deletion mutants without repression by large T antigen. J Mol Appl Genet. 1983;2(1):127–135. [PubMed] [Google Scholar]
  17. Fukasawa T., Obonai K., Segawa T., Nogi Y. The enzymes of the galactose cluster in Saccharomyces cerevisiae. II. Purification and characterization of uridine diphosphoglucose 4-epimerase. J Biol Chem. 1980 Apr 10;255(7):2705–2707. [PubMed] [Google Scholar]
  18. Guarente L., Lauer G., Roberts T. M., Ptashne M. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin. Cell. 1980 Jun;20(2):543–553. doi: 10.1016/0092-8674(80)90640-6. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Hopper J. E., Broach J. R., Rowe L. B. Regulation of the galactose pathway in Saccharomyces cerevisiae: induction of uridyl transferase mRNA and dependency on GAL4 gene function. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2878–2882. doi: 10.1073/pnas.75.6.2878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Johnson A. D., Poteete A. R., Lauer G., Sauer R. T., Ackers G. K., Ptashne M. lambda Repressor and cro--components of an efficient molecular switch. Nature. 1981 Nov 19;294(5838):217–223. doi: 10.1038/294217a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Kew O. M., Douglas H. C. Genetic co-regulation of galactose and melibiose utilization in Saccharomyces. J Bacteriol. 1976 Jan;125(1):33–41. doi: 10.1128/jb.125.1.33-41.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klar A. J., Halvorson H. O. Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae. Mol Gen Genet. 1974;135(3):203–212. doi: 10.1007/BF00268616. [DOI] [PubMed] [Google Scholar]
  29. LELOIR L. F. The enzymatic transformation of uridine diphosphate glucose into a galactose derivative. Arch Biochem Biophys. 1951 Sep;33(2):186–190. doi: 10.1016/0003-9861(51)90096-3. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. MANDELSTAM J. The repression of constitutive beta-galactosidase in Escherichia coli by glucose and other carbon sources. Biochem J. 1962 Mar;82:489–493. doi: 10.1042/bj0820489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matsumoto K., Adachi Y., Toh-e A., Oshima Y. Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein. J Bacteriol. 1980 Feb;141(2):508–527. doi: 10.1128/jb.141.2.508-527.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Matsumoto K., Toh-e A., Oshima Y. Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4. J Bacteriol. 1978 May;134(2):446–457. doi: 10.1128/jb.134.2.446-457.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matsumoto K., Toh-e A., Oshima Y. Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1981 Feb;1(2):83–93. doi: 10.1128/mcb.1.2.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matsumoto K., Uno I., Toh-E A., Ishikawa T., Oshima Y. Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source. J Bacteriol. 1982 Apr;150(1):277–285. doi: 10.1128/jb.150.1.277-285.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matsumoto K., Yoshimatsu T., Oshima Y. Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae. J Bacteriol. 1983 Mar;153(3):1405–1414. doi: 10.1128/jb.153.3.1405-1414.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Nogi Y., Fukasawa T. Nucleotide sequence of the transcriptional initiation region of the yeast GAL7 gene. Nucleic Acids Res. 1983 Dec 20;11(24):8555–8568. doi: 10.1093/nar/11.24.8555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nogi Y., Matsumoto K., Toh-e A., Oshima Y. Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae. Mol Gen Genet. 1977 Apr 29;152(3):137–144. doi: 10.1007/BF00268810. [DOI] [PubMed] [Google Scholar]
  42. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Rose M., Casadaban M. J., Botstein D. Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2460–2464. doi: 10.1073/pnas.78.4.2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Russell P. R. Evolutionary divergence of the mRNA transcription initiation mechanism in yeast. Nature. 1983 Jan 13;301(5896):167–169. doi: 10.1038/301167a0. [DOI] [PubMed] [Google Scholar]
  46. Schell M. A., Wilson D. B. Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid. Gene. 1979 Apr;5(4):291–303. doi: 10.1016/0378-1119(79)90104-5. [DOI] [PubMed] [Google Scholar]
  47. Schlesinger D. H., Schell M. A., Wilson D. B. The NH2-terminal sequences of galactokinase from Escherichia coli and Saccharomyces cerevisiae. FEBS Lett. 1977 Nov 1;83(1):45–47. doi: 10.1016/0014-5793(77)80638-8. [DOI] [PubMed] [Google Scholar]
  48. Sherman F., Stewart J. W., Schweingruber A. M. Mutants of yeast initiating translation of iso-1-cytochrome c within a region spanning 37 nucleotides. Cell. 1980 May;20(1):215–222. doi: 10.1016/0092-8674(80)90249-4. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. St John T. P., Scherer S., McDonell M. W., Davis R. W. Deletion analysis of the Saccharomyces GAL gene cluster. Transcription from three promoters. J Mol Biol. 1981 Oct 25;152(2):317–334. doi: 10.1016/0022-2836(81)90245-x. [DOI] [PubMed] [Google Scholar]
  52. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  53. Struhl K. The yeast his3 promoter contains at least two distinct elements. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7385–7389. doi: 10.1073/pnas.79.23.7385. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES