Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Feb 25;18(4):745–751. doi: 10.1093/nar/18.4.745

A mutation in the Zn-finger of the GAL4 homolog LAC9 results in glucose repression of its target genes.

P Kuger 1, A Gödecke 1, K D Breunig 1
PMCID: PMC330322  PMID: 2107531

Abstract

The transcriptional activator LAC9, a GAL4 homolog of Kluyveromyces lactis which mediates lactose and galactose-dependent activation of genes involved in the utilization of these sugars can also confer glucose repression to those genes. Here we report on the isolation and characterization of LAC9-2, an allele which encodes a glucose-sensitive activator in contrast to the one previously cloned. A single amino acid exchange of leu-104 to tryptophan is responsible for the glucose-insensitive phenotype. The mutation is located within the Zn-finger-like DNA binding domain which is highly conserved between LAC9 and GAL4. Glucose repression is also eliminated by duplication of the LAC9-2 allele. The data indicate that LAC9 is a limiting factor for beta-galactosidase gene expression under all growth conditions and that glucose reduces the activity of the activator.

Full text

PDF
745

Images in this article

747Image
on p.747
750Image
on p.750

Selected References

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

  1. Breunig K. D. Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9. Mol Gen Genet. 1989 Apr;216(2-3):422–427. doi: 10.1007/BF00334386. [DOI] [PubMed] [Google Scholar]
  2. Breunig K. D., Kuger P. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site. Mol Cell Biol. 1987 Dec;7(12):4400–4406. doi: 10.1128/mcb.7.12.4400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cherry J. R., Johnson T. R., Dollard C., Shuster J. R., Denis C. L. Cyclic AMP-dependent protein kinase phosphorylates and inactivates the yeast transcriptional activator ADR1. Cell. 1989 Feb 10;56(3):409–419. doi: 10.1016/0092-8674(89)90244-4. [DOI] [PubMed] [Google Scholar]
  4. Denis C. L., Gallo C. Constitutive RNA synthesis for the yeast activator ADR1 and identification of the ADR1-5c mutation: implications in posttranslational control of ADR1. Mol Cell Biol. 1986 Nov;6(11):4026–4030. doi: 10.1128/mcb.6.11.4026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dickson R. C., Markin J. S. Physiological studies of beta-galactosidase induction in Kluyveromyces lactis. J Bacteriol. 1980 Jun;142(3):777–785. doi: 10.1128/jb.142.3.777-785.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dickson R. C., Sheetz R. M., Lacy L. R. Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid. Mol Cell Biol. 1981 Nov;1(11):1048–1056. doi: 10.1128/mcb.1.11.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Entian K. D. Glucose repression: a complex regulatory system in yeast. Microbiol Sci. 1986 Dec;3(12):366–371. [PubMed] [Google Scholar]
  8. Eraso P., Gancedo J. M. Catabolite repression in yeasts is not associated with low levels of cAMP. Eur J Biochem. 1984 May 15;141(1):195–198. doi: 10.1111/j.1432-1033.1984.tb08174.x. [DOI] [PubMed] [Google Scholar]
  9. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  10. Fedor M. J., Lue N. F., Kornberg R. D. Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast. J Mol Biol. 1988 Nov 5;204(1):109–127. doi: 10.1016/0022-2836(88)90603-1. [DOI] [PubMed] [Google Scholar]
  11. Fedor M. J., Lue N. F., Kornberg R. D. Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast. J Mol Biol. 1988 Nov 5;204(1):109–127. doi: 10.1016/0022-2836(88)90603-1. [DOI] [PubMed] [Google Scholar]
  12. Forsburg S. L., Guarente L. Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev. 1989 Aug;3(8):1166–1178. doi: 10.1101/gad.3.8.1166. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnston M., Dover J. Mutations that inactivate a yeast transcriptional regulatory protein cluster in an evolutionarily conserved DNA binding domain. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2401–2405. doi: 10.1073/pnas.84.8.2401. [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. Kim K. S., Guarente L. Mutations that alter transcriptional activation but not DNA binding in the zinc finger of yeast activator HAPI. Nature. 1989 Nov 9;342(6246):200–203. doi: 10.1038/342200a0. [DOI] [PubMed] [Google Scholar]
  19. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Leonardo J. M., Bhairi S. M., Dickson R. C. Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis. Mol Cell Biol. 1987 Dec;7(12):4369–4376. doi: 10.1128/mcb.7.12.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. MAGASANIK B. Catabolite repression. Cold Spring Harb Symp Quant Biol. 1961;26:249–256. doi: 10.1101/sqb.1961.026.01.031. [DOI] [PubMed] [Google Scholar]
  24. Matsumoto K., Uno I., Ishikawa T., Oshima Y. Cyclic AMP may not be involved in catabolite repression in Saccharomyces cerevisiae: evidence from mutants unable to synthesize it. J Bacteriol. 1983 Nov;156(2):898–900. doi: 10.1128/jb.156.2.898-900.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  27. Mylin L. M., Bhat J. P., Hopper J. E. Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator. Genes Dev. 1989 Aug;3(8):1157–1165. doi: 10.1101/gad.3.8.1157. [DOI] [PubMed] [Google Scholar]
  28. Pfeifer K., Arcangioli B., Guarente L. Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell. 1987 Apr 10;49(1):9–18. doi: 10.1016/0092-8674(87)90750-1. [DOI] [PubMed] [Google Scholar]
  29. Riley M. I., Dickson R. C. Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis. J Bacteriol. 1984 May;158(2):705–712. doi: 10.1128/jb.158.2.705-712.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Riley M. I., Hopper J. E., Johnston S. A., Dickson R. C. GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon. Mol Cell Biol. 1987 Feb;7(2):780–786. doi: 10.1128/mcb.7.2.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  32. Rudolph H., Koenig-Rauseo I., Hinnen A. One-step gene replacement in yeast by cotransformation. Gene. 1985;36(1-2):87–95. doi: 10.1016/0378-1119(85)90072-1. [DOI] [PubMed] [Google Scholar]
  33. Ruzzi M., Breunig K. D., Ficca A. G., Hollenberg C. P. Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):991–997. doi: 10.1128/mcb.7.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Salmeron J. M., Jr, Johnston S. A. Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene. Nucleic Acids Res. 1986 Oct 10;14(19):7767–7781. doi: 10.1093/nar/14.19.7767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Salmeron J. M., Jr, Langdon S. D., Johnston S. A. Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80. Mol Cell Biol. 1989 Jul;9(7):2950–2956. doi: 10.1128/mcb.9.7.2950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Selleck S. B., Majors J. E. In vivo DNA-binding properties of a yeast transcription activator protein. Mol Cell Biol. 1987 Sep;7(9):3260–3267. doi: 10.1128/mcb.7.9.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sheetz R. M., Dickson R. C. Lac4 is the structural gene for beta-galactosidase in Kluyveromyces lactis. Genetics. 1981 Aug;98(4):729–745. doi: 10.1093/genetics/98.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sreekrishna K., Dickson R. C. Construction of strains of Saccharomyces cerevisiae that grow on lactose. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7909–7913. doi: 10.1073/pnas.82.23.7909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stanssens P., Opsomer C., McKeown Y. M., Kramer W., Zabeau M., Fritz H. J. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 1989 Jun 26;17(12):4441–4454. doi: 10.1093/nar/17.12.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Struhl K. Negative control at a distance mediates catabolite repression in yeast. 1985 Oct 31-Nov 6Nature. 317(6040):822–824. doi: 10.1038/317822a0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Webster T. D., Dickson R. C. The organization and transcription of the galactose gene cluster of Kluyveromyces lactis. Nucleic Acids Res. 1988 Aug 25;16(16):8011–8028. doi: 10.1093/nar/16.16.8011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winston F., Chumley F., Fink G. R. Eviction and transplacement of mutant genes in yeast. Methods Enzymol. 1983;101:211–228. doi: 10.1016/0076-6879(83)01016-2. [DOI] [PubMed] [Google Scholar]
  45. Witte M. M., Dickson R. C. Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis. Mol Cell Biol. 1988 Sep;8(9):3726–3733. doi: 10.1128/mcb.8.9.3726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wray L. V., Jr, Witte M. M., Dickson R. C., Riley M. I. Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):1111–1121. doi: 10.1128/mcb.7.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zell R., Fritz H. J. DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 1987 Jun;6(6):1809–1815. doi: 10.1002/j.1460-2075.1987.tb02435.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES