Summary
We have suggested previously from Northern blot analysis that transcription of the negative regulatory gene GAL80 was controlled positively by another regulatory gene GALA, and negatively by GAL80 itself, in similar way to GAL1, GAL7 and GAL10 genes encoding galactosemetabolizing enzymes in Saccharomyces cerevisiae. To study further the controlled expression of GAL80, we have exploited the gene fusion technique. We constructed gene fusions consisting of 5′ fragments of GAL80 and a 5′ truncated lacZ of Escherichia coli, and introduced the GAL80‘-’lacZ fusions into wild-type yeast or various GALA or GAL80 mutants using multiple-copy or single-copy plasmid vectors. We then studied β-galactosidase activity in the resultant transformants under uninduced, induced or glucose-repressed conditions. Expression of the GAL80‘-’lacZ fusions was clearly under the control of Gal4/Gal80. Next we constructed GAL7‘-’lacZ fusions, whose upstream activating sequence (UAS) from GAL7 was replaced with a GAL80 fragment containing a UAS-like sequence located in the 5′ flanking region of GAL80. Synthesis of β-galactosidase directed by the hybrid genes was inducible by galactose exactly like the original GAL7‘-’lacZ fusion with a UAS from GAL7. Finally we constructed a GAL7-GAL80 hybrid gene, in which the entire 5′ flanking region was derived from GAL7. When the chromosomal GAL80 gene in wild-type yeast was replaced with the hybrid gene, the uninduced level, but not the induced level, of the GAL10-encoded enzyme (uridine diphosphoglucose-4-epimerase) was significantly increased.
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References
Adams BG (1972) Induction of galactokinase in Saccharomyces cerevisiae: Kinetics of induction and glucose effects. J Bacteriol 111:308–313
Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW (1979) Sterile host yeast (SHY): A eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17–24
Bram RJ, Kornberg RD (1985) Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci USA 82:43–47
Bram RJ, Lue NF, Kornberg RD (1986) A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription. EMBO J 5:603–608
Casadaban MJ, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980
Casadaban M, Martinez-Arias A, Shapira SK, Chou J (1983) β-galactosidase gene fusions for analyzing gene expression of Escherichia coli and yeast. Methods Enzymol 100:293–308
Douglas HC, Hawthorne DC (1964) Enzymatic expression and genetic linkage of genes controlling galactose utilization in Saccharomyces cerevisiae. Genetics 49:837–844
Douglas HC, Hawthorne DC (1966) Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics 54:911–916
Douglas HC, Pelroy G (1963) Gene controlling inducibility of galactose pathway enzymes in Saccharomyces. Biochim Biophys Acta 52:155–156
Fukasawa T, Obonai K, Segawa T, Nogi Y (1980) The enzymes of the galactose cluster in Saccharomyces cerevisiae II. Purification and characterization of uridine diphosphoglucose-4-epimerase. J Biol Chem 255:2705–2707
Giniger E, Varnum SM, Ptashne M (1985) Specific DNA Binding of GAL4, a positive regulatory protein. Cell 40:767–774
Guarente L (1983) Yeast promoter and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol 101:181–191
Guarente L (1984) Yeast promoters: Positive and negative elements. Cell 36:799–800
Hashimoto H, Kikuchi Y, Nogi Y, Fukasawa T (1983) Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae: Isolation and characterization of the regulatory gene GAL4. Mol Gen Genet 191:31–38
Johnston SA, Hopper JE (1982) Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci USA 79:6971–6975
Laughon A, Gesteland RF (1982) Isolation and preliminary characterization of the GAL4 gene, an positive regulator of transcription in yeast. Proc Natl Acad Sci USA 79:6827–6831
Laughon A, Gesteland RF (1984) Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol 4:260–267
Martinez-Arias A, Yost HJ, Casadaban MJ (1984) Role of an upstream regulatory element in leucine repression of the Saccharomyces cerevisiae leu2 gene. Nature 307:740–742
Miller JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY pp 352–355
Nogi Y, Fukasawa T (1984) Nucleotide sequence of the yeast regulatory gene GAL80. Nuclei Acids Res 12:9287–9298
Nogi Y, Matsumoto K, Toh-e A, Oshima Y (1977) Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae. Mol Gen Genet 153:137–144
Nogi Y, Shimada H, Matsuzaki Y, Hashimoto H, Fukasawa T (1984) 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 195:29–34
Oshima Y (1982) Regulatory circuits for gene expression: The metabolism of galactose and phosphate. In: Strathern J, Jones E, Broach J (eds) The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY pp 159–180
Shimada H, Fukasawa T (1985) Controlled transcription of the yeast regulatory gene GAL80. Gene 39:1–9
Sripati CE, Warner JR (1978) Isolation, characterization, and translation of mRNA from yeast. Methods cell Biol 20:61–81
Stinchcomb DT, Mann C, Davis RW (1982) Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol 158:157–179
St John TP, Davis RW (1981) The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol 152:285–315
Tajima M, Nogi Y, Fukasawa T (1985) Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1:67–77
Tajima M, Nogi Y, Fukasawa T (1986) Duplicate upstream activating sequences in promoter region of the Saccharomyces cerevisiae GAL7 gene. Mol Cell Biol 6:246–256
Torchia TE, Hamilton RW, Cano CL, Hopper JE (1984) Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: Effects on carbon controlled regulation on the galactose/melibiose pathway genes. Mol Cell Biol 4:1521–1527
West RW Jr, Yocum RR, Ptashne M (1984) Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: Location and function of the upstream activating sequence UASG. Mol Cell Biol 4:2467–2478
Yocum RR, Hanley S, West R Jr, Ptashne M (1984) Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol 4:1985–1998
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Communicated by M. Takanami
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Igarashi, M., Segawa, T., Nogi, Y. et al. Autogenous regulation on the Saccharomyces cerevisiae regulatory gene GAL80 . Mol Gen Genet 207, 273–279 (1987). https://doi.org/10.1007/BF00331589
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DOI: https://doi.org/10.1007/BF00331589