Abstract
The methylotrophic yeast Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2. The AOX2 gene is transcribed at a much lower level than the AOX1 gene. Apart from this difference in expression levels, the two genes are regulated similarly. To study the role of cis-acting elements in the promoter region of the AOX2 gene, we constructed expression plasmids in which the human serum albumin (HSA) gene was placed under the control of various deleted or mutated AOX2 promoter derivatives. By analyzing the expression of HSA in P. pastoris transformants, we have identified three cis-acting regulatory elements in the AOX2 promoter. The positive cis-acting element AOX2-UAS, located between positions −337 and −313 (relative to the transcription initiation codon), is required for response to transcriptional induction by methanol in an orientation-independent manner, and artificial amplification of the AOX2-UAS resulted in an increase in the transcriptional activity of the promoter. A sequence homologous to AOX2-UAS was also found in the AOX1 promoter, and in methanol-regulated promoters in other methylotrophic yeast. Two negative cis-acting elements, AOX2-URS1 and AOX2-URS2 play a role in repressing transcription from the AOX2 promoter. The function of AOX2-UAS is completely repressed by this unique repression system when both the AOX2-URS1 and AOX2-URS2 are functional.
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References
Adler FL, Adler LT (1980) Passive hemagglutination and hemolysis for estimation of antigens and antibodies. Methods Enzymol 70:455–466
Anthony C (1982) The biochemistry of methylotrophs. Academic Press, New York
Couderc R, Baratti J (1980) Oxidation of methanol by the yeast Pichia pastoris. Purification and properties of alcohol oxidase. Agric Biol Chem 44:2279–2289
Cregg JM, Madden KR (1987) Development of yeast transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. In: Stewart GG, Russell I, Klein RD, Hiebsch RR (eds) Biological research on industrial yeasts, vol. II. CRC Press, Boca Raton, Florida, pp 1–18
Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as a host system for transformations. Mol Cell Biol 5:3376–3385
Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9:1316–1323
Denis CL, Young ET (1983) Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae. Mol Cell Biol 3:360–370
Denis CL, Ciriacy M, Young ET (1981) A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae. J Mol Biol 148:355–368
Digan ME, Tschopp J, Grinna L, Lair SV, Craig WS, Velicelebi G, Siegel R, Davis GR, Thill G (1988) Secretion of heterologous proteins from the methylotrophic yeast, Pichia pastoris. Dev Ind Microbiol 29:59–65
Eisen AW, Taylor E, Blumberg H, Young ET (1988) The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2. Mol Cell Biol 8:4552–4556
Ellis SB, Brust PF, Koutz PJ, Waters AF, Harpold MM, Gingeras TR (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol 5:1111–1121
Flick JS, Jonstone M (1990) Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae. Mol Cell Biol 10:4757–4769
Flick JS, Johnston M (1991) GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats. Mol Cell Biol 11:5101–5112
Finley RLJ, Chen, S, Ma J, Byrne P, West RWJ (1990). Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes. Mol Cell Biol 10:5663–5670
Ghosh D (1993) Transcription factor data base (TFD) ver. 6.2. NIH National Center for Biotechnology Information.
Giniger E, Varnum SM, Ptashne M (1985) Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell 40:767–774
Griggs DW, Johnston M (1991) Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression. Proc Natl Acad Sci USA 88:8597–8601
Guarente L, Hoar E (1984) Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the “TATA box”. Proc Natl Acad Sci USA 81:7860–7864
Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali canons. J Bacteriol 153:163–168
Ito W, Ishiguro H, Kurosawa Y (1991) A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene 102:67–70
Janowicz ZA, Eckart MR, Drewke C, Roggenkamp RO, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT (1985) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res 13:3043–3062
Koutz P, Davis GR, Stillman C, Barringer K, Cregg J, Thill G (1989) Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5:167–177
Kumagai MH, Sverlow GG, della-Cioppa G, Grill LK (1993) Conversion of starch to ethanol in a recombinant Saccharomyces cerevisiae strain expressing rise α -amylase from a novel Pichia pastoris alcohol oxidase promoter. Bio/Technology 11:606–610
Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz Z, Eckart M, Roggenkamp R, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13:3063–3082
Lorch Y, Kornberg RD (1985) A region flanking the GAL7 gene and a binding site for GAL4 protein as upstream activating sequences in yeast. J Mol Biol 186:821–824
Nehlin JO, Carlberg M, Ronne H (1991) Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J 10:3373–3377
Sakai Y, Tani Y (1992) Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast Candida boidinii S2. Gene 114:67–73
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schultz J, Carlson M (1987) Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae. Mol Cell Biol 7:3637–3645
Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Shuster J, Yu J, Cox D, Chan RVL, Smith M, Young E (1986) ADR1-mediated regulation of ADH2 requires an inverted repeat sequence. Mol Cell Biol 6:1894–1902
Trumbly RJ (1988) Cloning and characterization of the CYC8 gene mediating glucose repression in yeast. Gene 73:97–111
Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15:3859–3876
Veenhuis M, van Dijken JP, Harder W (1983) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol 24:1–82
Verdier JM (1990) Regulatory DNA-binding proteins in yeast: an overview. Yeast 6:271–297
Williams FE, Trumbly RJ (1990) Characterization of TUP1, a mediator for glucose respression in Saccharomyces cerevisiae. Mol Cell Biol 10:6500–6511
Williamson VM, Cox D, Young ET, Russell DW, Smith M (1983) Characterization of transposable element-associated mutations that alter yeast alcohol dehydrogenase II expression. Mol Cell Biol 3:20–31
Yu J, Donoviel MS, Young ET (1989) Adjacent upstream activation sequence elements synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae. Mol Cell Biol 9:34–42
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Ohi, H., Miura, M., Hiramatsu, R. et al. The positive and negative cis-acting elements for methanol regulation in the Pichia pastoris AOX2 gene. Molec. Gen. Genet. 243, 489–499 (1994). https://doi.org/10.1007/BF00284196
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DOI: https://doi.org/10.1007/BF00284196