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. 1987 May 11;15(9):3859–3876. doi: 10.1093/nar/15.9.3859

Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris.

J F Tschopp, P F Brust, J M Cregg, C A Stillman, T R Gingeras
PMCID: PMC340787  PMID: 3108861

Abstract

Two DNA fragments containing putative control regions regulating the expression of the alcohol oxidase (AOX) and dihydroxy-acetone synthase (DAS) genes from the methylotrophic yeast Pichia pastoris were used in the construction of vectors for the expression of the Escherichia coli lacZ gene. These vectors were transformed into P. pastoris host cells and employed in experiments to measure the control mechanisms employed by each promoter in the production of beta-galactosidase fusion products. Results in P. pastoris suggest that the processes used to regulate the expression of these gene fusions involve both repression/derepression and induction mechanisms. Expression of the AOX-lacZ and DAS-lacZ fusions was examined in Saccharomyces cerevisiae as well. Interestingly, beta-galactosidase was expressed in a regulated manner in the heterologous host.

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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. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  3. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen C. Y., Oppermann H., Hitzeman R. A. Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 1984 Dec 11;12(23):8951–8970. doi: 10.1093/nar/12.23.8951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cregg J. M., Barringer K. J., Hessler A. Y., Madden K. R. Pichia pastoris as a host system for transformations. Mol Cell Biol. 1985 Dec;5(12):3376–3385. doi: 10.1128/mcb.5.12.3376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Douglas M. G., Geller B. L., Emr S. D. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983–3987. doi: 10.1073/pnas.81.13.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duntze W., Neumann D., Gancedo J. M., Atzpodien W., Holzer H. Studies on the regulation and localization of the glyoxylate cycle enzymes in Saccharomyces cerevisiae. Eur J Biochem. 1969 Aug;10(1):83–89. doi: 10.1111/j.1432-1033.1969.tb00658.x. [DOI] [PubMed] [Google Scholar]
  10. Eggeling L., Sahm H. Regulation of alcohol oxidase synthesis in Hansenula polymorpha: oversynthesis during growth on mixed substrates and induction by methanol. Arch Microbiol. 1980 Sep;127(2):119–124. doi: 10.1007/BF00428015. [DOI] [PubMed] [Google Scholar]
  11. Ellis S. B., Brust P. F., Koutz P. J., Waters A. F., Harpold M. M., Gingeras T. R. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol. 1985 May;5(5):1111–1121. doi: 10.1128/mcb.5.5.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Emr S. D., Schauer I., Hansen W., Esmon P., Schekman R. Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2347–2355. doi: 10.1128/mcb.4.11.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Girvitz S. C., Bacchetti S., Rainbow A. J., Graham F. L. A rapid and efficient procedure for the purification of DNA from agarose gels. Anal Biochem. 1980 Aug;106(2):492–496. doi: 10.1016/0003-2697(80)90553-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  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. 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]
  20. 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]
  21. Mellor J., Dobson M. J., Roberts N. A., Kingsman A. J., Kingsman S. M. Factors affecting heterologous gene expression in Saccharomyces cerevisiae. Gene. 1985;33(2):215–226. doi: 10.1016/0378-1119(85)90096-4. [DOI] [PubMed] [Google Scholar]
  22. Roggenkamp R., Janowicz Z., Stanikowski B., Hollenberg C. P. Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet. 1984;194(3):489–493. doi: 10.1007/BF00425563. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  25. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tschopp J. F., Emr S. D., Field C., Schekman R. GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J Bacteriol. 1986 Apr;166(1):313–318. doi: 10.1128/jb.166.1.313-318.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Veenhuis M., Van Dijken J. P., Harder W. The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol. 1983;24:1–82. doi: 10.1016/s0065-2911(08)60384-7. [DOI] [PubMed] [Google Scholar]
  29. Zitomer R. S., Montgomery D. L., Nichols D. L., Hall B. D. Transcriptional regulation of the yeast cytochrome c gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3627–3631. doi: 10.1073/pnas.76.8.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. ten Berge A. M., Zoutewelle G., Needleman R. B. Regulation of maltose fermentation in Saccharomyces carlsbergensis. 3. Constitutive mutations at the MAL6-locus and suppressors changing a constitutive phenotype into a maltose negative phenotype. Mol Gen Genet. 1974;131(2):113–121. doi: 10.1007/BF00266147. [DOI] [PubMed] [Google Scholar]
  31. van Dijken J. P., Otto R., Harder W. Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol. 1976 Dec 1;111(1-2):137–144. doi: 10.1007/BF00446560. [DOI] [PubMed] [Google Scholar]

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