Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 May;5(5):1111–1121. doi: 10.1128/mcb.5.5.1111

Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris.

S B Ellis, P F Brust, P J Koutz, A F Waters, M M Harpold, T R Gingeras
PMCID: PMC366829  PMID: 3889590

Abstract

The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.

Full text

PDF
1111

Images in this article

1114Image
on p.1114
1115Image
on p.1115
1116Image
on p.1116
1117Image
on p.1117
1118Image
on p.1118

Selected References

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

  1. Arima K., Oshima T., Kubota I., Nakamura N., Mizunaga T., Toh-e A. The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide. Nucleic Acids Res. 1983 Mar 25;11(6):1657–1672. doi: 10.1093/nar/11.6.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooney C. L., Levine D. W. Microbial utilization of methanol. Adv Appl Microbiol. 1972;15:337–365. doi: 10.1016/s0065-2164(08)70096-0. [DOI] [PubMed] [Google Scholar]
  6. Dobson M. J., Tuite M. F., Roberts N. A., Kingsman A. J., Kingsman S. M., Perkins R. E., Conroy S. C., Fothergill L. A. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982 Apr 24;10(8):2625–2637. doi: 10.1093/nar/10.8.2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fukui S., Tanaka A., Kawamoto S., Yasuhara S., Teranishi Y., Osumi M. Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity. J Bacteriol. 1975 Jul;123(1):317–328. doi: 10.1128/jb.123.1.317-328.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldman B. M., Blobel G. Biogenesis of peroxisomes: intracellular site of synthesis of catalase and uricase. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5066–5070. doi: 10.1073/pnas.75.10.5066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goodman J. M., Scott C. W., Donahue P. N., Atherton J. P. Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii. J Biol Chem. 1984 Jul 10;259(13):8485–8493. [PubMed] [Google Scholar]
  10. Hanahan D., Meselson M. Plasmid screening at high colony density. Gene. 1980 Jun;10(1):63–67. doi: 10.1016/0378-1119(80)90144-4. [DOI] [PubMed] [Google Scholar]
  11. Harpold M. M., Dobner P. R., Evans R. M., Bancroft F. C. Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence. Nucleic Acids Res. 1978 Jun;5(6):2039–2053. doi: 10.1093/nar/5.6.2039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harpold M. M., Evans R. M., Salditt-Georgieff M., Darnell J. E. Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell. 1979 Aug;17(4):1025–1035. doi: 10.1016/0092-8674(79)90341-6. [DOI] [PubMed] [Google Scholar]
  13. Hazeu W., de Bruyn J. C., Bos P. Methanol assimilation by yeasts. Arch Mikrobiol. 1972;87(2):185–188. doi: 10.1007/BF00425000. [DOI] [PubMed] [Google Scholar]
  14. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  15. Kato N., Sakazawa C., Nishizawa T., Tani Y., Yamada H. Purificaton and characterization of S-formylglutathione hydrolase from a methanol-utilizing yeast, Kloeckera sp. No. 2201. Biochim Biophys Acta. 1980 Feb 14;611(2):323–332. doi: 10.1016/0005-2744(80)90068-6. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 1981 Oct 24;9(20):5233–5252. doi: 10.1093/nar/9.20.5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Montgomery D. L., Leung D. W., Smith M., Shalit P., Faye G., Hall B. D. Isolation and sequence of the gene for iso-2-cytochrome c in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jan;77(1):541–545. doi: 10.1073/pnas.77.1.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Neben I., Sahm H., Kula M. R. Studies on an enzyme, S-formylglutathione hydrolase, of the dissimilatory pathway of methanol in Candida boidinii. Biochim Biophys Acta. 1980 Jul 10;614(1):81–91. doi: 10.1016/0005-2744(80)90169-2. [DOI] [PubMed] [Google Scholar]
  21. Payvar F., Schimke R. T. Methylmercury hydroxide enhancement of translation and transcription of ovalbumin and conalbumin mRNA's. J Biol Chem. 1979 Aug 25;254(16):7636–7642. [PubMed] [Google Scholar]
  22. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Roa M., Blobel G. Biosynthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6872–6876. doi: 10.1073/pnas.80.22.6872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robbi M., Lazarow P. B. Synthesis of catalase in two cell-free protein-synthesizing systems and in rat liver. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4344–4348. doi: 10.1073/pnas.75.9.4344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Sahm H., Roggenkamp R., Wagner F., Hinkelmann W. Microbiodies in methanol-grown Candida boidinii. J Gen Microbiol. 1975 Jun;88(2):218–222. doi: 10.1099/00221287-88-2-218. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  28. Shields D., Blobel G. Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes. Proc Natl Acad Sci U S A. 1977 May;74(5):2059–2063. doi: 10.1073/pnas.74.5.2059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Tschumper G., Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980 Jul;10(2):157–166. doi: 10.1016/0378-1119(80)90133-x. [DOI] [PubMed] [Google Scholar]
  31. Veenhuis M., van Dijken J. P., Pilon S. A., Harder W. Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions. Arch Microbiol. 1978 May 30;117(2):153–163. doi: 10.1007/BF00402303. [DOI] [PubMed] [Google Scholar]
  32. van Dijkan J. P., Veenhuis M., Harder W. Peroxisomes of methanol-grown yeasts. Ann N Y Acad Sci. 1982;386:200–216. doi: 10.1111/j.1749-6632.1982.tb21417.x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. van Dijken J. P., Veenhuis M., Kreger-van Rij N. J., Harder W. Microbodies in methanol-assimilating yeasts. Arch Microbiol. 1975;102(1):41–44. doi: 10.1007/BF00428343. [DOI] [PubMed] [Google Scholar]

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

RESOURCES