Lipids

, Volume 35, Issue 3, pp 257–262

Cloning and sequencing of the Candida albicans C-4 sterol methyl oxidase gene (ERG25) and expression of an ERG25 conditional lethal mutation in Saccharomyces cerevisiae

  • Matthew A. Kennedy
  • Theresa A. Johnson
  • N. Douglas Lees
  • Robert Barbuch
  • James A. Eckstein
  • Martin Bard
Article

Abstract

The ERG25 gene encoding the Candida albicans C-4 sterol methyl oxidase was cloned and sequenced by complementing a Saccharomyces cerevisiae erg25 mutant with a C. albicans genomic library. The Erg25p is comprised of 308 amino acids and shows 65 and 38% homology to the enzymes from S. cerevisiae and Homo sapiens, respectively. The protein contains three histidine clusters common to nonheme iron-binding enzymes and an endoplasmic reticulum retrieval signal as do the proteins from S. cerevisiae and humans. A temperature-sensitive (ts) conditional lethal mutation of the C. albicans ERG25 was isolated and expressed in S. cerevisiae. Sequence analysis of the ts mutant indicated an amino acid substitution within the region of the protein encompassed by the histidine clusters involved in iron binding. Results indicate that plasmid-borne conditional lethal mutants of target genes have potential use in the rescue of Candida mutations in genes that are essential for viability.

Abbreviations

ERG25

C-4 methyl oxidase gene

ERG26

C-3 sterol dehydrogenase

GC

gas chromatography

plasmid pCERG25

the original complementing clone of 4kb in the Candida albicans shuttle vector YPB1

SD

synthetic dropout

ts

temperature-sensitive

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References

  1. 1.
    White, T.C., Marr, K.A., and Bowden, R.A. (1998) Clinical, Cellular, and Molecular Factors That Contribute to Antifungal Resistance, Clin. Microbiol. Rev. 11, 382–402.PubMedGoogle Scholar
  2. 2.
    Daum, G., Lees, N.D., Bard, M., and Dickson, R. (1998) Biochemistry, Cell Biology, and Molecular Biology of Lipids of Saccharomyces cerevisiae, Yeast 14, 1471–1510.PubMedCrossRefGoogle Scholar
  3. 3.
    Lees, N.D., Bard, M., and Kirsch, D.R. (1997) Biochemistry and Molecular Biology of Sterol Synthesis in Saccharomyces cerevisiae, in Biochemistry and Function of Sterols (Parish, E.J., and Nes, W.D., eds.) pp. 85–99, CRC Press, Boca Raton.Google Scholar
  4. 4.
    Jensen-Pergakes, K.L., Kennedy, M.A., Lees, N.D., Barbuch, R., Koegel, C., and Bard, M. (1998) Sequencing, Disruption, and Characterization of the Candida albicans Sterol Methyltransferase (ERG6) Gene: Drug Susceptibility Studies in erg6 Mutants, Antimicrob. Agents Chemother. 42, 1160–1167.PubMedGoogle Scholar
  5. 5.
    Gaber, R.F., Copple, D.M., Kennedy, B.K., Vidal, M., and Bard, M. (1989) The Yeast Gene ERG6 Is Required for Normal Membrane Function but Is Not Essential for Biosynthesis of the Cell-Cycle Sparking Sterol, Mol. Cell Biol. 9, 3447–3456.PubMedGoogle Scholar
  6. 6.
    Lees, N.D., Lofton, S.L., Woods, R.A., and Bard, M. (1980) The Effects of Varied Energy Source and Detergent on the Growth of Sterol Mutants of Saccharomyces cerevisiae, J. Gen. Microbiol. 118, 209–214.Google Scholar
  7. 7.
    Bard, M., Bruner, D.A., Pierson, C.A., Lees, N.D., Biermann, B., Frye, L., Koegel, C., and Barbuch, R. (1996) Cloning and Characterization of ERG25, the Saccharomyces cerevisiae Gene Encoding the C-4 Sterol Methyl Transferase, Proc. Natl. Acad. Sci. USA 93, 186–190.PubMedCrossRefGoogle Scholar
  8. 8.
    Gachotte, D., Barbuch, R., Gaylor, J., Nickel, E., and Bard, M. (1998) Characterization of the Saccharomyces cerevisiae ERG26 Gene Encoding the C-3 Sterol Dehydrogenase (C-4 decarboxylase) Involved in Sterol Biosynthesis, Proc. Natl. Acad. Sci. USA 95, 13794–13799.PubMedCrossRefGoogle Scholar
  9. 9.
    Fonzi, W.A., and Irwin, M.Y. (1993) Isogenic Strain Construction and Gene Mapping in Candida albicans, Genetics 134, 717–728.PubMedGoogle Scholar
  10. 10.
    Gietz, R.D., Schiestl, R.H., Willems, A.R., and Woods, R.A. (1995) Studies on the Transformation of Intact Yeast Cells by the LiAc/SS-DNA/PEG Procedure, Yeast 11, 355–360.PubMedCrossRefGoogle Scholar
  11. 11.
    Greener, A., Callahan, M., and Jerpseth, B. (1996) An Efficient Random Mutagenesis Technique Using an E. coli Mutator Strain, Methods Mol. Biol. 57, 375–385.PubMedGoogle Scholar
  12. 12.
    Goshorn, A.K., Grindle, S.M., and Scherer, S. (1992) Gene Isolation by Complementation in Candida albicans and Applications to Physical and Genetic Mapping, Infect. Immun. 60, 876–884.PubMedGoogle Scholar
  13. 13.
    Santos, M.A.S., Perreau, V.M., and Tuite, M.F. (1996) Transfer RNA Structural Change Is a Key Element in the Reassignment of the CUG Codon in Candida albicans, EMBO J. 15, 5060–5068.PubMedGoogle Scholar
  14. 14.
    Li, L., and Kaplan, J. (1996) Characterization of Yeast Methyl Sterol Oxidase (ERG25) and Identification of a Human Homologue, J. Biol. Chem. 271, 16927–16933.PubMedCrossRefGoogle Scholar
  15. 15.
    Shanklin, J., Achim, C., Schmidt, H., Fox, B.G., and Munck, E. (1997) Mossbauer Studies of Alkane ω-Hydroxylase: Evidence for a Diiron Cluster in an Integral-Membrane Protein, Proc. Natl. Acad. Sci. USA 94, 2981–2986.PubMedCrossRefGoogle Scholar
  16. 16.
    Arthington, B.A., Bennett, L.G., Skatrud, P.L., Guynn, C.J., Barbuch, R.J., Ulbright, C.E., and Bard, M. (1991) Cloning, Disruption, and Sequence of the Gene Encoding Yeast C-5 Desaturase, Gene 102, 39–44.PubMedCrossRefGoogle Scholar
  17. 17.
    Bard, M., Lees, N.D., Turi, T., Craft, D., Coffrin, L., Barbuch, R., Koegel, C., and Loper, J.C. (1993) Sterol Synthesis and Viability of erg11 (cytochrome P-450 lanosterol demethylase) Mutations in Saccharomyces cerevisiae and Candida albicans, Lipids 28, 963–967.PubMedGoogle Scholar
  18. 18.
    Lorenz, R.T., and Parks, L.W. (1992) Cloning, Sequencing, and Disruption of the Gene Encoding the C-14 Reductase in Saccharomyces cerevisiae, DNA Cell Biol. 11, 685–692.PubMedCrossRefGoogle Scholar
  19. 19.
    Kurtz, M.B., and Marrinan, J. (1989) Isolation of hem3 Mutants from Candida albicans by Sequential Gene Disruptions, Mol. Gen. Genet. 217, 47–52.PubMedCrossRefGoogle Scholar

Copyright information

© AOCS Press 2000

Authors and Affiliations

  • Matthew A. Kennedy
    • 2
  • Theresa A. Johnson
    • 2
  • N. Douglas Lees
    • 2
  • Robert Barbuch
    • 1
  • James A. Eckstein
    • 1
  • Martin Bard
    • 2
  1. 1.Department of Drug Disposition, Eli Lilly and CompanyLilly Corporate CenterIndianapolis
  2. 2.Department of BiologyIndiana University Purdue University IndianapolisIndianapolis

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