Skip to main content
SpringerLink
Log in

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including β-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Assaraf YG, Schimke RT (1987) Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry. Proc Nat Acad Sci USA 84:7154–7158

    Google Scholar 

  • Baccanari DP, Tansik RL, Joyner SS, Fling ME, Smith PL, Freisheim JH (1989) Characterization of Candida albicans dihydrofolate reductase. J Biol Chem 264:1100–1107

    Google Scholar 

  • Baldari C, Cesareni G (1985) Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene 35:27–32

    Google Scholar 

  • Balzi E, Chen W, Ulaszewski S, Capicaux E, Goffeau A (1987) The multidrug resistance gene PDR1 from Saccharomyces cerevisiae. J Biol Chem 262:16871–16879

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    CAS  PubMed  Google Scholar 

  • Cowan KH, Jolivet J (1984) A methotrexate-resistant human breast cancer line with multiple defects, including diminished formation of methotrexate polyglutamates. J Biol Chem 259:10793–10800

    Google Scholar 

  • Davidse LC, Flach W (1978) Differential binding of methyl benzimidazole-1-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J Cell Biol 72:174–193

    Google Scholar 

  • Endicott JA, Ling V (1989) The biochemistry of p-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 58:137–171

    Article  CAS  PubMed  Google Scholar 

  • Falco SC, Dumas KS (1985) The genetic analysis of mutants of Saccharomyces cerevisiae resistant to the herbicide sulfo-meturon methyl. Genetics 109:21–35

    Google Scholar 

  • Fling ME, Kopf J, Richards CA (1988) Nucleotide sequence of the dihydrofolate reductase gene of Saccharomyces cerevisiae. Gene 63:165–174

    Google Scholar 

  • Flintoff WF, Nagainis CR (1983) Transport of methotrexate in Chinese hamster kidney cells: A mutant defective in methotrexate uptake and cell binding. Arch Bioch Biophys 223:433–440

    Google Scholar 

  • Goldie JH, Krystal G, Hartley D, Gudauskas G, Dedhar S (1980) A methotrexate insensitive variant of folate reductase present in two lines of methotrexate-resistant L5178Y cells. Eur J Cancer 16:1539–1546

    Google Scholar 

  • Gömpel-Klein P, Brendel M (1990) Allelism of SNQ1 and ATR1, genes of the yeast Saccharomyces cerevisiae required for controlling sensitivity to 4-nitroquinoline-N-oxide and aminotriazole. Curr Genet 18:93–96

    Google Scholar 

  • Gorman JA, Dove WF, Warren N (1981) Isolation of Physarum DNA segments that support autonomous replication in yeast. Mol Gen Genet 183:306–313

    Google Scholar 

  • Haber DA, Beverley SM, Kiely ML, Schimke RT (1981) Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem 256:9501–9510

    Google Scholar 

  • Henderson GB, Suresh MR, Vitals KS, Huennekens FM (1986) Transport of folate compounds in L1210 cells: Kinetic evidence that folate influx proceeds via the high affinity transport system for 5-methylhydrofolate and methotrexate. Cancer Res 46:1639–1643

    Google Scholar 

  • Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359

    Google Scholar 

  • Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167

    Google Scholar 

  • Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272

    Article  CAS  PubMed  Google Scholar 

  • Hoffman W (1985) Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae. J Biol Chem 260:11831–11837

    Google Scholar 

  • Huffaker TC, Thomas JH, Botstein D (1987) Diverse effects of β-tubulin mutations on microtubule formation and function. J Cell Biol 106:1997–2010

    Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    Google Scholar 

  • Kanazawa S, Driscoll M, Struhl K (1988) ATR1, a Saccharomyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance. Mol Cell Biol 8:664–673

    Google Scholar 

  • Khan MI, Ecker DJ, Butt T, Gorman JA, Crooke ST (1987) A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. Plasmid 17:171–172

    Google Scholar 

  • Kuchler K, Sterne RE, Thorner J (1989) Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells. EMBO J 8:3973–3984

    Google Scholar 

  • Kwon-Chung KJ, Hill HB (1970) Studies on the pink adenine deficient strain of Candida albicans. 1. Cultural and morphological characteristics. Sabouraudia 8:48–54

    Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    CAS  PubMed  Google Scholar 

  • Lai MH, Kirsch DR (1989) Nucleotide sequence of cytochrome P450 L1A1 (lanosterol 14α-demethylase) from Candida albicans. Nucleic Acids Res 17:804

    Google Scholar 

  • Leppert G, McDevitt R, Falco SC, Van Dyk TK, Ficke MB, Golin J (1990) Cloning by gene amplification of two loci conferring multiple drug resistance in Saccharomyces. Genetics 125:13–20

    Google Scholar 

  • Lorian V (ed) (1986) Gradient plate technique. In: Antibiotics and laboratory medicine, 2nd edn, p 10

  • Lorincz A (1984) Quick preparation of plasmid DNA from yeast. Focus 6:11

    Google Scholar 

  • Losberger C, Ernst JF (1989a) Sequences of the Candida albicans gene encoding actin. Nucleic Acids Res 17:9488

    Google Scholar 

  • Losberger C, Ernst JF (1989b) Sequence and transcript analysis of the C. albicans URA3 gene encoding orotidine-5′-phosphate decarboxylase. Curr Genet 16:153–157

    Google Scholar 

  • Lott TJ, Page LS, Boiron P, Benson J, Reiss E (1989) Nucleotide sequence of the Candida albicans aspartyl proteinase gene. Nucleic Acids Res 17:1779

    Google Scholar 

  • Magee BB, Koltin Y, Gorman JA, Magee PT (1988) Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes. Mol Cell Biol 8:4721–4726

    Google Scholar 

  • McGrath JP, Varshavsky A (1989) The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature 340:400–404

    Google Scholar 

  • Nakamaye K, Eckstein F (1986) Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res 14:9679–9698

    Google Scholar 

  • National Committee for Clinical Laboratory Standards (1980) Proposed Standard PSM-7. Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa.

    Google Scholar 

  • Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for beta-tubulin from a benoyml-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461

    Google Scholar 

  • Quinlan RA, Pogson CI, Gull K (1980) The influence of the microtubule inhibitor methyl benzimidazole-2-yl-carbamate (MBC) on nuclear division and cell cycle in Saccharomyces cerevisiae. J Cell Sci 46:341–352

    Google Scholar 

  • Rine J, Hansen W, Hardeman E, Davis RW (1983) Targeted selection of recombinant clones through gene dosage effects. Proc Natl Acad Sci USA 80:6750–6754

    Google Scholar 

  • Rosenbluh A, Mevarech M, Koltin Y, Gorman JA (1985) Isolation of genes from Candida albicans by complementation in Saccharomyces cerevisiae. Mol Gen Genet 200:500–502

    Google Scholar 

  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

    CAS  PubMed  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    CAS  PubMed  Google Scholar 

  • Saunders GW, Rank GH (1982) Allelism of pleiotropic drug resistance in Saccharomyces cerevisiae. Can J Genet Cytol 24:493–502

    Google Scholar 

  • Schimke RT, Kaufman RJ, Alt FW, Kellems RF (1978) Gene amplification and drug resistance in cultured murine cells. Science 202:1057–1055

    Google Scholar 

  • Sharp PM, Tuohy TMF, Mosurski KR (1986) Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res 14:5125–5143

    Google Scholar 

  • Sharp PM, Cowe E, Higgins DG, Shields DC, Wolfe KH, Wright F (1988) Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity. Nucleic Acids Res 16:8207–8211

    Google Scholar 

  • Sheir-Neiss G, Lai MH, Morris NR (1978) Identification of a gene for beta-tubulin in Aspergillus nidulans. Cell 15:639–647

    Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Short JM, Fernandez JM, Sorge JA, Huse WD (1988) Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600

    CAS  PubMed  Google Scholar 

  • Singer SC, Richards CA, Ferone R, Benedict D, Ray P (1989) Cloning, purification, and properties of Candida albicans thymidylate synthase. J Bacteriol 171:1372–1378

    Google Scholar 

  • Sirotnak FM, Moccino DM, Kelleher LE, Goutas LJ (1981) Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant L1210 clonal cell lines derived in vivo. Cancer Res 41:4447–4452

    Google Scholar 

  • Smith HA, Allaudeen HS, Whitman MH, Koltin Y, Gorman JA (1988) Isolation and characterization of a β-tubulin gene from Candida albicans. Gene 63:53–63

    Google Scholar 

  • Smith HA, Gorman JW, Koltin Y, Gorman JA (1990) Functional expression of the Candida albicans β-tubulin gene in Saccharomyces cerevisiae. Gene 90:115–123

    Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    CAS  PubMed  Google Scholar 

  • Stearns T, Botstein D (1988) Unlinked noncomplementation: Isolation of new conditional-lethal mutations in each of the tubulin genes of Saccharomyces cerevisiae. Genetics 119:249–260

    Google Scholar 

  • Subik J, Ulaszewski S, Goffeau A (1986) Genetic mapping mucidin resistance mutations in Saccharomyces cerevisiae: a new PDR locus on chromosome II. Curr Genet 10:665–670

    Google Scholar 

  • Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771

    Google Scholar 

  • Taylor MW, Schmidt W, Cosstick R, Okruszek A, Eckstein F (1985a) The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res 13:8749–8764

    Google Scholar 

  • Taylor JW, Ott J, Eckstein F (1985b) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13:8764–8785

    Google Scholar 

  • Thomas JH, Neff NF, Botstein D (1985) Isolation and characterization of mutations in the β-tubulin gene of Saccharomyces cerevisiae. Genetics 112:715–734

    Google Scholar 

  • Umesono K, Toda T, Hayashi S, Yanagida M (1983) Two cell cycle genes NDA2 and NDA3 of the fission yeast Schizosaccharomyces pombe control microtubular organization and sensitivity to anti-mitotic benzimidazole compounds. J Mol Biol 168:271–284

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequence of the Ml3mpl8 and pUC19 vectors. Gene 33:103–119

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Jessica A. Gorman

    Present address: Department of Microbial Molecular Biology, Bristol-Myers Squibb Institute for Pharmaceutical Research, P.O. Box 4000, 08543, Princeton, NJ, USA

  2. Herbert A. Smith

    Present address: Office of Biologic and Investigational New Drugs, Food and Drug Administration, 9304 Parklawn Building, 5600 Fishers Lane, 20852, Rockville, MD, USA

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Burroughs Wellcome Co., 3030 Cornwallis Rd., 27709, Research Triangle Park, NC, USA

    Mary E. Fling & Jan Kopf

  2. Department of Microbiology, Tel-Aviv University, Ramat-Aviv, 69978, Tel Aviv, Israel

    Aviva Tamarkin & Yigal Koltin

  3. Department of Molecular Genetics, SmithKline Beecham, 709 Swedeland Rd., P.O. Box 1539, 19406, King of Prussia, PA, USA

    Jessica A. Gorman & Herbert A. Smith

Authors
  1. Mary E. Fling
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jan Kopf
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aviva Tamarkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jessica A. Gorman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Herbert A. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yigal Koltin
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by C.P. Hollenberg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fling, M.E., Kopf, J., Tamarkin, A. et al. Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate. Molec. Gen. Genet. 227, 318–329 (1991). https://doi.org/10.1007/BF00259685

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00259685

Key words

Search

Navigation