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Current Genetics

, Volume 45, Issue 6, pp 390–398 | Cite as

Shuttle vectors for Candida albicans: control of plasmid copy number and elevated expression of cloned genes

  • Wenjin Du
  • Melisa Coaker
  • Jack D. Sobel
  • Robert A. Akins
Technical Note

Abstract

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.

Keywords

Candida albicans CaIMH3 Mycophenolic acid Shuttle vector 

References

  1. Backen AC, Broadbent ID, Fetherston RW, Rosamond JD, Schnell NF, Stark MJ (2000) Evaluation of the CaMAL2 promoter for regulated expression of genes in Candida albicans. Yeast 16:1121–1129CrossRefPubMedGoogle Scholar
  2. Beckerman J, Chibana H, Turner J, Magee PT (2001) Single-copy IMH3 allele is sufficient to confer resistance to mycophenolic acid in Candida albicans and to mediate transformation of clinical Candida species. Infect Immun 69:108–114CrossRefPubMedGoogle Scholar
  3. Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109PubMedGoogle Scholar
  4. Berman J, Sudbery PE (2002) Candida albicans: a molecular revolution built on lessons from budding yeast. Nat Rev Genet 3:918–930CrossRefPubMedGoogle Scholar
  5. Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133PubMedGoogle Scholar
  6. Brown DH Jr, Slobodkin IV, Kumamoto CA (1996) Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme-mediated integration. Mol Gen Genet 251:75–80PubMedGoogle Scholar
  7. Cannon RD, Jenkinson HF, Shepherd MG (1992) Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae. Mol Genet Genomics 235:453–457Google Scholar
  8. Castano I, et al (2003) Tn7-based genome-wide random insertional mutagenesis of Candida glabrata. Genome Res 13:905–915CrossRefPubMedGoogle Scholar
  9. De Backer MD, Magee PT, Pla J (2000) Recent developments in molecular genetics of Candida albicans. Annu Rev Microbiol 54:463–498PubMedGoogle Scholar
  10. Geber A, Williamson PR, Rex JH, Sweeney EC, Bennett JE (1992) Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization. J Bacteriol 174:6992–6996PubMedGoogle Scholar
  11. Horrocks P, Lanzer M (1999) Differences in nucleosome organization over episomally located plasmids coincides with aberrant promoter activity in P. falciparum. Parasitol Int 48:55–61CrossRefPubMedGoogle Scholar
  12. Kohler GA, White TC, Agabian N (1997) Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid. J Bacteriol 179:2331–2338PubMedGoogle Scholar
  13. Kontoyiannis DP, Sagar N, Hirschi KD (1999) Overexpression of Erg11p by the regulatable GAL1 promoter confers fluconazole resistance in Saccharomyces cerevisiae. Antimicrob Agents Chemother 43:2798–2800PubMedGoogle Scholar
  14. Kurtz MB, Cortelyou MW, Miller SM, Lai M, Kirsch DR (1987) Development of autonomously replicating plasmids for Candida albicans. Mol Cell Biol 7:209–217PubMedGoogle Scholar
  15. Lopez-Ribot JL, et al (1998) Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother 42:2932–2937PubMedGoogle Scholar
  16. Maebashi K, et al (2001) Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients. J Antimicrob Chemother 47:527–536CrossRefPubMedGoogle Scholar
  17. Mann BJ, Akins RA, Lambowitz AM, Metzenberg RL (1988) The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1. Mol Cell Biol 8:1376–1379PubMedGoogle Scholar
  18. Pla J, Perez-Diaz RM, Navarro-Garcia F, Sanchez M, Nombela C (1995) Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector. Gene 165:115–120PubMedGoogle Scholar
  19. Rine J (1991) Gene overexpression in studies of Saccharomyces cerevisiae. Methods Enzymol 194:239–251PubMedGoogle Scholar
  20. Rustchenko EP, Curran TM, Sherman F (1993) Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae. J Bacteriol 175:7189–7199PubMedGoogle Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  22. Sanchez-Martinez C, Perez-Martin J (2002) Site-specific targeting of exogenous DNA into the genome of Candida albicans using the FLP recombinase. Mol Genet Genomics 268:418–424CrossRefPubMedGoogle Scholar
  23. Staib P, et al (1999) Host-induced, stage-specific virulence gene activation in Candida albicans during infection. Mol Microbiol 32:533–546PubMedGoogle Scholar
  24. Staib P, Michel S, Kohler G, Morschhauser J (2000) A molecular genetic system for the pathogenic yeast Candida dubliniensis. Gene 242:393–398CrossRefPubMedGoogle Scholar
  25. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039PubMedGoogle Scholar
  26. Versaw WK, Metzenberg RL (1996) Activator-independent gene expression in Neurospora crassa. Genetics 142:417–423PubMedGoogle Scholar
  27. White TC (1997) Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41:1482–1487PubMedGoogle Scholar
  28. White TC, Holleman S, Dy F, Mirels LF, Stevens DA (2002) Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 46:1704–1713CrossRefPubMedGoogle Scholar
  29. Wirsching S, Michel S, Kohler G, Morschhauser J (2000) Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor (in process citation). J Bacteriol 182:400–404CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Wenjin Du
    • 1
  • Melisa Coaker
    • 2
  • Jack D. Sobel
    • 2
  • Robert A. Akins
    • 1
  1. 1.Department of Biochemistry and Molecular biologyWayne State University School of MedicineDetroitUSA
  2. 2.Division of Infectious Diseases, Department of Internal MedicineWayne State University School of MedicineDetroitUSA

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