Molecular and General Genetics MGG

, Volume 198, Issue 1, pp 179–182 | Cite as

Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations

  • Amanda M. Gillum
  • Emma Y. H. Tsay
  • Donald R. Kirsch
Short Communication

Summary

A gene bank of Sau3A partially digested Candida albicans DNA in vector YEp13 was used to complement a ura3 mutation (orotidine-5′-phosphate decarboxylase, OMPdecase) in S. cerevisiae. Two plasmids which complemented ura3 and showed clear linkage of Ura+ and plasmid markers were selected for further study. Both plasmids also complemented the corresponding OMPdecase mutation (pyrF) in E. coli. Restriction mapping and subcloning studies localized the OMPdecase complementing activity to a region common to both plasmids. Probes prepared from this common region hybridized specifically to C. albicans DNA and not to E. coli or S. cerevisiae DNA. Southern blot analysis also showed that the restriction map of the ura3 complementing region of one plasmid was colinear with C. albicans genomic DNA. Expression of the OMPdecase complementing gene in E. coli and S. cerevisiae was not dependent upon orientation relative to vector sequences, suggesting that promotion could be occurring within the C. albicans fragment. Expression was sufficient to allow complementation in S. cerevisiae with integrating as well as high copy number vectors.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bach ML, Lacroute F, Botstein D (1979) Evidence for transcriptional regulation of orotidine-5′-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli. Proc Natl Acad Sci USA 76:386–390Google Scholar
  2. Bolivar F (1978) Construction and characterization of new cloning vehicles III. Derivatives of plasmid pBR 322 carrying unique EcoRI sites for selection of EcoRI generated recombinant molecules. Gene 4:121–136Google Scholar
  3. Botstein D, Falco SC, Stewart SE, Brennan M Scherer S, Stinchcomb DT, Struhl K, Davis RW (1979) Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene, 8:17–24Google Scholar
  4. Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene Gene 8:121–133Google Scholar
  5. Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 174–176Google Scholar
  6. Grindley NDF Joyce CM (1980) Analysis of the structure and function of the kanamycin resistance transposon Tn 903. Cold Spring Harbor Symp Quant Biol 44125–133Google Scholar
  7. Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933Google Scholar
  8. Kakar SN, Magee PT (1982) A genetic analysis of Candida albicans: identification of different isoleucine-valine, methionine, and arginine alleles by complementation. J Bacteriol 151:1247–1252Google Scholar
  9. Kakar SN, PartridgeRN, Magee PT (1983) A genetic analysis of Candida albicans isolation of a wide variety of auxotrophs and demonstration of linkage and complementation. Genetics 104:241–255Google Scholar
  10. Petes TD, Broach JR, Wensink PC, Hereford LM, Fink GR, Botstein D (1978) Isolation and analysis of recombinant DNA molecules containing yeast DNA Gene 4:37–49Google Scholar
  11. Poulter R, Jeffery K, Hubbard MJ, Shepherd MG, Sullivan PA (1981) Parasexual genetic analysis of Candida albicans by spheroplast fusion. J Bacteriol 146:833–840Google Scholar
  12. Poulter R, Hanrahan V, Jeffery K, Markie D, Shepherd MG, Sullivan PA (1982) Recombination analysis of naturally diploid Cadida alibcans. J Bacteriol 152:969–975Google Scholar
  13. Poulter RTM, Rikkerink EHA (1983) Genetic analysis of red, adenine-requiring mutants of Candida albicans. J Bacteriol 156:1066–1077Google Scholar
  14. Rigby PWJ, Diekmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251Google Scholar
  15. Rose M, Casadaban MJ, Botstein D (1981) Yeast genes fused to β-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci USA 75:2460–2564Google Scholar
  16. Sarachek A, Rhoads DD, Schwarzhoff RH (1981) Hybridisation of Candida albicans through fusion of protoplasts. Arch Microbiol 129:1–8Google Scholar
  17. Sherman F, Fink GR, Lawrence CW (1979) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 90–92Google Scholar
  18. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  19. Szostak JW, Wu R (1979) Insertion of a genetic marker into the ribosomal DNA of yeast. Plasmid 2:536–554Google Scholar
  20. Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA segments from agarose gels to diazobenzloxymethal paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683–3687Google Scholar
  21. Whelan WL Magee PT (1981) Natural heterozygosity in Candida albicans. J Bacteriol 145:896–903Google Scholar
  22. Whelan WL, Partridge RM, Magee PT (1980) Heterozygosity and Segregation in Candida albicans Mol Gen Genet 180:107–113Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • Amanda M. Gillum
    • 1
  • Emma Y. H. Tsay
    • 1
  • Donald R. Kirsch
    • 1
  1. 1.The Squibb Institute for Medical ResearchPrincetonUSA

Personalised recommendations