Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus

Alexandra Carolyn Brand, Donna Margaret MacCallum, Alistair James Petersen Brown, Neil Andrew Robert Gow, Frank Christopher Odds

Research output: Contribution to journalArticle

214 Citations (Scopus)

Abstract

Uridine auxotrophy, based on disruption of both URA3 alleles in diploid Candida albicans strain SC5314, has been widely used to select gene deletion mutants created in this fungus by "Ura-blasting" and PCR-mediated disruption. We compared wild-type URA3 expression with levels in mutant strains where URA3 was positioned either within deleted genes or at the highly expressed RPS10 locus. URA3 expression levels differed significantly and correlated with the specific activity of Ura3p, orotidine 5'-monophosphate decarboxylase. Reduced URA3 expression following integration at the GCN4 locus was associated with an attenuation of virulence. Furthermore, a comparison of the SC5314 (URA3) and CAI-4 (ura3) proteomes revealed that inactivation of URA3 caused significant changes in the levels of 14 other proteins. The protein levels of all except one were partially or fully restored by the reintegration of a single copy of URA3 at the RPS10 locus. Transcript levels of genes expressed ectopically at this locus in reconstituted heterozygous mutants also matched the levels found when the genes were expressed at their native loci. Therefore, phenotypic changes in C. albicans can be associated with the selectable marker rather than the target gene. Reintegration of URA3 at an appropriate expression locus such as RPS10 can offset most problems related to the phenotypic changes associated with gene knockout methodologies.
Original languageEnglish
Pages (from-to)900-909
Number of pages10
JournalEukaryotic Cell
Volume3
Issue number4
DOIs
Publication statusPublished - 1 Aug 2004

Keywords

  • Animals
  • Candida albicans
  • Chromosomes, Fungal
  • Female
  • Fungal Proteins
  • Gene Expression Regulation, Fungal
  • Gene Targeting
  • Mice
  • Mice, Inbred DBA
  • Orotidine-5'-Phosphate Decarboxylase
  • Phenotype
  • RNA, Messenger
  • Survival Rate

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