Eukaryotic Cell IAI Online 2003
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Eukaryotic Cell, August 2004, p. 900-909, Vol. 3, No. 4
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.4.900-909.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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 Brand,{dagger} Donna M. MacCallum,{dagger} Alistair J. P. Brown, Neil A. R. Gow, and Frank C. Odds*

Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, Aberdeen AB25 2ZD, United Kingdom

Received 27 February 2004/ Accepted 23 April 2004

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.

* Corresponding author. Mailing address: School of Medical Sciences, Institute of Medical Sciences, Aberdeen AB25 2ZD, United Kingdom. Phone and fax: 44 1224 555828. E-mail: f.odds{at}abdn.ac.uk.

{dagger} A.B. and D.M.M. contributed equally to this work.

Eukaryotic Cell, August 2004, p. 900-909, Vol. 3, No. 4
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.4.900-909.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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