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Journal of Bacteriology, October 2000, p. 5730-5736, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Single-Transformation Gene Function Test in Diploid Candida albicans

Brian Enloe, Aviva Diamond, and Aaron P. Mitchell*

Department of Microbiology and Institute of Cancer Research, Columbia University, New York, New York 10032

Received 22 May 2000/Accepted 24 July 2000

The fungal pathogen Candida albicans is naturally diploid, and current gene disruption strategies require two successive transformations. We describe here a genetic construct (UAU1) for which two copies may be selected. Insertion of UAU1 into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion. Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele. Thus nonessential and essential genes may be distinguished rapidly through PCR tests for homozygosis and triplication. We find that homozygous mutations may be isolated at three nonessential loci (ADE2, RIM20, and YGR189), while only allelic triplications were found at two essential loci (SNF1 and CDC28). We have unexpectedly isolated homozygous mutants with mutations at CDC25; they are viable but defective in filamentation on serum-containing medium. The UAU1 cassette is thus useful to assess rapidly the essentiality of C. albicans genes.

* Corresponding author. Mailing address: Department of Microbiology, Columbia University, 701 West 168th St., New York, NY 10032. Phone: (212) 305-8251. Fax: (212) 305-1741. E-mail: apm4{at}columbia.edu.

Journal of Bacteriology, October 2000, p. 5730-5736, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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