Random mutagenesis of an essential Candida albicans gene

Glen Palmer, Joy E. Sturtevant

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

A method for the analysis of Candida albicans gene function, which involves random mutagenesis of the open reading frame, is described. This method is especially suited for the study of essential and multi-functional genes, with several advantages over regulatable promoters more commonly used to study essential gene function. These advantages include expression from the endogenous promoter, which should yield a more appropriate transcript expression and abrogate the need for shifts in carbon or amino acid sources necessary with the use of regulatable promoters. Furthermore, there is potential for isolating individual functions of multi-functional genes. To verify this experimental approach, we randomly mutated the essential C. albicans gene, BMH1. The resulting "pool" of putative mutant alleles was then introduced into a BMH1/bmh1Δ strain of C. albicans, such that only the mutagenized BMH1 sequences could be expressed. Transformants were screened for rapamycin sensitivity, defects in filamentation on M199 agar, and growth at 42°C. In this way, we identified six non-lethal mutant alleles of BMH1 with altered amino acid sequences. Further phenotypic analysis of these mutant strains enabled us to segregate individual functions of C. albicans BMH1. The relative merits of Escherichia coli versus PCR-mediated mutagenesis are discussed.

Original languageEnglish (US)
Pages (from-to)343-356
Number of pages14
JournalCurrent Genetics
Volume46
Issue number6
DOIs
StatePublished - Dec 1 2004

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Candida albicans
Mutagenesis
Genes
Alleles
Essential Genes
Sirolimus
Open Reading Frames
Agar
Amino Acid Sequence
Carbon
Escherichia coli
Amino Acids
Polymerase Chain Reaction
Growth

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Random mutagenesis of an essential Candida albicans gene. / Palmer, Glen; Sturtevant, Joy E.

In: Current Genetics, Vol. 46, No. 6, 01.12.2004, p. 343-356.

Research output: Contribution to journalArticle

Palmer, Glen ; Sturtevant, Joy E. / Random mutagenesis of an essential Candida albicans gene. In: Current Genetics. 2004 ; Vol. 46, No. 6. pp. 343-356.
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