Tn7-based genome-wide random insertional mutagenesis of Candida glabrata

Irene Castaño, Rupinder Kaur, Shihjung Pan, Robert Cregg, Alejandro De Las Peñas, Nini Guo, Matthew C. Biery, Nancy L. Craig, Brendan P. Cormack

Research output: Contribution to journalArticlepeer-review

Abstract

We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7. Tn7 was used to mutagenize a C. glabrata genomic fosmid library. Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli. Subsequently, these were introduced by recombination into the C. glabrata genome. We found that C. glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement. We were able to generate a highly representative set of ∼104 allele replacements in C. glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis. Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen. In addition, the method might be broadly applicable to mutational analysis of other organisms.

Original languageEnglish (US)
Pages (from-to)905-915
Number of pages11
JournalGenome research
Volume13
Issue number5
DOIs
StatePublished - May 1 2003

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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