title: Tn7-based genome-wide random insertional mutagenesis of Candida glabrata.
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 approximately 10(4) 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.
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Castano
authors.forename: Rupinder
authors.initials: R
authors.lastname: Kaur
authors.forename: Shihjung
authors.initials: S
authors.lastname: Pan
authors.forename: Robert
authors.initials: R
authors.lastname: Cregg
authors.forename: Alejandro De Las
authors.initials: Ade L
authors.lastname: Penas
authors.forename: Nini
authors.initials: N
authors.lastname: Guo
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Biery
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Craig
authors.forename: Brendan P
authors.initials: BP
authors.lastname: Cormack
mesh_terms: D041221:Candida glabrata / Q000145:classification / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D016681:Genome, Fungal; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010641:Phenotype; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics; D014498:Uracil / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004271:DNA, Fungal; D005819:Genetic Markers; D014498:Uracil