title: Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum.
abstract: Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.
authors: Department of Microbiology, Ohio State University, Columbus, OH 43210, USA.
authors: Megan M
authors: MM
authors: Kemski
authors: Bryan
authors: B
authors: Stevens
authors: Chad A
authors: CA
authors: Rappleye
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D006658:Histoplasma / Q000235:genetics* / Q000254:growth & development; D032023:Integrons / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA