Development of a Shuttle Vector That Transforms at High Frequency for the Emerging Human Fungal Pathogen: .

Routine molecular manipulation of any organism is inefficient and difficult without the existence of a plasmid. Although transformation is possible in , no plasmids are available that can serve as cloning or shuttle vectors. centromeres have been well characterized but have not been explored further as molecular tools. We tested centromeric sequences to identify which, if any, could be used to create a plasmid that was stably maintained after transformation. We cloned all seven centromeric sequences and tested them for transformation frequency and stability. Transformation frequency varied significantly; however, one was found to transform at a very high frequency. A 1.7 Kb subclone of this sequence was used to construct a shuttle vector. The vector was stable with selection and maintained at ~1 copy per cell but could be easily lost when selection was removed, which suggested that the properties of the centromeric sequence were more Autonomously Replicating Sequence (ARS)-like than centromere-like when part of a plasmid. Rescue of this plasmid from transformed cells into revealed that it remained intact after the initial transformation, even when carrying large inserts. The plasmid was found to be able to transform all four clades of , with varying frequencies. This plasmid is an important new reagent in the molecular toolbox, which will enhance the investigation of this human fungal pathogen.