Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in .

The ability to generate autonomously replicating plasmids has been elusive in , a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively). and a heterologous marker, yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologous marker was highly dependent upon the addition of a origin sequence. Several chromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin, , from , drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner. Circular plasmids are important tools for molecular manipulation in model fungi such as baker's yeast, yet, in , an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a origin and an origin sequence from a distantly related yeast can both function as origins in Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between and baker's yeast.