Molecular and genetic analysis of URA5 transformants of Cryptococcus neoformans

Abstract

Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.