Construction of a complete URA5 deletion strain of a human pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic human pathogen, which infects the central nervous system causing the fatal disease, meningitis. In order to understand the genetic background of this human pathogen, the basic molecular manipulation techniques of deletion, overexpression, and so on have been developed. URA5, a gene encoding orotate phosphoribosyltransferase, has frequently been used to introduce foreign gene fragments by complementing ura5 mutant strains, which are not, however, stable; reversion to uracil prototroph is thus frequently observed on selective condition. The high possibility of reversion makes it inconvenient to use this mutation to identify appropriate transformants and thus, manipulation in molecular genetics. We report here the isolation of a stable ura5 mutant of C. neoformans, designated as TAD1, by eliminating the URA5 gene by homologous recombination using the biolistic DNA delivery system. The availability of the stable ura5 mutant offers the advantage that no spontaneous reversion occurs so that a satisfactory rate of homologous recombination can be achieved. The strain will allow efficient genomic analysis in C. neoformans.