Identification of genomic differences between Cryptococcus neoformans and Cryptococcus gattii by Representational Difference Analysis (RDA).

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous yeasts that cause cryptococcosis in immunocompromised and immunocompetent individuals, respectively. In this study, Representational Difference Analysis (RDA) was applied to isolate sequences representing genomic differences between C. neoformans var. grubii and C. gattii. RDA is a PCR based subtractive enrichment procedure for isolating the differences between two nearly identical genomes. We have approximately 200 clones sequenced leading to the identification of 19 different sequences with significant similarities (E(value)<10(-5)) to the completely sequenced genome of the C. neoformans var. neoformans JEC21 strain. Southern blot experiments using five selected clones confirmed the presence of polymorphisms for three sequences (a putative Insulin Degrading Enzyme (IDE), a chitin synthase and an endoplasmic reticulum protein). This is the first report of a genomic RDA application for pathogenic yeasts and the results may be useful to develop markers for other applications, such as specific primers and probes.