The pathogenic yeast cryptococcus neoformans (Cn) causes cryptococcosis, a life-threatening disease of the brain. Molecular studies of Cn variety gattii have lagged behind other two varieties (var. grubii and var. neoformans) although they have distinct biology and disease patterns. We focused on gene discovery in MATalpha locus because it predominates in clinical strains. A var. gattii cosmid library was screened with DNA probes from other two varieties. Two positive clones were sequenced to identify ORFs based on similarities to known proteins, and to ESTs using bioinformatics, and manually by a curator. Approximately 76 kb sequenced DNA revealed 23 genes and ORFs. The existence of predicted genes was verified by RT-PCR analyses designed to amplify spliced sequences. The results confirmed that the transcripts were expressed both at 30 and 37 degreesC. The var. gattii MATalpha locus genes showed rearrangements in order and orientation vis-A-vis other two varieties. Mating-specific genes showed higher nonsynonymous mutation rates. and gene trees showed var. gattii strains in a distinct clade. The identification of the largest number, thus far, of var. gattii structural genes should set the stage for future molecular pathogenesis studies. (C) 2004 Elsevier Inc. All rights reserved.