Development Of Tools For Molecular Study Of Cryptococcus

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Abstract

Although there is much interest in Cryptococcal research, genetic analysis in C. neoformans is less well developed than in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ability to control the expression of a gene of interest can be a useful tool for functional analysis, requiring fusion of the coding region to a promoter which functions in the organism being studied and which can be conveniently regulated. In C. neoformans, such regulated expression would ordinarily require a strain which lacks or has a disrupted copy of the endogenous gene of interest but which carries a fusion of the gene to a regulatable promoter. In C. neoformans, gene disruptions can be achieved by homologous integration using biolistic transformation. To be generally useful, any regulated expression system must be introduced in such a way as to complement the disrupted gene for the promoter used in the construct. The maltase gene, when induced by growth of the organism on alpha-linked carbon sources, encodes and secretes an alpha-glucosidase capable of hydrolyzing the alpha linkages of carbon sources such as maltose, sucrose etc. Inclusion of synthetic substrates such as X-Glu in the growth medium can serve as a color selection marker to indicate expression of the alpha-glucosidase by the transformed strain. During the past year, we have cloned the MAL-1 gene from the genomic libraries of a serotype D reference strain. The ORF of the MAL-1 gene in C. neoformans consists 1812 base pairs and contains 7 introns. This year, a MAL-1 disruption construct was created and the gene was disrupted. Since the MAL-1 gene encodes an alpha-glucosidase and its expression is induced under derepressing conditions if grown in the absence of glucose, its promoter sequence can be utilized to regulate the expression of heterologous genes under said conditions.