THE CRYPTOCOCCUS-NEOFORMANS GAL7 GENE AND ITS USE AS AN INDUCIBLE PROMOTER

A Cryptococcus neoformans galactose auxotroph was created by ultraviol et light mutagenesis and complemented with a C. neoformans genomic lib rary. The translated sequence of the complementing DNA revealed a high degree of similarity to a number of UDP glucose-D-galactose-1-phospha te uridylyltransferases. Expression of C. neoformans GAL7 mRNA followe d a pattern similar to Saccharomyces cerevisiae expression; it was fir st observed within 2.5 min of induction and fully induced by 30 min. T he gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two g enes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MF alpha, which enco des a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, beta-glucuronidase (gus A), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7p::GUS fusion was used to quantify induc ibility of the GAL7 promoter, the level of enzyme activity was at leas t 500-fold greater for cells grown in galactose than for cells grown i n glucose. The GAL7 promoter is the first inducible promoter character ized in C. neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.