This project proposes to clone the C. albicans RAM2 gene specifying a common subunit of prenylation enzymes. Several methods are considered and the method given priority is to use PCR with degenerate oligonucleotides based on database sequences of the protein in other organisms. If this method is not successful then other methods will be used sequentially until success is achieved. In order these methods are: 1) complementation in S. cerevisiae with a C. albicans genomic library in an appropriate vector; 2) preparation of antibodies to the S. cerevisiae fusion protein, confirmation of reactivity with a candidal extract and screening of a C. albicans expression library; and 3) screening the candidal library with the S. cerevisiae gene.
Aim 2 has multiple components to characterize RAM2. The gene including promoter will be sequenced. If the original cloning does not produce the whole gene, the entire sequence will be isolated from a genomic library or if a larger clone is isolated, the gene will be subcloned. The chromosomal location and number of alleles will be determined by chromosomal and Southern blotting. The expression of the gene in different strains, under different growth, morphology, and switch phenotypes will be determined by Northern analysis. If not already analyzed, the ability of the gene to complement a ram2 strain of S. cerevisiae will be determined. A candidal fusion protein will be produced and used to elicit antibodies. These antibodies will be used to monitor protein levels. If time permits, the gene will be disrupted.
