GENOMIC APPROACHES TO CANDIDA VIRULENCE

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The long term goal of this proposal is to use post-genomics era technologies and approaches to increase our understanding of the molecular basis of pathogenesis in HIV-related oral candidiasis. Our immediate goal is to fabricate DNA micro arrays using the C. albicans genomic DNA sequence data generated by the Stanford DNA Sequencing and Technology Center (Stanford Center). Using this technology the patterns of C. albicans gene expression in various disease contexts will be assessed. This information will contribute to the development of specific diagnostics, drug targets and insights into the mechanism of C. albicans pathogenesis in immunocompromised individuals. The Specific Aims of this proposal are to:(1) fabricate DNA micro arrays using both PCR-product based and oligonucleotide-based technologies. Fabrication of whole genome DNA micro arrays will be completed before the termination of this grant. Fabrication will be an iterative process, beginning immediately with the construction of PCR based DNA micro arrays and using a subset of available C. albicans genomic sequences. At an early stage in these studies, protocols to make the micro arrays generally available to the Candida research community will be developed. (2) The first generation of micro arrays will be used to analyze patterns of gene expression in C. albicans obtained from a variety of environments. This will be the first approach to cataloging the function and coordinate expression of gene sequences, both known and unknown. One of the many key issues in the study of pathogenesis is the validation of animal models of infection as these will provide the basis for further analysis of genes whose functions are as yet unknown. We are therefore particularly interested in comparing the patterns of expression of C. albicans genes in the oral mucosa, with those expressed in the extant rodent models. (3) initiate the functional analysis of individual genes of interest. Comparison of the patterns of gene expression using micro arrays will identify genetic sequences which merit further study. Those of unknown function will be targeted for further genetic analysis, including the construction of specific knockout mutations and the analysis of ensuing phenotype. In this way it will be possible to begin a complete characterization of the range of gene expression which contributes to the pathogenesis of Candida in different host contexts.