Aspergillus parasiticus and related fungi frequently produce aflatoxin contamination in food and feed crops in the US and in other areas of the world. Aflatoxin B1 (AFB1) I hepatotoxic, mutagenic, teratogenic, immunotoxic, and is one of the most potent naturally occurring carcinogens known in certain animals. Data also strongly suggest that AFB1 is a contributory risk factor in primary liver cancer in humans. The long term goal of this research is to eliminate AFB1 from the food chain. The short term goal is to understand the molecular mechanisms which regulate the expression of key genes involved in the biosynthesis of AFB1. The proposed studies are designed to identify several control points in the AFB1 biosynthetic pathway which provide targets for inhibition of toxin synthesis by compounds produced by or introduced onto the host plant. The following specific aims are proposed to develop an in-depth understanding of the mechanisms which regulate gene expression at the level of transcription and protein localization. 1. Identify specific cis-acting sites and trans-acting regulatory factors (TAF) which mediate the regulation of AFB1 biosynthesis. 2. Analyze the subcellular localization and interaction of enzymes which catalyze late pathway functions involved in AFB1 synthesis. To achieve Specific aim 1, Gel mobility shift and DNAse I footprint analyses will map the location of cis-acting sites in the promoters of key genes involved in AFB1 synthesis. The functional significance of these sites will be analyzed in vivo by measuring the effects of directed mutagenesis on the expression of beta-glucuroindase (GUS) reporter constructs under appropriate growth conditions. Genes encoding TAF will be cloned using established technologies and the influence of nutrients, environmental factors, and fungal development on TAF expression/activity will be analyzed. To achieve Specific aim 2, polyclonal antibodies will be used to localize key enzymes involved in AFB1 synthesis by cell fractionation, immunolocalization, and electron microscopy. These same protocols plus immunoprecipitation will be utilized to study the possible physical interaction and co-localization of these enzymes in fungal cells.
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