Aflatoxins are biologically active secondary metabolites synthesized by Aspergillus flavus and A. parasiticus. These fungi are ubiquitous and frequently produce aflatoxin contamination in food and feed crops in the US and throughout the world. In animal systems aflatoxin B1 (AFB1) is hepatotoxic, mutagenic, teratogenic, immunotoxic, and carcinogenic. AFB1 is the most potent naturally occurring carcinogen known. Epidemiological studies on human populations suggest that AFB1 is a contributory risk factor in primary liver cancer. The long term goal of this research is to eliminate AFB1 from the food chain. The short term goal of this research proposal is to understand the molecular mechanisms which regulate the expression of key genes (UVM8, nor-1, and ver-1) 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 by compounds synthesized by or introduced onto the host plant. The following specific aims are proposed to develop an in depth understanding of the mechanisms which regulate expression of UVM8, nor-1, and ver-1 at the level of transcription, translation, and protein localization. 1. Identify specific trans-acting factors and their cis- acting sites in the promoters of nor-1, ver-1, and UVM8 which regulate their timing and level of expression. 2. Identify the timing of expression and subcellular localization of the proteins encoded by these three genes. To accomplish specific aim 1, deletion analyses of the nor-1, ver-1 and UVM8 promoters will be conducted on beta glucuronidase (GUS) reporter fusion constructs to determine the number and types of regulatory sites. Gel shift and methylation interference analyses will precisely map these sites and provide a mechanism for purification of trans-acting regulatory factors. To accomplish specific aim 2, antibodies (Ab) raised to nor-1, ver-1 and UVM8 proteins will be utilized to determine the timing of their expression by Western blot analyses of cell extracts. The intracellular localization of these proteins will be determined in cryosections by immunolocalization with fluorescent Ab. Localization of protein expression in whole fungal cells or colonies will be accomplished by localizing GUS reporter protein activity with chromogenic or fluorescent substrates.
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