Characterization of Novel Genes Expressed During Aflatoxin Biosynthesis

DNA microarrays made from Aspergillus flavus expressed sequence tags will be used to identify a set of genes coordinately regulated with aflatoxin under a number of conditions. The function of these genes will be determined by characterization of strains with mutated copies of the genes or with copies of the genes with altered regulation. A 14-3-3 homolog in A. flavus will be studied for its role in aflatoxin biosynthesis. The effect of induced mutation will be studied under a number of physiological conditions using microarray technology to compare and contrast expression profiles of the mutant and wild type. The yeast two-hybrid system will be used to identify proteins that may interact with 14-3-3.
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Aflatoxins are toxic and carcinogenic compounds produced in food and feeds by the filamentous fungus Aspergillus flavus. Their synthesis by the fungus is regulated by environmental, developmental, and nutritional cues. The overall objective of the research outlined in this study is to identify the genes involved in this regulation with the long-term goal of using the information gained to design strategies to block aflatoxin contamination. Emerging data show that aflatoxin biosynthesis is controlled by a complex regulatory network. To identify the sets of genes involved in the regulatory network, gene expression profiling, which allows one to measure simultaneously the expression of several genes at once, will be used. Such a strategy has already identified one gene that is involved in aflatoxin biosynthesis.
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The specific objectives of this proposal are: <ol> <Li>Determine the function of an A. flavus 14-3-3 homolog in aflatoxin biosynthesis
<Li>Use DNA microarrays to identify a common set of differentially expressed during aflatoxin biosynthesis <Li>Analyze the function of the above set of genes with putative roles in aflatoxin biosynthesis by gene disruption</ol>
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Results from these studies will not only provide information useful in the control of aflatoxin contamination, but may also contribute to our understanding of the biosynthesis of other mycotoxins in food. A further outcome of this research is the beginning of a database of genes expressed during secondary metabolism.