Submitted to: Biochemistry and Biophysics Research Communication
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 10, 2000
Publication Date: N/A
Interpretive Summary: Aflatoxins are well known hepato-carcinogens that are produced by the fungi, Aspergillus flavus and Aspergillus parasiticus. When these fungi invade a crop, they commonly produce aflatoxins and render the crop unsalable. But, when these aflatoxin-producing fungi grow in media containing nitrate, they stop producing aflatoxins. In this report, we studied the major protein factor that regulates the use of nitrogen compounds by these fungi. This nitrogen regulatory protein contained various functional domains; it bound to regions of DNA important for the function of genes involved in the synthesis of aflatoxins. Thus, manipulation of fungal nitrogen metabolism may be a reasonable strategy to minimize aflatoxin contamination of food and feed.
Technical Abstract: The major nitrogen regulatory gene, areA, that mediates nitrogen metabolite repression in many fungi, was cloned from Aspergillus parasiticus. The A. parasiticus areA gene encoded a polypeptide of 864 amino acids which contained a nuclear localization signal (NLS), a highly acidic region from positions 497 to 542, a Cys-X2-Cys-X17-Cys-X2-Cys DNA-binding motif and a conserved carboxyl terminus. Electrophoretic mobility shift assays suggested that the A. parasiticus AREA DNA-binding domain fusion protein bound cooperatively to single GATA elements in the A. parasiticus niaD-niiA intergenic region. The formation of AREA and DNA complexes varied depending on the protein concentration and the number of GATA elements in the probes; at higher AREA concentrations larger molecular weight and more complexes were formed. A. parasiticus AREA also bound to the aflR-aflJ intergenic region of the aflatoxin biosynthesis gene cluster. Various regions of areA were fused to a yeast GAL4 DNA-binding domain coding region to localize putative transcription activation domain(s) of AREA based on activation of the GAL1(p)::lacZ reporter gene expression. The amino portion of AREA had no activation activity. The carboxyl portion and the portion containing the acidic region only had very weak activation activities. In comparison, the portion between NLS and the acidic domain demonstrated 16 to 20-fold higher activation activities, which suggests that the transcription activation domain of A. parasiticus AREA is located in this region.