|Liang, Shun-Hsin - U OF MICHIGAN MED SCHOOL|
|Linz, John - MICHIGAN STATE UNIVERSITY|
Submitted to: Biochem Biophys Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 16, 2002
Publication Date: N/A
Interpretive Summary: This work describes experiments that have been conducted in an effort to better understand the mechanisms by which aflatoxins are produced by fungi. Aflatoxins are toxic and carcinogenic compounds often produced by the fungi, Aspergillus flavus and Aspergillus parasiticus, during growth on crops such as corn, peanuts, cottonseed, and treenuts. Because of the potential health risks, aflatoxin contamination of food and feed crops is also of great economic importance to farmers who cannot sell their crops due to strict domestic and international regulatory guidelines with regards to aflatoxin contamination. ARS scientists have succeeded in developing strains of Aspergillus parasiticus that no longer produce aflatoxins. These strains will serve as tools to aid in unraveling the mechanisms responsible for production of aflatoxin, which will in turn help to devise strategies for eliminating aflatoxin contamination of food and feed crops.
Technical Abstract: The genes required for the synthesis of aflatoxin (AF) in Aspergillus flavus, Aspergillus parasiticus, and sterigmatocystin (ST) in Aspergillus nidulans have been shown to be clustered on a chromosome in these fungi. Transcription of these genes is dependent upon the activity of the aflR gene, also present on the AF/ST gene cluster, which encodes a zinc binuclear cluster DNA-binding protein. While many strains of A. parasiticus have only one copy of aflR (aflR-1), a commonly used laboratory strain, SU-1, and others contain a second copy of this gene (aflR-2) which resides on a duplicated region of the aflatoxin gene cluster. Here we report on a series of experiments designed to determine if the second, non-cluster copy of aflR, designated aflR-2, is transcribed and translated as a functional protein. Transformation of niaD mutants of A. parasiticus SU-1 with a vector containing the aflatoxin cluster aflR gene disrupted by the niaD gene resulted in the isolation of numerous niaD+, aflatoxin-nonproducing transformants. Southern and northern hybridization analyses, thin layer chromatographic analysis of aflatoxins and aflatoxin precursors as well as ELISA assays of aflatoxin-nonproducers demonstrated that the aflatoxin gene cluster copy of aflR had been successfully inactivated. DNA sequence analysis showed that aflR-2 was capable of encoding a deduced 47 kDa protein; but northern and RT-PCR analysis indicated that aflR-2 was transcribed at extremely low levels compared to aflR-1 and mRNAs of aflatoxin pathway genes were not processed to their mature forms. Functional analysis of AflR-2 protein in a yeast system showed that it was not activating transcription.