The aflR gene of Aspergillus parasiticus and A. flavus encodes a binuclear
zinc-finger, DNA-binding protein, AflR, responsible for activating the tran
scription of all known aflatoxin biosynthetic genes including itself. Studi
es to determine how environmental and nutritional factors affect aflR expre
ssion and hence aflatoxin production in A. parasiticus have been difficult
to perform due to the lack of aflR "knockout" mutants. Transformation of an
O-methylsterigmatocystin (OMST)-accumulating strain of A. parasiticus with
an aflR-niaD gene disruption vector resulted in clones harboring a recombi
nationally inactivated aflR gene which no longer produced OMST or aflR tran
script. By transformation of this aflR disruptant strain with constructs co
ntaining mutated versions of the aflR promoter, we identified three cis-act
ing sites that were necessary for aflR function: an AflR-binding site, a Pa
cC-binding site, and a G + A-rich site near the transcription start site of
aflR.