Submitted to: Biochem Biophys Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 10, 2000
Publication Date: N/A
Interpretive Summary: Two fungi, Aspergillus flavus and Aspergillus parasiticus, produce a compound called aflatoxin during their growth on such crops as corn, peanut, and cotton that is toxic to animals and humans upon ingestion. Numerous strategies have been put forward to prevent aflatoxin contamination in the field or to remove aflatoxin from contaminated crops. In this paper we studied a gene, avnA, that is involved in aflatoxin production in the fungus. AvnA is under the control of a master switch protein, AFLR, that is responsible for "turning on" all of the genes involved in aflatoxin production. We identified a region of DNA upstream of avnA to which AFLR interacts thus turning on activity of avnA. We also identified regions in the avnA upstream gene region that appeared to be involved in "turning off" or reducing the activity of avnA. From these results we hope to be able to identify under what environmental conditions the fungus produces a factor(s) that negatively affects avnA activity and hence aflatoxin production. Identification of the factor(s) and the environmental conditions that result in negative regulation of avnA production and possibly other aflatoxin genes might provide us with information that will allow us to develop strategies to eliminate aflatoxin contamination of crops.
Technical Abstract: One of the key early genes in aflatoxin biosynthesis, avnA, encodes a pathway-specific cytochrome P450 monoxygenase that catalyzes the hydroxylation of the polyketide anthraquinone, averantin. B-Glucuronidase (GUS) reporter assays were used to determine which sites in the 367-bp verB-avnA intergenic region of avnA are involved in transcription control. Prior studies suggested that AFLR, a Zn2Cys6-type sequence specific DNA binding protein, is required for expression of itself and other genes in the aflatoxin biosynthetic pathway and binds to promoter regions with the consensus requences 5'-TGGN5CGR-3'. Truncation of the avnA promoter at -118 bp increased activity 3-fold, a result suggesting that 3 possible upstream AFLR-binding sites are not required for gene activity and that negative regulatory elements may be present in the deleted region. GUS activity was reduced 6-fold when the promoter was further truncated at -100, thereby deleting a putative AFLR binding site at -100 to -100. Mutation of bases at -108 and -109 within the AFLR binding site reduced promoter activity 10-fold, while mutation of similar bases at two other upstream sites had no effect on promoter activity. These results indicate that the -100 to -111 AFLR binding site is the main positive regulatory element required for avnA expression. Proteins in nuclear extracts from fungi grown on a medium which does not support aflatoxin gene expression, but not in medium that supports aflatoxin production, bound to two sites 5' to the AFLR binding site. These sites could be involved in negative regulation of avnA gene expression.