Transcriptional regulation of the Aspergillus parasiticus aflatoxin biosynthetic pathway gene nor-1

Aflatoxin is a potent hepatocarcinogen produced predominantly by Aspergillus flavus and A. parasiticus that can contaminate several commodities including corn, cotton, peanuts and certain tree nuts. Aflatoxin biosynthesis is a complex process that requires several genes that all reside in an aflatoxin gene cluster.; The specific objectives of these studies were to: (1) demonstrate that transcriptional activation of nor-1 (and presumably the other aflatoxin structural genes) is at least partly responsible for the increase in aflatoxin production under inducing conditions; (2) develop a nor-1 reporter system for use in identifying cis-acting sites in the nor-1 promoter; (3) determine the role of an aflatoxin pathway regulator, AflR, in the regulation of nor-1 ; and (4) identify additional possible cis-acting sites in the nor-1 promoter. To measure nor-1 trancriptional activation, plasmids that contained the nor-1 promoter fused to the beta-glucuronidase gene were transformed into A. parasiticus. Preliminary experiments utilized A. parasiticus D8D3, a strain that carries a 3.0 kb nor-1 promoter fragment fused to GUS that integrated at the nor-1 terminator. The transcriptional activation of nor-1 mirrored the accumulation of aflatoxin, nor-1 transcript and Nor-1 protein. In addition, under culture conditions that generated the most aflatoxin, the highest GUS activities were recorded. A new nor-1::GUS reporter plasmid was constructed that enabled easy nor-1 promoter insertions. However, the site of integration of the nor-1::GUS plasmid was demonstrated to be an important consideration. Integration outside of the aflatoxin gene cluster resulted in significantly reduced nor-1 transcription. Of the three putative AflR binding sites located in the nor-1 promoter area (AflR1, AflR2 and AflR3), only AflR1 is clearly necessary for nor-1 transcriptional activation. AflR2 may also be involved in nor-1 transcriptional activation but the presence of the ORF3 gene (unknown function), which is located between nor-1 and AflR2, prevents a clear conclusion. AflR3 is not necessary for nor-1 transcriptional activation because deletion of AflR3 in a nor-1::GUS reporter strain resulted in no change in nor-1 transcriptional activation. Substitution of a putative TATA box in the nor-1 promoter in the context of a larger promoter demonstrated the requirement for the TATA box in nor-1 transcriptional activation. Evidence was also presented for two additional cis-acting sites in the nor-1 promoter, nor-1 and CRE. (Abstract shortened by UMI.)...