STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE NOR-1 GENE INVOLVED IN THE BIOSYNTHESIS OF AFLATOXINS BY ASPERGILLUS-PARASITICUS

The nor-1 gene was cloned previously by complementation of a mutation (nor-1) in Aspergillus parasiticus SU-1 which blocked aflatoxin B1 bio synthesis, resulting in the accumulation of norsolorinic acid (NA). In this study, the nucleotide sequences of the cDNA and genomic DNA clon es encompassing the coding region of the nor-1 gene were determined. T he transcription initiation and polyadenylation sites of nor-1 were lo cated by primer extension and RNase protection analyses and by compari son of the nucleotide sequences of the nor-1 genomic and cDNA clones. A plasmid, pNA51-82, was created for one-step disruption of the nor-1 gene by inserting a functional copy of the nitrate reductase (niaD) ge ne from A. parasiticus into the coding region of the nor-1 gene. Trans formation of A. parasiticus NR-3 (niaD Afl(+)) with pNA51-82 resulted in niaD(+) transformants that accumulated NA and produced reduced leve ls of aflatoxin as determined by thin-layer chromatography and enzyme- linked immunosorbent assay analyses of extracts from mycelia and the g rowth medium. Southern analysis of genomic DNA isolated from the NA-ac cumulating transformants indicated that the wild-type nor-1 gene in th e chromosome had been replaced by the nonfunctional allele carried on pNA51-82. This recombinational inactivation event provides direct evid ence that the nor-1 gene is functionally involved in aflatoxin biosynt hesis. Comparison of the predicted nor-1 amino acid sequence with sequ ences in the GenBank and EMBL databases suggested that the protein is a member of the family of short-chain alcohol dehydrogenases, consiste nt with its proposed function as a keto reductase.