PHYSICAL AND TRANSCRIPTIONAL MAP OF AN AFLATOXIN GENE-CLUSTER IN ASPERGILLUS-PARASITICUS AND FUNCTIONAL DISRUPTION OF A GENE INVOLVED EARLYIN THE AFLATOXIN PATHWAY

Two genes involved in aflatoxin B-1 (AFB1) biosynthesis in Aspergillus parasiticus; nor-1 and ver-1, were localized to a 35-kb region on one A. parasiticus chromosome and to the genomic DNA fragment carried on a single cosmid, NorA. A physical and transcriptional map of the 35-kb genomic DNA insert in cosmid NorA was prepared to help determine whet her other genes located in the nor-1-ver-1 region were involved in afl atoxin synthesis. Northern (RNA) analysis performed on RNA isolated fr om A. parasiticus SU1 grown in aflatoxin-inducing medium localized 14 RNA transcripts encoded by this region. Eight of these transcripts, pr eviously unidentified, showed a pattern of accumulation similar to tha t of nor-1 and ver-1, suggesting possible involvement in AFB1 synthesi s. To directly test this hypothesis, gene-1, encoding one of the eight transcripts, was disrupted in A. parasiticus CS10, which accumulates the aflatoxin precursor versicolorin A, by insertion of plasmid pAPNVE S4. Thin-layer chromatography revealed that gene-1 disruptant clones n o longer accumulated versicolorin A. Southern hybridization analysis o f these clones indicated that gene-1 had been disrupted by insertion o f the disruption vector. These data confirmed that gene-1 is directly involved in AFB1 synthesis. The predicted amino acid sequence of two r egions of gene-1 showed a high degree of identity and similarity with the beta-ketoacyl-synthase and acyltransferase functional domains of p olyketide synthases, consistent with a proposed role for gene-1 in pol yketide backbone synthesis.