Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in the aflatoxin pathway.
Two genes involved in aflatoxin B1 (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 whether other genes located in the nor-1-ver-1 region were involved in aflatoxin synthesis. Northern (RNA) analysis performed on RNA isolated from A. parasiticus SU1 grown in aflatoxin-inducing medium localized 14 RNA transcripts encoded by this region. Eight of these transcripts, previously unidentified, showed a pattern of accumulation similar to that of nor-1 and ver-1, suggesting possible involvement in AFB1 synthesis. 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 pAPNVES4. Thin-layer chromatography revealed that gene-1 disruptant clones no longer accumulated versicolorin A. Southern hybridization analysis of these clones indicated that gene-1 had been disrupted by insertion of the disruption vector. These data confirmed that gene-1 is directly involved in AFB1 synthesis. The predicted amino acid sequence of two regions of gene-1 showed a high degree of identity and similarity with the beta-ketoacyl-synthase and acyltransferase functional domains of polyketide synthases, consistent with a proposed role for gene-1 in polyketide backbone synthesis.
Trail
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F
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824, USA.
Mahanti
N
N
Rarick
M
M
Mehigh
R
R
Liang
S H
SH
Zhou
R
R
Linz
J E
JE
D016604:Aflatoxin B1 / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D002874:Chromosome Mapping; D003470:Culture Media; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D005810:Multigene Family; D014158:Transcription, Genetic
D003470:Culture Media; D016604:Aflatoxin B1