title: A novel cAMP-response element, CRE1, modulates expression of nor-1 in Aspergillus parasiticus.
abstract: The level of aflatoxin accumulation in the filamentous fungus Aspergillus parasiticus is modulated by a variety of environmental cues. The presence of glucose (a preferred carbon source) in liquid and solid glucose minimal salts (GMS) growth media strongly stimulated aflatoxin accumulation. Peptone (a non-preferred carbon source) in peptone minimal salts (PMS) media stimulated only low levels of aflatoxin accumulation. Glucose stimulated transcription of the aflatoxin structural genes ver-1 and nor-1 to similar intermediate levels in liquid GMS, while on solid media, ver-1 transcription was stimulated to 20-fold higher levels than nor-1. PMS liquid and solid media stimulated very low or non-detectable levels of transcription of both genes. Electrophoretic mobility shift analysis using a nor-1 promoter fragment (norR) and A. parasiticus cell protein extracts revealed specific DNA-protein complexes of different mobility on GMS and PMS solid and liquid media. An imperfect cAMP-response element, CRE1, was identified in norR that mediated formation of the specific DNA-protein complexes. Mutation in CRE1 or AflR1 (AflR cis-acting site) caused up to a 3-fold decrease in cAMP-mediated stimulation of nor-1 promoter activity on GMS agar. South-Western blot analysis identified a 32-kDa protein that specifically bound to norR. p32 could be co-immunoprecipitated by anti-AflR antibody and co-purified with an AflR-maltose-binding protein fusion demonstrating a physical interaction between AflR and p32 in vitro. We hypothesize that p32 assists AflR in binding to the nor-1 promoter, thereby modulating nor-1 gene expression in response to environmental cues.
authors.affiliation: Department of Food Science and Human Nutrition, Michigan State University, East Lasing 48824, USA.
authors.forename: Ludmila V
authors.initials: LV
authors.lastname: Roze
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Miller
authors.forename: Matthew
authors.initials: M
authors.lastname: Rarick
authors.forename: Nibedita
authors.initials: N
authors.lastname: Mahanti
authors.forename: John E
authors.initials: JE
authors.lastname: Linz
mesh_terms: D000348:Aflatoxins / Q000096:biosynthesis / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics*; D001230:Aspergillus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins; D003470:Culture Media / Q000737:chemistry; D000242:Cyclic AMP / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D024101:Mitochondrial Proteins; D009154:Mutation; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: C113837:AFLR protein, Aspergillus; D000348:Aflatoxins; C087299:C1QBP protein, human; D023081:CCAAT-Binding Factor; D002352:Carrier Proteins; D003470:Culture Media; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D000242:Cyclic AMP; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C062609:norsolorinic acid ketoreductase