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J. Biol. Chem., Vol. 279, Issue 26, 27428-27439, June 25, 2004

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A Novel cAMP-response Element, CRE1, Modulates Expression of nor-1 in Aspergillus parasiticus^*

Ludmila V. Roze, Michael J. Miller¶, Matthew Rarick, Nibedita Mahanti||, and John E. Linz**

From the Departments of Food Science and Human Nutrition and Microbiology and Molecular Genetics, the ^**National Food Safety and Toxicology Center, and the Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824

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.

Received for publication, January 5, 2004 , and in revised form, March 23, 2004.

^* This work was supported by the National Institutes of Health Grant CA52003-12, a Michigan Life Sciences Research Corridor grant, and the Michigan State University Intramural Research Grants Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Dept. of Food Science, North Carolina State University, Raleigh, NC 27695.

^|| Present address: Biology/DNA Laboratory, Michigan State Police, East Lansing, MI 48823.

To whom correspondence should be addressed: Dept. of Food Science and Human Nutrition, 234B GM Trout Bldg., E. Lansing, MI 48824. Tel.: 517-355-8474; Fax: 517-353-8963; E-mail: jlinz{at}msu.edu.

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				L. V. Roze, R. M. Beaudry, A. E. Arthur, A. M. Calvo, and J. E. Linz Aspergillus Volatiles Regulate Aflatoxin Synthesis and Asexual Sporulation in Aspergillus parasiticus Appl. Envir. Microbiol., November 15, 2007; 73(22): 7268 - 7276. [Abstract] [Full Text] [PDF]

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