Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans

Peters, David G.; Caddick, Mark X.

doi:10.1093/nar/22.24.5164

Cited by 21 publications

(17 citation statements)

References 54 publications

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“…Preparation of Protein Extracts for EMSA-Cellular protein was extracted from A. parasiticus cultures using our modifications (31) of the method of Peters and Perez-Esteban (40,41). Briefly 1-liter flasks containing 10 6-mm glass beads and 500 ml of medium (GMS or PMS) were inoculated with 1 ϫ 10 8 spores.…”

Section: Strains Growth Media and Growth Conditions-escherichia Colmentioning

confidence: 99%

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

Roze

Miller

Rarick

et al. 2004

Journal of Biological Chemistry

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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.Aflatoxins are secondary metabolites produced by Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus on food and feed crops (1). Aflatoxins have a broad range of toxic effects on humans and animals including carcinogenicity, neurotoxicity, and reproductive and developmental toxicity (for a review, see Ref.2). Therefore, aflatoxin contamination raises serious concerns related to environmental safety, food quality, and human and animal health.

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Section: Strains Growth Media and Growth Conditions-escherichia Colmentioning

confidence: 99%

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

Roze

Miller

Rarick

et al. 2004

Journal of Biological Chemistry

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“…The activity of NIT2 is modulated by the negative acting factor NMR through direct protein-protein interaction in N. crassa (13). Employing electrophoretic mobility shift assay analysis, at least three GATA binding activities other than AREA have been distinguished in A. nidulans (14). This suggests the existence of additional GATA factors possibly involved in nitrogen metabolite repression, thus resembling the situation in S. cerevisiae.…”

mentioning

confidence: 85%

“…In such a model, overproduction of NREB leads to repression of nitrogen metabolite regulated genes by disturbing the balance between NRE and NREB. Recently, a negative-acting factor from A. nidulans has been identified genetically that is involved in determining the functional specificity of AREA and is proposed to operate through direct interaction with the zinc finger domain of AREA (14,49).…”

Section: Nreb Binds To Gata Motifs Present In Its Own Promoter and Inmentioning

confidence: 99%

Overexpression of nreB, a New GATA Factor-encoding Gene of Penicillium chrysogenum, Leads to Repression of the Nitrate Assimilatory Gene Cluster

Haas

Angermayr

Zadra

et al. 1997

Journal of Biological Chemistry

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To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungus Penicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein. Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae. All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus. Northern blot analysis revealed the presence of two nreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites. The steady state level of both transcripts is subject to nitrogen metabolite repression. The putative DNA binding domain of NREB, expressed as a fusion protein in Escherichia coli, binds in vitro to GATA sites of its own 5-upstream region as well as in the promoter of the nitrate assimilation gene cluster. Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes. Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

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“…(i) Protein extraction. Cell protein extracts were generated by using modifications of the procedures of Peters and Caddick (25) and Peres-Esteban et al (24). Conidiospores (1 ϫ 10 7 ) were inoculated into 1-liter flasks containing 500 ml of appropriate growth medium and 10 glass beads.…”

Section: Gus Reporter Assaysmentioning

confidence: 99%

Role of cis -Acting Sites NorL , a TATA Box, and AflR1 in nor-1 Transcriptional Activation in Aspergillus parasiticus

Miller

Roze²,

Trail³

et al. 2005

Appl Environ Microbiol

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The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus Aspergillus. nor-1 encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (AflR1). Proteins in Aspergillus parasiticus cell extracts and AflR expressed in Escherichia coli do not bind to A. parasiticus AflR1 in vitro, so it was not clear if this site was required for nor-1 expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in nor-1 expression in A. parasiticus and identified additional cis-acting sites required for maximum nor-1 transcriptional activation. Deletion and substitution of AflR1 in the nor-1 promoter in A. parasiticus nor-1::GUS reporter strains showed that this site is required for nor-1 transcriptional activation in vivo. Substitution of a putative TATA box in the nor-1 promoter resulted in nondetectable ␤-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel cis-acting site, designated NorL, between residues ؊210 and ؊238 that was required for maximum nor-1 transcriptional activation in A. parasiticus grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific NorL-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the NorL site appears only once in the aflatoxin gene cluster, its association with the nor-1 promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.

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Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans

Abstract: ABSTRACT

Cited by 21 publications

References 54 publications

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

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

Overexpression of nreB, a New GATA Factor-encoding Gene of Penicillium chrysogenum, Leads to Repression of the Nitrate Assimilatory Gene Cluster

Role of cis -Acting Sites NorL , a TATA Box, and AflR1 in nor-1 Transcriptional Activation in Aspergillus parasiticus

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