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TheamdARegulatory Gene ofAspergillus nidulans:Characterization of Gain-of-Function Mutations and Identification of Binding Sites for the Gene Product

1997; Academic Press; Volume: 21; Issue: 1 Linguagem: Inglês

10.1006/fgbi.1997.0968

1096-0937

Alex Andrianopoulos, Janynke F. Brons, Meryl A. Davis, Michael J. Hynes,

RNA and protein synthesis mechanisms

Genetic evidence suggests that the amdA gene of Aspergillus nidulans encodes a protein which controls the expression of the amdS and aciA structural genes. The amd166 and amd1666 mutations in the 5' regulatory region of amdS lead to higher levels of amdA-dependent amdS expression. We show here that the putative DNA binding domain of amdA is capable of binding specific regions of the amdS and aciA promoters in vitro and this region includes sequences duplicated and triplicated in the amd166 and amd1666 mutations, respectively. Footprinting analysis has shown that AmdA binds to two sites in this region represented by the sequences 5'-GCGGGG-3' and 5'-GAGGGG-3'. A number of gain-of-function mutations in amdA were localized to a region rich in acidic and hydrophobic amino acid residues and shown to be involved in transcriptional activation by studies of fusions with the GAL4 DNA binding domain in Saccharomyces cerevisiae. Therefore, both an increased probability of AmdA binding to the amdS promoter and an increased activation potential of AmdA can result in higher levels of expression.