Genetics - Theses

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Subject: Aspergillus -- Genetics × Author: Todd, Richard B. ×

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    Analysis of the facB gene of Aspergillus nidulans
    Todd, Richard B. (University of Melbourne, 1995)
    The facB gene of Aspergillus nidulans encodes a transcriptional activator which contains a Zn(II)2Cys6 DNA binding cluster, a putative leucine zipper-like dimerization motif and potential acidic activation domains. facB mediates acetate induction of amdS (acetamidase) and the genes of acetate utilisation (facA, acuD, and acuE). This thesis describes an analysis of the facB gene product using a mutational approach and in vitro DNA binding studies. A facB null mutant was constructed by gene replacement This mutant is unable to grow when acetate is the sole carbon source, indicating that facB is required for utilisation of acetate. Important functional regions of FacB were identified by the localisation of lesions in facB mutant alleles. Regions of FacB likely to be involved in DNA binding, dimerization, transactivation, and a possible structural function in acetate metabolism were identified. The Zn(II)2Cys6 motif was shown to be necessary for facB function by in vitro mutagenesis of specific cysteine residues. A facB allele harbouring this Zn(H)2Cys6 mutation failed to complement for growth on acetate upon transformation into a facB null mutant Transformation of this facB allele into a wildtype strain resulted in decreased growth on acetate and acetamide media. The DNA binding function of FacB was examined in detail. A FacB fusion protein was expressed in Escherichia coli and was shown to bind DNA fragments from facB-regulated promoters in vitro in a sequence-specific manner. Abolition of in vitro binding by an expressed FacB fusion protein containing mutated cysteines in the Zn(II)2Cys6 cluster confirmed that this motif was required for DNA binding activity. Expressed FacB proteins with mutations flanking the Zn(II)2Cys6 motif showed altered DNA binding specificity, implicating these regions in determination of DNA binding site specificity. FacB binding sites were defined by Gel Mobility Shift Assay (GMSA), DNase I and Missing Contact Interference Footprinting analyses. FacB binding sites fall into two dissimilar classes. Mutant binding sites from the amdS promoter, known to modulate expression, were shown to alter FacB binding in vitro.