Genetics - Theses
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ItemCloning and characterisation of the regulatory gene, amdA, of Aspergillus nidulans(University of Melbourne, 1993)Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdI66 and amdI666, have been isolated which specifically affect amdA activation of amdS. These mutations affect an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. The work presented in this thesis describes the cloning and initial functional characterisation of the amdA gene and three of its mutant alleles. The wild-type amdA gene has been cloned by undertaking a chromosome walk from genes gatA and alcC on linkage group VII and localised by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed at low levels and constitutively under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880 amino acid protein which contains two C2H2 zinc fingers, similar to those found in Xenopus laevis transcription factor TFIIIA, a potential nuclear localisation sequence and two potential transcriptional activation domains. Functional analysis of in vitro-generated deletion mutations and of the semi-dominant mutant allele, amdA7, indicate that the acidic activation domain identified in the protein sequence is functionally significant. Interestingly, the C2H2 zinc finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognises sequences which coincide with the proposed site of action for AmdA within the promoter of this structural gene.