Genetics - Theses

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End date: 2009 × Type: PhD thesis × Subject: Aspergillus nidulans × Subject: Genetic transcription ×

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    Characterisation of the facB88 translocation of Aspergillus nidulans
    Murphy, Rachael L. (University of Melbourne, 1996)
    The positively acting regulatory gene facB of Aspergillus nidulans mediates acetate induction of the amdS (acetamidase) gene and genes required for acetate metabolism (facA, acuD and acuE). facB encodes a gene product with a Zn(II)2Cys6 DNA binding cluster, heptad repeats and potential activation domains, and binds to sequences 5' of amdS, facA, acuD and acuE. facB orthologues from A. oryzae and A. niger have been compared to the A. nidulans facB gene at the level of DNA and predicted protein sequences. Highly conserved regions in the predicted translation products have been identified and discussed in terms of their relevance to protein function. Putative transcription factor binding sites were identified in the 5' non-coding regions of each orthologue. The facB88 reciprocal translocation results in high-level constitutive amdS expression (superactivation) and this is mediated by a chimeric gene formed by the translocation event. This chimeric gene was found to encode the N-terminal half of FacB, including the DNA binding domain, fused to a new gene encoding two C2H2 zinc finger DNA binding motifs. The new gene was designated amdX, the cloning, sequencing and transcriptional analysis of which is reported here. Inactivation of amdX and creation of multicopy strains by transformation revealed that amdX is a minor positive regulator of amdS expression. The DNA binding function of AmdX was examined, using an Escherichia coli-expressed AmdX fusion protein, by gel mobility shift assays and DNase I footprinting. AmdX was shown to bind to two sites in the amdS 5' region which overlap the binding sites for the AmdA and CreA regulatory proteins. The facB88 chimeric gene mediating amdS superactivation was designated facB-amdX. Molecular dissection of facB-amdX showed that both the FacB and AmdX DNA binding domains are required for amdS superactivation and that they contribute to amdS expression in a synergistic manner. Cooperative DNA binding of the FacB and AmdX DNA binding domains to the amdS 5' region is proposed to mediate the superactivation ability of the facB-amdX gene product.