Genetics - Theses

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End date: 1999 × Start date: 1990 × Subject: Aspergillus nidulans ×

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    The isolation and analysis of the hap genes of Aspergillus nidulans
    Papagiannopoulos, Peter. (University of Melbourne, 1996)
    The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence required for setting the basal level of transcription. Mobility shift assays have identified a factor in A. nidulans nuclear extracts that binds specifically to this CCAAT sequence. In Saccharomyces cerevisiae, the HAP3 and HAP5 genes encode components of a highly conserved multi subunit complex which is able to bind CCAAT sequences. The identification, cloning and sequencing of genes from A. nidulans with homology to HAP3 and HAP5, known as hapC and hapE respectively, is described here. The predicted amino acid sequences of the proteins encoded by the hapC and hapE genes share extensive sequence identity to conserved regions in HAP3 and HAP5 respectively. Furthermore, they both show identity to the histone-fold motif, a motif used widely as a means for protein-protein and DNA- protein interactions. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially poorly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, the hapC deletion results in reduced levels of amdS expression, particularly under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT specific binding to the amdS or gatA promoter, indicating that hapC encodes a component of the complex binding at this sequence. In the presence of the hapC deletion growth on acetamide and amdS
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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.