Molecular characterisation of mutations in the qutA activator gene for quinic acid utilisation in Aspergillus nidulans
2014-12-15T10:38:03Z (GMT) by
Aspergillus nidulans utilises quinic acid as a sole carbon source for growth. Genetic and biochemical analysis has shown that the inducible quinic acid utilisation (qut) pathway shares intermediates with the constitutive biosynthetic shikimate pathway. Molecular analysis suggests that the qutA and qutB genes evolved by duplication and cleavage of the aromA gene, the activator protein shows identity to the two amino terminal domains and the repressor protein shows identity to the three carboxyl terminal domains.;A contribution to this analysis is detailed in this thesis which extends the identification of functional motifs in the qutA activator. A genetic map of non-inducible qutA mutant strains has been constructed. The mutants yield either recessive, semi-dominant or dominant phenotypes in diploid strains with the wild type activator. Oligonucleotide primers were designed to amplify specific regions of the qutA gene, by the Polymerase Chain Reaction (PCR), from wild type and mutant genomic DNA. Single Stranded Conformational Polymorphism (SSCP) analysis of the PCR amplified DNA was used to align the genetic and physical maps. The PCR amplified DNA was used in sequencing reactions to characterise seventeen mutations at the nucleotide level and infer changes in the protein.;The genetic and physical maps proved to be co-linear. All the non-inducible qutA mutations mapped to the 3' half of the gene, clearly implying that mutations in the 5' half of the gene produce a transcriptionally active protein, indicating a centrally located transcriptional activation motif. The two dominant non-inducible mutations mapped to the junction of the two putative domains of the qutA protein predicted by its homology to aromA, strongly supporting the proposed bi-domain structure for the activator. The results presented in this thesis together with other recent work discussed enable a revision of the molecular model, and provide a strong foundation for the in vitro study of the functions of the two regulatory proteins.