Genetics - Theses

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Type: PhD thesis × Subject: Aspergillus nidulans -- Genetics × Author: Small, Anna J. ×

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    Characterization of the tamA gene of Aspergillus nidulans
    Small, Anna J. (University of Melbourne, 2000)
    In Aspergillus nidulans, the GATA zinc finger protein AreA activates the expression of enzymes that metabolize less favoured nitrogen sources in the absence of preferred nitrogen sources, such as ammonium or glutamine. The amount and activity of AreA are modulated by a number of mechanisms, including an interaction with the NmrA protein through the GATA zinc finger and C-terminal regions of AreA to inhibit DNA binding under nitrogen-sufficient conditions. The TamA protein has also been implicated in nitrogen regulation, with mutants described as having reduced levels of a number of nitrogen metabolic enzymes. This thesis describes the characterization of the tamA gene and investigates its role in nitrogen regulation. tamA encodes a 739 amino acid protein that contains features common to DNA-binding transcription factors, including a potential Zn(II)2Cys6 DNA-binding domain. Uga35p of S. cerevisiae shows some similarity in both structure and function to TamA, and remarkably the Zn(II)2Cys6-like domains of both proteins are not required for function. To define important regions of TamA, sequence changes in a number of tamA mutants were determined and constructs containing deletions of various regions were tested for function. While the most N-terminal and C-terminal regions of TamA were dispensable for function, changes affecting even small parts of other regions of the protein abolished function. This suggests that the overall protein conformation is critical. Constructs encoding the TamA protein fused to DNA-binding domains were shown to activate gene expression in A. nidulans by recruitment of AreA. The Aspergillus oryzae AreA and Neurospora crassa NIT2 proteins were able to substitute for A. nidulans AreA in this interaction. Although the GATA zinc finger did not seem to be involved, the 12 amino acids at the AreA C-terminus were essential for interaction with TamA. This region is also involved in the interaction with NmrA, suggesting that competition for binding to the AreA C-terminus may be a part of the function of TamA. Uga35p was not able to interact with AreA and also could not complement a tamA? mutation, demonstrating differences in the coevolution of nitrogen regulatory mechanisms between different species.