Genetics - Theses

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End date: 2003 × Subject: Aspergillus -- Genetics × Author: Xiao, Wenwang. ×

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    Structural and functional analysis of the amdR positively-acting regulatory gene in Aspergillus oryzae
    Xiao, Wenwang. (University of Melbourne, 1992)
    The mechanism of the amdR regulation has been well characterized in Aspergillus nidulans. The corresponding A.oryzae gene was cloned and characterized in this study, which provides an insight into the conservation of the regulatory proteins during evolution. The A.oryzae homologue was isolated by cross hybridization with the A.nidulans amdR gene, and shown to complement the A.nidulans amdR44- mutant. Induction studies indicated that the A.oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A.nidulans protein. Inactivation of the A.oryzae amdR gene further confirmed its regulatory role in GABA utilization. Transformation of the A.nidulans amdR gene restored the amdR activity in the gene inactivation mutant of A.oryzae, indicating again that these two amdR genes are functionally interchangeable. Sequence comparison of the two amdR genes showed that the overall homology of DNA sequence throughout the coding region was 64%; and that the conservation of the two polypeptides was 85%, with 76% identity. The conserved amino acid sequences are concentrated in the putative functional domains, such as DNA binding, nuclear localization, transcription activation, and inducer interaction. Two stretches of amino acid sequences which are outside the known functional domains are significantly divergent between two species, suggesting that these represent regions of little functional importance. The A.nidulans amdR gene contains three introns, and the first and third introns have been found at identical positions within the A.oryzae gene. Intron II region is probably a coding region in A.oryzae due to its lack of consensus 5' splice site and a high degree of homology with the corresponding A.nidulans sequence. The transcriptional initiation, termination and splice signals are similar between these two amdR genes, although the overall DNA sequences in these regions have diverged. The extent of sequence divergence between these two amdR genes indicates that the species have evolved separately for a period of time. However, the sequence divergence has been restrained to those changes which have little effect on the functional activity of the amdR gene products.