Genetics - Theses
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ItemGenetic and molecular analysis of a positive regulatory gene in Aspergillus nidulans(University of Melbourne, 1989)In the ascomycete fungus Aspergillus nidulans, utilization of certain amides, omega-amino acids and cyclic amides (lactams) as carbon and/or nitrogen sources requires the structural genes of amdS, gatA, gabA, lamA and lamB, whose expression is coordinately controlled by the positively-acting regulatory gene amdR. Transcriptional activation by amdR is dependent on omega-amino acid inducers (ligands) such as GABA and ?-alanine. To understand the mechanisms by which amdR exerts its regulatory control over structural gene expression, a program was initiated to clone and characterize the amdR gene. Using DNA- mediated transformation of A.nidulans, the amdR regulatory gene was cloned from a genomic cosmid library. Transcript analysis showed that the 2.7kb amdR mRNA is constitutively transcribed at a very low level under all tested conditions. Sequence and transcriptional analysis of amdR showed that it contains three small introns, heterogeneous 5' and 3' transcription sites and multiple codons prior to the major AUG initiator. In addition, the semidominant amdR6c allele was cloned and its lesion identified. The predicted amdR protein sequence has a cysteine-rich "zinc-finger" DNA binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl- terminal half of the product and two sequences homologous to the SV40 large T antigen nuclear localization motif. A series of 5', 3� and internal deletions of amdR were examined in vivo for transcription activator function, showing that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wildtype levels of transcription activation. A number of the amdR deletion products were also shown to compete with the wildtype amdR product in vivo. Dosage phenomena studies using amdR yielded transformants which exhibited stronger growth than the wildtype, indicating increased expression of the relevant structural genes. This suggests that the low constitutive level of amdR product sets the upper limits of basal and induced transcription of the structural genes. Further increases in amdR product concentrations in vivo, through overexpression of the amdR gene, yielded transformants with phenotypic abnormalities. From the molecular and genetic studies presented, a model for amdR-mediated regulation of structural gene expression was formulated.