Site-directed mutagenesis of the -144 activator-binding site of qa-1F in neurospora crassa /

Abstract

The quinic acid cluster of Neurospora crassa provides an interesting model for studying genetics. Its unique operon-like organization provides a good opportunity to study the eukaryotic mechanism of activation and repression of the cluster responsible for utilizing quinic acid as a carbon source. The activator protein, qa-1F, elevates levels of transcription by binding at a conserved sequence that occurs at least 14 times within the cluster. The activator, qa-1F, has one of its highest affinities for the activator-binding site that regulates its own transcription. This site occurs at -144 in relationship to the start site of transcription of qa-lF . By isolating this sequence using peR, and cloning into a RF M13mp18 baculovirus vector, the vector will be utilized to produce single-stranded infectious phage particles with our cloned binding site. Once these infectious phage particles are isolated, site-directed mutagenesis will be performed using the kunkel method to change specific conserved bases. These mutants can be transformed back into Neurospora where the significance of these changes will be better understood.