Site-directed mutagenesis of the -127 activator binding site of the qa-2 gene of neurospora crassa /

Abstract

The quinic acid (qa) gene cluster is a positively regulated system. In the absence of the inducer, quinic acid, the repressor protein binds to the activator protein, blocking transcription of the qa genes. Addition of quinic acid releases the activator protein, which is then free to bind to its activator binding sites, increasing the levels of transcription of the qa genes. The activator binding sites are composed of a 16 base pair (bp) conserved sequence, but the importance of the individual bases in the sites is currently unknown. To determine the importance of the bases, the DNA containing the activator binding site was cloned and isolated. The -127 binding site of the qa-2 gene was chosen due to its high binding affinity for the activator protein. A plasmid known to contain the entire qa cluster was digested with Pst! and the fragment containing the qa-2-qa-x intergenic region was cloned into pBR322 to form plasmid 177. This was digested further with the enzymes EcoRI and Pst! and the desired' fragment cloned into pBluescript to form pEP, which was then digested with EcoRI and HindUI and the appropriate fragment was cloned into pBluescript to form pEH. PEH was finally digested with KpnI to give a 500 bp fragment that was cloned into M13. Using the method described by Kunkel, et al (1991), site-directed mutagenesis was performed on one of the most highly conserved bases in the site. A clone was then sequenced to determine if the mutagenesis was successful. While the clone chosen did not contained the desired mutation, future students will study the remaining prepared to clones to attempt to isolate a mutant.