This project aims specifically to recruit and train minority students to succeed in molecular biological research in my laboratory, in order to encourage and facilitate the entry of these students into the biomedical professions. Minority and women students graduating from my relatively new lab are already doing very well in research positions at graduate schools and Biotechnology companies. The long term objectives of research in this laboratory are to study how transcription from eukaryotic promoters is regulated by DNA binding of protein transcription factors having zinc-fingers for DNA sequence recognition. Prediction of the precise amino acid sequence required by a zinc-finger protein to bind to any given DNA sequence element is a goal which molecular biologists believe will have profound implications for medical therapy by regulation of gene expression. While this research is fundamental, the biomedical relevance of being able to turn on or off certain oncogenes by gene therapy cannot be overlooked. We will contribute to this work by using molecular modelling, site-directed mutagenesis of the transcript ion factor gene, and DNA binding studies of the mutant protein-DNA complexes in vivo and in vitro. The plasmids for this project have already been assembled in my laboratory by student researchers using the ADR1 zinc-finger protein in yeast as a model system which is easily manipulated genetically. The experimental design for our project uses a set of plasmids in the yeast Saccharomyces cerevisiae to determine how ADR1 binds specifically to the 22 base-pair UAS1 DNA sequence, turning on transcription of the ADH2 gene for alcohol dehydrogenase II. By site-directed mutagenesis of the zinc-finger region of the ADR1 gene, it will be possible to systematically modify the three crucial base-contacting residues (15, 18, and 21) of the 30 amino acids in each of two zinc-finger loops of the protein, then test if a given mutant abolishes or changes the specificity of DNA binding to UAS1 or derivative sequences. ADR1 mutants made in pTZ-ADR1 can be expressed in E. coli, then tested in vitro for binding to synthetic UAS sequences. After cloning this ADR1 mutant as a DNA casette into yeast plasmid YEpGAL-ADR1, it will be expressed in yeast cells containing plasmid YEpADH2+UAS1, to test if the predicted mutant UAS sequence inserted in the promoter of the reporter gene ADH2 is activated by ADR1 binding. Thus, DNA binding rules may be elucidated.
