SBIR-STTR Award

Our goal is to identify genes that are regulated by the putative transcription factor encoded by the human PAX-6 (aniridia) locus. The specific aim of Phase I is to use bacterially produced pas-6 protein to identify specific binding sites in human genomic DNA. Isolation of sequences bound by pax-6 will be achieved by making fusion protons of pax-6 with the maltose binding protein, and purifying protein/DNA complexes on amylose columns. Genomic fragments that include recognition sites for pax-6 will be screened for the presence of sequences expressed in the retina and the developing brain.At the end of Phase I, clones representing tissue specific targets of pax-6 binding will be obtained. These materials will facilitate cloning of novel genes that are regulated by pax-6. Since PAX-6 and other PAX genes are known to be mutated in human development disorders, cloning of target genes will permit the study of transcriptional mechanisms involved in the molecular pathology of these disorders. In addition to furthering knowledge of disease mechanisms, this research has important implications for manipulating gene expression in the nervous system.National Institute of Neurological Disorders and Stroke (NINDS)

Pax-6 is an essential nervous system transcription factor that is involved in basic pattern formation during development, as well as in several inherited disorders. The goal of this research is to clone pax-6 regulated promoters from the human genome, and to identify genes associated with the promoters, Potential pax-6 regulated promoters will be cloned using a bacterially produced pax-6 fusion protein to isolate and clone genomic DNA fragments that bind to the paired box and homeobox of pax-6. Individual clones from pax-6 binding site libraries established during Phase I and Phase II of this research will be screened for specific binding to pax-6 by high throughput electrophoretic mobility shift assays. Clones with specific binding activity' will be sequenced at an in-house automated sequencing facility, and submitted to database searches and sequence analyses to identify clones that share potential binding sites and/or illustrate critical features of eukaryotic promoters. This set of sequences will be used for the sequence-based identification of potential pax-6 binding sites, as well as binding sites for other transcription factors associated with pax-regulated promoters. Detailed analyses of the binding site clones, including deletion mutagenesis and DNAse I footprint analysis to identify specific sites of protein/DNA interaction, and identification of cDNAs for genes associated with the promoters, will be performed on the best candidates. The most outstanding of the putative pax-6-regulated promoters identified by this series of experiments will be used to study the transcriptional activity of pax-6 in cultured cells. PROPOSED COMMERCIAL APPLICATION: The identification of genes that are regulated by pax transcription factors will lead to an understanding of the molecular pathology of inherited disorders such as aniridia and Waardenburg's syndrome, thereby providing a direction for gene therapy research. Since pax genes are involved in neural development, an understanding of their transcriptional mechanisms will be of general applicability to therapeutic programs requiring targeted gene expression