Novel Transcription Factor Selection System

The expression of many inducible genes is regulated at the level of mRNA transcription. Thepromoter regions of these genes are comprised of regulatory sequences that bind a specific combinationof transcription factors. Our aim is to identify and clone novel transcription factors specific forindividual members of the TNF/CD4O ligand superfamily. We will use a novel selection system to screencDNA libraries for proteins which can transactivate expression of antibiotic resistance in the fissionyeast, S. pombe, from a target TNF/NGF receptor superfamily promoter. In phase I, the concept of thisnovel transcription factor screen will be reduced to practice. Reporter constructs utilizing the TNFpromoter to drive the neomycin phosphotransferase gene will be constructed and transformed into S.pombe to determine baseline sensitivity to the neomycin analog G418. Next the gene for SP1transcription factor will be cloned into our cDNA library expression vector and its ability to confer G418resistance on the strain bearing the TNF promoter construct will be demonstrated. in Phase II this systemwill be used to identify and clone novel TNF/CD4O ligand superfamily transcription factors starting withthe CD4O ligand. Inhibition of these transcription factors offers a novel means for immune intervention.