This proposal is designed to determine the precise role of two distinct estrogen receptors (X, Kd = 0.1 nM and Y, Kd = 2nM) in chick oviduct cytosol on estrogen specific nuclear events. The two receptors will be purified to homogeneity and their effects of RNA polymerase I and RNA polymerase II activities will be defined by adding them to isolated nuclei. These nuclei will be prepared from oviducts isolated from chicks which have been withdrawn from estrogen for various time periods (24, 48, and 72 hours). The effects of the two receptors on increasing the efficiency of transcription of the ovalbumin gene using a truncated ovalbumin gene and a HeLa cell extract in an in vitro transcription system will be determined. The two receptors will also be affinity labeled using [3H]-tamoxifen aziridine and the molecular weights and isoelectric points of proteolytic degradation products and subunits will be determined. This information will be used to study the intracellular regulation of the two receptors under various conditions of steroid treatment. The implied role of GTP and ATP on the activation of receptor precursors to X and Y respectively will be investigated. Finally, as an aid in the purification and quantitation of the two receptors, attempts will be made to generate receptor specific polyclonal and monoclonal antibodies. Overall these studies will prove invaluable in understanding how steroid hormones mediate growth and differentiated function in normal and neoplasmic tissues.
