The general goal of this project is to characterize upstream promoter factors that regulate murine alpha-globin gene transcription. In published work, four such factors, termed CP1, CP2, GATA-1, and IRP have been described. Each has been purified to apparent homogeneity by DNA sequence affinity chromatography, and the function of each of factor has been assessed using an in vitro transcription assay. In addition cDNA and genomic clones encoding CP1, CP2 and GATA-1 have been obtained. Each of the cloned factors has been expressed in bacteria and factor specific antisera have been (cP2, GATA-1) or are being (CP1) prepared. Because this laboratory has been most recently involved in the cloning of CP2, and because this factor has been less well studied, the specific aims of this proposal emphasize the further characterization of this factor. Our specific goals for the next three years are to: 1. Determine the DNA binding and transcription activation domains of the cloned transcription factor CP2. 2. Obtain complete murine and human genomic clones for CP2, localize the genes to their appropriate chromosomes, determine their intron/exon structures, identify and characterize their promoters. 3. Examine the tissue distribution and expression of CP2 and obtain CDNA clones that encode antigenically related factor present in Xenopus oocytes and yeast. 4. Use antisera against GATA-1 and CP2 and columns of GATA-1 and CP2 affixed to sepharose beads to determine if these factors interact with additional nuclear proteins. Prepare antisera specific for CP1 and determine if additional subunits of this factor must be cloned.
