We propose to continue to study the regulation of differential gene expression using the model of avian red blood cell development. We are studying and have characterized (or are in the process of characterizing) 14 different erythroid-specific genes, and have generated antibody reagents for most of these. Furthermore, we have been able to directly demonstrate erythroid-specific control of two of these genes (beta-globin and histone H5) after transfection into precursor erythroid cells, and that the contol is elicited in beta-globin in a temporally-specific manner during erythropoiesis. During the term of this proposal, we wish to study the regulatory mechanism which confer the temporal-, abundance- and tissue- specificity of these genes by mutational analysis and by correlation of these findings on in vivo expression (both by translient assay and using a retrovirus vector) with the specific cis-acting sequences and the regulatory molecules, we will study the interaction of the regulatory proteins with their cognate gene-specific sequences in vitro. The model system examined in detail in this proposal is concerned with attempts to precisely delimit the cis-acting regulatory sequences making up the beta-globin enhancer, and, if possible, to separately define the sequences responsible for biochemical methods. Using the results of those studies, we plan to isolate the beta-globin regulatory molecule(s), and then clone those regulatory genes as a prelude to studying the interaction of purfied tissue-specific transcription factors with other erythroid- specific genes.
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