Many biological processes are regulated at the level of initiation of transcription of genes. The goal of the experiments in this proposal is to increase our understanding of the mechanisms by which transcription is regulated in eukaryoptic cells. To this end, an in-depth study of the cis-acting DNA sequences and the trans-acting components required for transcription of mammalian Beta-globin genes will be performed. Cloned globin genes with mutant regulatory sequences will be introduced into tissue culture cells to delineate both promoter elements and DNA sequences responsible for the tissue-specific transcription of Beta-globin genes in erythroid cells. Cell-free transcription experiments will be used to confirm the in vivo analysis, and both in vitro transcription and DNA binding assays will be used to identify the trans-acting promoter and erythroid cell-specific factors responsible for the regulated transcription of the gene. These assays will be used to purify the transcription components and to study in detail their interactions with wild type and mutant DNA templates. The identification of the DNA sequences and factors required for transcription of the Beta-globin gene and the establishment of a reconstituted regulated transcription system in vitro will provide a useful model for analyzing the detailed molecular mechanisms of eukaryotic gene regulation.
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