The long term goal of this grant application is to understand the molecular evolution of the primate alpha-like globin gene clusters (alpha cluster), and the mechanisms of transcriptional regulation of the human alpha cluster. The research plan for the next five years consists of five specific aims. First, we will continue our studies of the sequence organizations of the adult alpha globin loci in orangutan, olive baboon, rhesus macaque and human, and extend them to gibbon and New World Monkeys. The studies will also be extended to the evolution of the embryonic epsilon genes. These experiments will be carried out by molecular cloning (phage vector, yeast vector and PCR technique) and nucleotide sequencing. Second, we will study the mechanisms of transcriptional regulation of the human theta 1 globin gene whose promoter consists of CCAAT-TATA boxes embedded within a novel arrangement of Alu family repeat sequences, and a 30 by GC-rich motif conserved at upstream of all mammalian theta 1 genes. This will be done by both in vitro transcription and in vivo transient expression assays. The possible functions(s) and structure of the putative theta 1 polypeptide will also be investigated following high level expression of the polypeptide in mammalian, yeast and bacteria cells. Thirdly, we will study the molecular mechanisms of transcriptional regulation of human alpha and epsilon globin genes by their upstream promoter in human erythroid cells. This will be accomplished by in vivo transcriptional assays of wild type and mutagenized promoter elements in human erythroid cell cultures, by studying specific nuclear factor - promoter DNA interactions, by purification of the transcription factors, and by molecular cloning of the factor genes. Fourth, we will study the molecular mechanisms of the erythroid cell-specific interactions between a 5'-beta locus enhancer and upstream promoter elements that are involved in transcriptional activation of human globin genes in erythroid cells. Transient expression assays, and transcription analysis of transfected and stably integrated recombinant clones will be used to study the mode of the enhancer functioning upon individual human globin promoters in human erythroid cell cultures. the competitions among different human globin promoters, wild type of mutagenized ones, for this enhancer in vivo will also be investigated. Finally, we will study the effects of DNA methylation on the transcriptional regulation of human alpha-like globin genes. This will be accomplished by globin gene transcription analysis of transfected plasmids methylated at different sequences of the globin genes and their flanking regions.
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