The long term goal of this laboratory is to understand the molecular mechanisms of eukaryotic gene evolution and expression. We are particularly interested in studying human multigene families that are associated with genetic diseases. I propose to study, in the next 5 years, the sequence organization and transcriptional regulation of the Alpha-like globin gene clusters (Alpha-cluster) of four primate species including human. First, we will extend our study of the sequence organization of the human Alpha-cluster to three other primates (gibbon, rhesus and baboon). This will provide us with extremely important information on the molecular evolution of this gene cluster. Secondly, we will investigate in detail the in vivo regulation of transcription of the Alu family repeats interspersed throughout the human Alpha-cluster. These repetitive sequences will be cloned into different plasmids and somatic cell DNA vectors and transfected into mammalian cell cultures. Their transcriptional initiations and terminations, and promoter sequence requirements in vivo will be studied. The in vivo assembled ribonucleoprotein particles will be isolated for structural characterization. Thirdly, we will purify RNA polymerase III and other protein factors involved in the RNA polymerase III-dependent transcription of the Alu family repeats. Antibodies directed against these purified proteins will be used to clone the genes coding for either the subunits of the polymerase or the protein factors from cDNA-expression libraries. The cloned genes of these proteins can be used to study their own transcriptional regulation in various human cells. Fourth, we will study the relationship between chromatin structure and the transcriptional regulation of the Alpha-like globin genes in vivo. In particular, we will examine the DNAase I- and S1-nuclease-hypersensitivities of the Alpha-cluster inside erythroid and non-erythroid tissues, and on episomal DNA in somatic cell cultures. lastly, we will use recombinant DNA clones containing different regions of the human Alpha-cluster as substrates and probes for studying non-globin, non-repeat transcriptional units in the Alpha-cluster.
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