The goal is to elucidate the molecular events involved in the developmentally regulated expression of rabbit globin genes. Both direct biochemical assays and interspecies structural comparisons (with their evolutionary inferences) are used to achieved this goal. These studies are helpful in understanding human hemoglobinopathies such as thalassemias and are essential to the design of potential therapeutic regimens. The mechanisms discovered for the regulation of the globin multigene family may also be applicable to other gene families which in a defective form cause a variety of genetic and developmental diseases. This proposal is specifically designed to continue the studies of the structure and expression of rabbit Alpha- and Beta-globin gene families in the following six projects. (1) The complete linkage map of the Zeta and Alpha globin genes will be established, the genes and flanking regions sequenced, and the developmental time of expression determined. (2) The regulation of Alpha1 gene expression will be studied, and the possible involvement of GC boxes and a conserved 3' flanking sequence will be tested by in vitro mutagenesis and expression in transfected cells. (3) The mechanism of attenuation and termination of transcription will be analyzed for both Alpha and Beta genes. Our model for transcriptional attenuation involving stern- and loop formation in the RNA and antisense transcription will be tested by placing the proposed Beta1 attenuator in viral transcription units and by appropriate deletion mutagenesis experiments. The complex transcription unit of Alpha1 will be mapped in detail for a comparison with Beta 1. (4) Nucleases will be used to dissect aspects of globin chromatin structure in nucleic from erythroid cells. The sequence requirements for adopting an unusual DNA conformation sensitive to nuclease S1 will be determined, and spectroscopic methods will be used to study this unusual conformation. (5) Structural studies on long L1 repeats will be completed, and the transcription of both long and short repeats will be studied in heterologous cells. (6) The sequence of the entire rabbit Beta-like globin gene cluster (44 kb) will be completed and compared to the homologous region in humans and mice.
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