The ultimate goal of this research is to define and characterize the biologic role of murine DNA binding protein A (dbpA) in hematopoiesis and to elucidate the molecular mechanisms by which these biologic effects are mediated. Dr. Ley's laboratory has a longstanding interest in the regulation of hematopoiesis, especially understanding the molecular events that mediate fetal to adult hemoglobin switching. dbpA is a single stranded DNA binding protein and a member of the family of cold shock proteins. It was cloned by screening a bone marrow expression library in an effort to identify regulatory proteins that may contribute to the developmentally regulated hemoglobin switch. Currently, the biologic function of dbpA is unknown. Our preliminary data suggests that dbpA expression is restricted to bone marrow and spleen. The tissue specific expression of dbpA suggests it may play an important role in hematopoiesis or in mature hematopoietic cell function. To establish the fundamental knowledge of the murine dbpA gene locus, gene expression, and protein structure and function that will serve as a foundation for understanding mdbpA, we propose the following specific aims: 1. We will completely characterize the genomic dbpA loci by generating a restriction map of each locus, cloning, sequencing, and defining the chromosomal location for the murine and human dbpA genes. 2. We will characterize the normal pattern of dbpA expression in the adult mouse and during embryonic development by Northern analyses, in situ hybridization, and immunohistochemistry using an antibody that we will develop. 3. We will define the DNA binding specificity of recombinant dbpA and characterize the protein domains responsible for activity by creating specific deletions and mutations in the dbpA protein. 4. We will create and characterize a loss of function model for murine dbpA by targeted gene disruption in embryonic stem cells. The dbpA -/- mice will be assessed for developmental, morphologic, and hematopoietic defects.
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