COLD SHOCK PROTEINS AND HEMATOPOIESIS/DEVELOPMENT

本研究的最终目标是定义和表征 小鼠DNA结合蛋白A（dbpA）在造血和 为了阐明这些生物效应的分子机制， 调解。莱伊博士的实验室长期以来对 造血调控，特别是了解分子事件 调节胎儿到成人血红蛋白的转换。dbpA是单个 链DNA结合蛋白和冷休克家族成员 proteins.它是通过筛选骨髓表达文库克隆的， 一项鉴定调节蛋白质的研究， 发育调节的血红蛋白转换目前，生物 dbpA的功能未知。我们的初步数据表明，dbpA 表达仅限于骨髓和脾。组织特异性 dbpA的表达提示其可能在造血过程中起重要作用 或在成熟造血细胞功能中。建立基本的 了解鼠dbpA基因位点、基因表达和蛋白质 结构和功能，将作为理解的基础 mdbpA，我们提出以下具体目标： 1.我们将通过生成一个 每个基因座的限制性图谱，克隆，测序，并定义 鼠和人dbpA基因的染色体定位。 2.我们将描述dbpA在成人中表达的正常模式， 小鼠和胚胎发育期间通过北方原位分析 杂交和免疫组织化学使用的抗体，我们将 开发. 3.我们将确定重组dbpA的DNA结合特异性， 通过创建负责活性的蛋白质结构域， dbpA蛋白中的特异性缺失和突变。 4.我们将建立和表征小鼠功能丧失模型， dbpA通过在胚胎干细胞中靶向基因破坏。dbpA -/- 将评估小鼠的发育、形态和造血 缺陷

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.