COLD SHOCK PROTEINS AND HEMATOPOIESIS/DEVELOPMENT

这项研究的最终目标是定义和描述 小鼠DNA结合蛋白A在造血祖细胞中的生物学作用 为了阐明这些生物效应产生的分子机制 调解过的。莱伊博士的实验室长期以来一直对 造血调控，尤其是对分子事件的理解 来调节胎儿到成人的血红蛋白转换。DbpA是一个 DNA结合蛋白与冷休克家族的一员 蛋白质。通过筛选小鼠骨髓表达文库，克隆了该基因。 一项努力确定可能有助于 发育调节的血红蛋白开关。目前，生物界 DbpA的功能未知。我们的初步数据显示， 表达仅限于骨髓和脾。组织特异性 DbpA的表达提示其在造血中可能起重要作用 或处于成熟的造血细胞功能中。要确立根本 对小鼠dbpA基因位点、基因表达和蛋白的了解 将作为理解的基础的结构和功能 MdbpA，我们提出了以下具体目标： 1.我们将通过生成一个 每个基因座的限制图、克隆、测序和定义 小鼠和人的dbpA基因的染色体定位。 2.我们将描述在成人中的正常表达模式 小鼠和胚胎发育过程中的Northern分析，原位 杂交和免疫组织化学使用的抗体，我们将 发展。 3.我们将确定重组DBpA与DNA结合的特异性 通过创建对活性负责的蛋白质结构域的特征 DbpA蛋白中的特定缺失和突变。 4.我们将建立和表征小鼠的功能丧失模型 在胚胎干细胞中通过靶向基因破坏的方法。DbpA-/- 将对小鼠进行发育、形态和造血方面的评估 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.