ERYTHROPOIETIN RECEPTOR REGULATION OF ERYTHROPOIESIS

描述(取自应用程序) 这项建议的研究目标是研究分子和细胞 促红细胞生成调节机制的研究 促红细胞生成素受体(EPOR)的表达 正常红系发育。拟议的研究包括定性 人类EPOR中非启动子相关的顺式作用调控元件 基因及其相互作用的反式作用因子的鉴定 调控元件，并可能指导组织特异性转录调控 这种基因的。该基因的染色质结构将在组织中进行检查 像培养小鼠组织一样培养细胞以鉴定组织和发育 监管要素的阶段性变化。重组DNA 技术将用于克隆EPOR基因的基因组DNA片段 与其表达相关的基因转移/表达研究 组织培养细胞以及DNA-蛋白质相互作用的研究。另一个 这项建议的目的是研究细胞的信号转导功能 以两种人类红系疾病为模型的人类EPOR家族性 红细胞增多症和真性红细胞增多症。一种新的人类EPOR的作用 引起显性遗传性红细胞增多症的突变 激活的EPOR的下游靶点，如JAK2、STAT5、Shc和MAP 激酶，将被研究以表征相关的信号异常 与红系祖细胞的EPO高反应性有关。组织培养 表达突变型和/或野生型EpoRs和红系祖细胞的细胞 将在EPO DoS反应分析中进行研究，DNA 碎裂研究，凝胶迁移率变化分析和 免疫沉淀和免疫印迹实验。在另一个模型中， 真性红细胞增多症患者红系祖细胞的纯化 细胞内信号分子激活动力学的研究 是激活的EPOR的目标，如JAK2和Stats， 它的过度活跃可能与体外增强有关 对促红细胞生成素的反应性。这项实验室研究将由 首席研究员木拉提·O·阿卡索伊博士与一位说教 项目由赞助商Bernard G.Forget博士和 咨询委员会。这项提议将允许候选人制定 技术专长、基础知识和智力科学 独立和富有成效的调查职业生涯所必需的方法 分子和细胞生物学及其在发病机制研究中的应用 血液系统疾病。

DESCRIPTION (taken from application) The research goal of this proposal is to study the molecular and cellular mechanisms involved in the regulation of erythropoiesis mediated by the erythropoietin receptor (EpoR), the expression of which is essential for norma erythroid development. The proposed studies include characterization of nonpromoter-related, cis-acting regulatory elements within the human EpoR gene and identification of trans-acting factors which interact with the regulatory elements and may direct tissue-specific transcriptional control of the gene. The chromatin structure of the gene will be examined in tissue culture cells a well as mouse tissues to identify tissue-and developmental stage-specific changes in the regulatory elements. Recombinant DNA technology will be used in cloning of genomic DNA segments of the EpoR gene relevant to its expression followed by gene transfer/expression studies in tissue culture cells as well a studies of DNA-protein interactions. Another goal of this proposal is to study the signal transduction function of the human EpoR using two human disorders o erythropoiesis as models, familial erythrocytosis and polycythemia vera. The effect of a novel human EpoR mutant causing dominantly-inherited erythrocytosi on the activation of downstream targets of the activated EpoR, such as Jak2, Stat5, Shc and MAP kinase, will be studied to characterize signaling abnormalities associated with Epo hyper-responsiveness of erythroid progenitors. Tissue culture cells expressing the mutant and/or wild type EpoRs and erythroid progenitors from affected individuals will be studied in Epo dos response assays, DNA fragmentation studies, electrophoretic gel mobility shift assays and immunoprecipitation and Western blotting experiments. In another model, purified erythroid progenitors from patients with polycythemia vera wil be studied for the activation kinetics of intracellular signaling molecules that are targets for the activated EpoR, such as Jak2 and Stats, hyperactivity of which may be associated with increased in vitro responsiveness to Epo. This laboratory research will be performed by the principal investigator Dr. Murat O. Arcasoy in conjunction with a didactic program under the guidance of a sponsor, Dr. Bernard G. Forget and an advisory committee. This proposal will allow the candidate to develop the technical expertise, fundamental knowledge and intellectual scientific approach necessary for an independent and productive career in investigative molecular and cellular biology and its application to pathogenesis of hematologic disorders.