FUNCTION OF A PUTATIVE DETERMINANT IN HEMATOPOIESIS

描述：(改编自申请者摘要)EKLF为红系 红系早期表达的特异性转录因子 发展。它将序列元素“CACCC”绑定并激活 β-珠蛋白的发起人。其中一些残基的点突变， 包括那些已知会导致β-地中海贫血的人，废除 EKLF的反式激活能力。EKLF基因对小鼠的破坏作用 会导致严重的β-地中海贫血，从而导致胚胎死亡。这 更新建议将研究分子机制的作用 通过评估磷酸化是否调节EKLF活性并通过 识别与EKLF相互作用的蛋白质。此外，一项评估 未明确诊断的β-地中海贫血患者的EKLF基因突变 将会被执行。 之前来自首席调查员实验室的研究已经 测定了EKLF的DNA结合部位，表明EKLF 具有启动子和细胞类型特异性的功能。中的缺失突变体 EKLF的反式活化区显示出一个抑制区和 与积极行为相互作用可能需要的域 因素。 作者提出，MEL中CAC结合活性之一 提取物，CAC-D为EKLF。CIP处理提取物破坏结合 通过凝胶位移分析这种活性。CAC-D频段可能是 被一种EKLF抗体破坏。然而，凝胶从EKLF- 缺陷小鼠已被用来至少证明CAC-D是EKLF。 成功地引入了一种可诱导的、标记为EKLF的表位 进入MEL细胞进行纯化实验。此外，Poly-His标记 EKLF已在细菌中表达，用于产生抗体 (数据未显示)。部分EKLF已在酵母2中表达 混合载体。最终，人类EKLF基因被克隆出来。

DESCRIPTION: (Adapted from Applicant's Abstract) EKLF is an erythroid specific transcription factor that is expressed early in erythroid development. It binds and transactivates a sequence element "CACCC" in the beta-globin promoter. Point mutations in some of these residues, including those known to lead to beta-thalassemia, abolish the transactivating ability of EKLF. Genetic disruption of EKLF in mice causes a severe beta-thalassemia that leads to embryonic lethality. This renewal proposal will investigate the molecular mechanism of action of EKLF by assessing whether phosphorylation regulates EKLF activity and by identifying proteins that interact with EKLF. Further, an evaluation of EKLF mutations in previously undefined beta-thalassemia patients will be performed. Previous studies from the principal investigator's laboratory have determined the DNA binding site of EKLF and have shown that EKLF functions with promoter and cell type specificity. Deletion mutants in the transactivating region of EKLF demonstrate an inhibitory domain and a domain that may be required for interaction with a positive acting factor. The author proposes that one of the CAC binding activities in MEL extracts, CAC-D is EKLF. Treatment of extracts with CIP disrupts binding of this activity as analyzed by gel shift. The CAC-D band could be disrupted with an EKLF antibody. However, gel shifts from EKLF- deficient mice have been used to show at least that CAC-D is EKLF. An inducible, epitope tagged EKLF has been successfully been introduced into MEL cells for purification experiments. Further, a poly-his tagged version of EKLF has been expressed in bacteria for antibody production (data not shown). Portions of EKLF have been expressed in a yeast two hybrid vector. Finally, the human EKLF has been cloned.