FUNCTION OF A PUTATIVE DETERMINANT IN HEMATOPOIESIS

描述：（改编自申请人的摘要）EKLF是一种红系细胞 一种在红细胞早期表达的特异性转录因子 发展它结合并反式激活一个序列元件“CACCC”， β-珠蛋白启动子其中一些残基的点突变， 包括那些已知会导致β地中海贫血的疾病， EKLF的反式激活能力。小鼠EKLF的遗传破坏 会导致严重的β地中海贫血，导致胚胎死亡。这 更新建议将调查的分子机制的行动， 通过评估磷酸化是否调节EKLF活性， 识别与EKLF相互作用的蛋白质。 此外，评价 既往未明确的β-地中海贫血患者中的EKLF突变 将被执行。 主要研究者实验室的先前研究 确定了EKLF的DNA结合位点，并表明EKLF 具有启动子和细胞类型特异性。缺失突变体 EKLF反式激活区表现出抑制性结构域， 一个可能需要与积极行为互动的领域 因子 作者认为MEL中CAC的结合活性之一 提取物，CAC-D是EKLF。用CIP处理浸提液会破坏结合 通过凝胶位移分析该活性。CAC-D波段可能是 用EKLF抗体破坏。 然而，凝胶从EKLF- 缺陷型小鼠已被用于表明至少CAC-D是EKLF。 一种可诱导的，表位标记的EKLF已成功地引入 用于纯化实验。此外，多组氨酸标记的 EKLF的一个版本已经在细菌中表达用于抗体生产 (data未示出）。 部分EKLF已在酵母中表达， 杂交载体最后，人类EKLF已经被克隆。

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.