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REGULATION OF ZETA GLOBIN GENE EXPRESSION

Zeta 珠蛋白基因表达的调控

基本信息

批准号：
2799711
负责人：
DANIEL E SABATH
金额：
$ 9.82万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-01-01 至 1999-03-31
项目状态：
已结题

项目摘要

The broad, long-term objectives of this project are to determine the molecular basis for regulation of globin gene expression, and to extend these studies to the dissection of pathways involved in hematopoietic commitment during development. The specific aims of this project are to: 1. Test the hypothesis that development regulation of zeta-globin expression is due to differences between embryonic and non-embryonic erythroid cells in protein-DNA-interactions occurring within the proximal zeta-globin promoter, 2. Test the functional significance of the above protein-DNA interactions within the proximal zeta-globin promoter by introducing altered promoters into both erythroid cell lines and transgenic mice, 3. Identify the features of a novel upstream positive regulatory region that are responsible for high-level zeta-globin expression in the embryonic erythroid cell line K562, 4. Characterize a trans-acting factor important for regulation of zeta- globin expression by isolating a cDNA clone using expression cloning strategies and/or purifying the factor to homogeneity. The health-relatedness of this project is threefold. First, the study of zeta-globin gene regulation will contribute to overall understanding of regulation of globin expression in normal individuals and thalassemic patients. Second, since mechanisms that regulate zeta-globin expression are distinct from those used by other globulin genes, novel regulatory elements will be identified that can be adapted for somatic gene therapy expression constructs. Third, since zeta-globin is one of the first hematopoietic genes expressed during development, understanding its regulation will contribute to better understanding of normal hematopoiesis and abnormal hematopoietic conditions such as leukemia, in which inappropriate embryonic gene expression occurs. The research design and methods include characterization of protein-DNA interactions within the zeta-globin promoter by electrophoretic mobility shift assays, DNAaseI footprinting, and protein-DNA cross-linking studies, functional analysis of regulatory elements by testing the effects of site-directed mutations on zeta-globin promoter function in leukemic cell lines, transgenic mice, and embryoid bodies, and molecular cloning and/or purification of DNA-binding activities responsible for developmental regulation of zeta-globin expression.

该项目的广泛、长期目标是确定珠蛋白基因表达调控的分子基础及推广这些研究对参与造血的通路进行了解剖在发展过程中的承诺。该项目的具体目标是： 1.检验Zeta-珠蛋白的发育调控假说表达是由于胚胎和非胚胎之间的差异红系细胞近端蛋白质-DNA-相互作用的研究 Zeta-珠蛋白发起人， 2.检测上述蛋白质-DNA的功能意义通过引入Zeta-珠蛋白启动子近端的相互作用将启动子改变为红系细胞系和转基因小鼠， 3.确定一个新的上游正向调节区的特征它们负责高水平的zeta-珠蛋白在胚胎红系细胞系K562， 4.描述对Zeta-调控重要的反式作用因子利用表达克隆技术分离表达珠蛋白的基因克隆策略和/或将该因素提纯到同质性。这个项目与健康有关有三个方面。第一，研究对Zeta-珠蛋白基因调控的研究将有助于全面理解正常人和地中海贫血患者珠蛋白表达的调节病人。第二，由于调控Zeta-珠蛋白表达的机制与其他球蛋白基因使用的不同，新的调节将确定可用于体细胞基因治疗的元素表达式构造。第三，因为哲塔-珠蛋白是第一批造血基因在发育过程中的表达，了解其监管将有助于更好地理解正常造血和异常的造血条件，如白血病，在发生了不适当的胚胎基因表达。研究设计和方法包括蛋白质-DNA的表征 Zeta-珠蛋白启动子内的相互作用转移分析、DNAaseI足迹和蛋白质-DNA交联法研究，通过测试调节元件的功能分析基因定点突变对Zeta-珠蛋白启动子功能的影响白血病细胞系，转基因小鼠，拟胚体和分子负责DNA结合活性的克隆和/或纯化 Zeta-珠蛋白表达的发育调控。