Molecular Biology of Human Erythrocyte alpha-Spectrin

人红细胞α-血影蛋白的分子生物学

• 批准号: 6826132
• 负责人: PATRICK G GALLAGHER
• 金额: $ 36.79万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6826132
• 关键词: DNA footprinting; chromatin immunoprecipitation; clinical research; congenital hemolytic anemia; erythrocyte membrane; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genetic regulation; genetically modified animals; hematopoietic stem cells; hereditary spherocytosis; human subject; laboratory mouse; membrane proteins; molecular biology; patient oriented research; polymerase chain reaction; protein structure function; site directed mutagenesis; spectrin; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the erythrocyte membrane protein alpha-spectrin. Erythrocyte alpha-spectrin is a critical component of the erythrocyte membrane skeleton. The first aim of this proposal is to identify alpha- spectrin mutations in patients with recessively inherited hereditary spherocytosis (rHS) and hereditary pyropoikilocytosis (HPP), severe hemolytic anemias, and to characterize the effect of these mutations on spectrin structure, function, and/or gene regulation using genetic, biochemical, and molecular techniques, including a novel in vivo, lentivirus-based model of spectrin function. The second aim is to identify and characterize key regulatory factors that control expression of the erythrocyte alpha-spectrin gene. These results will be applied to the study of the role of alpha-spectrin gene transcription in erythropoiesis and membrane biogenesis and to the genetic study of patients with hemolytic anemia with mutations in these regulatory elements. The third aim is to analyze the regulation of erythrocyte membrane protein gene expression by the factor EKLF (Erythroid Krupple-Like Factor). The general methodology to be utilized includes: study of genomic DNA from patients with alpha-spectrin linked rHS and HPP using PCR-based DHPLC followed by nucleotide sequence analysis; cloning and structural analysis of the cDNA and genomic fragments of the alpha-spectrin gene relevant to its expression and regulation; study of cis-acting sequences by gene manipulation followed by gene transfer/expression studies in tissue culture cells; studies of trans-acting factors by electrophoretic mobility shift assays, DNAse-I footprinting, methylation interference techniques and site-directed mutagenesis followed by in vitro and in vivo analyses, and guanine-adenine ligation-mediated PCR dimethyl sulfate in vivo footprinting; in vivo chromatin immunoprecipitation studies; tissue- and developmental-specific studies of the regulatory sequences of the alpha-spectrin gene in transgenic mice; in vitro transduction of alpha- spectrin deficient MEL cells with a lentivirus containing the alpha-spectrin cDNA; transduction of hematopoietic stem and progenitor cells from alpha-spectrin deficient sph/sph mice with the same lentivirus, followed by stem and progenitor cell-lentiviral gene transplant into W/Wv and sph/sph mice. These studies will provide important insights into the role of spectrin in normal and disease states.

描述（由申请人提供）：该提案的长期目标是阐明红细胞膜蛋白α-血影蛋白正常和异常表达所涉及的分子机制。红细胞α-血影蛋白是红细胞膜骨架的重要组成部分。该提案的首要目的是鉴定患有隐性遗传性遗传性球形红细胞增多症（rHS）和遗传性焦变红细胞增多症（HPP）、严重溶血性贫血患者的α-血影蛋白突变，并利用遗传、生化和分子技术（包括一种新颖的体内、 基于慢病毒的血影蛋白功能模型。第二个目标是识别和表征控制红细胞α-血影蛋白基因表达的关键调节因子。这些结果将应用于研究α-血影蛋白基因转录在红细胞生成和膜生物发生中的作用，以及这些调节元件突变的溶血性贫血患者的遗传研究。第三个目的是分析因子EKLF（红细胞Krupple样因子）对红细胞膜蛋白基因表达的调节。使用的一般方法包括：使用基于 PCR 的 DHPLC 来研究患有 α-血影蛋白相关的 rHS 和 HPP 的患者的基因组 DNA，然后进行核苷酸序列分析；与其表达和调控相关的α-血影蛋白基因的cDNA和基因组片段的克隆和结构分析；通过基因操作研究顺式作用序列，然后在组织培养细胞中进行基因转移/表达研究；通过电泳迁移率变动分析、DNAse-I 足迹、甲基化干扰技术和定点突变进行反式作用因子研究，然后进行体外和体内分析，以及鸟嘌呤-腺嘌呤连接介导的 PCR 硫酸二甲酯体内足迹；体内染色质免疫沉淀研究；转基因小鼠中α-血影蛋白基因调控序列的组织和发育特异性研究；用含有α-血影蛋白cDNA的慢病毒体外转导α-血影蛋白缺陷的MEL细胞；用相同的慢病毒转导α-血影蛋白缺陷的 sph/sph 小鼠的造血干细胞和祖细胞，然后将干细胞和祖细胞慢病毒基因移植到 W/Wv 和 sph/sph 小鼠中。这些研究将为血影蛋白在正常和疾病状态下的作用提供重要的见解。