MOLECULAR BIOLOGY OF HUMAN ERYTHROCYTE ALPHA-SPECTRIN

描述：(研究人员摘要)这项提议的长期目标是 阐明正常和异常的分子机制 红细胞膜蛋白a-Spectrin的表达。红细胞 A-血影蛋白是红细胞膜骨架的重要组成部分。这个 这项建议的第一个目标是确定慢性粒细胞白血病患者的a-幽灵蛋白突变。 隐性遗传性球形红细胞增多症与遗传性 嗜热性红细胞增多症，严重溶血性贫血，并分析其结构 和/或这些异常的功能意义。这样做的第二个目的是 建议是确定和表征发起人和其他关键监管机构 控制红细胞a-血影蛋白基因表达的因素。这些 研究结果将用于研究a-血影蛋白基因的作用。 转录在红细胞生成和膜生物发生中的作用及其对遗传学的影响 已发现突变的溶血性贫血患者的研究 在推测的α-幽灵蛋白基因启动子中。这项建议的第三个目标是 纠正a血影蛋白缺陷型小鼠红白血病细胞系的缺陷 在Sph/Sph小鼠的红系细胞中，α-幽灵蛋白缺陷模型 遗传性溶血性贫血，通过逆转录病毒转导a-spectrin cdna。 本研究所采用的一般方法包括：研究 应用聚合酶链式反应技术检测a-Spectrin连锁RHS和HPP患者的基因组DNA 单链构象多态(SSCP)分析 该基因的核苷酸序列分析、克隆和结构分析 α-血影蛋白基因与其表达相关的基因组片段 利用重组DNA技术进行调控；顺式作用的研究 基因操作后的序列b，基因转移/表达研究 组织培养细胞.用电泳法研究反式作用因子 迁移率变化分析、DNase-I足迹、甲基化干扰 体外和体内的定点诱变技术和技术 分析和鸟嘌呤-腺嘌呤连接介导的聚合酶链式反应(GA-LMPCR)硫酸二甲酯 在体足迹、发育和组织特异性研究 转基因小鼠中α-血影蛋白基因启动子的调控序列 逆转录病毒体外转导α-血影蛋白缺陷型MEL细胞 含a-Spectrin基因的造血干细胞(HSCs)的基因转导 来自a-血影蛋白缺陷的sph/sph小鼠，携带含有 A-spectrin基因，然后将HSC-逆转录病毒基因移植到 Sph/sph小鼠。这些研究将为我们的研究提供重要的见解 A-血影蛋白在正常和疾病状态下的作用。

DESCRIPTION: (Investigator's abstract) The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the erythrocyte membrane protein a-spectrin. Erythrocyte a-spectrin is an important component of the erythrocyte membrane skeleton. The first aim of this proposal is to identify a-spectrin mutations in patients with recessively inherited hereditary spherocytosis and hereditary pyropoikilocytosis, severe hemolytic anemias, and to analyze the structural and/or functional significance of these abnormalities. The second aim of this proposal is to identify and characterize the promoter and other key regulatory factors that control expression of the erythrocyte a spectrin gene. These results will be applied to the study of the role of a-spectrin gene transcription in erythropoiesis and membrane biogenesis and to the genetic study of patients with hemolytic anemia who have been found to have mutations in the putative a-spectrin gene promoter. The third aim of this proposal is to correct the defect in an a spectrin deficient murine erythroleukemia cell line and in the erythroid cells of sph/sph mice, an a-spectrin deficieny model of inherited hemolytic anemia, via retroviral transduction of the a-spectrin cDNA. The general methodology tc be utilized in this research includes: study of genomic DNA from patients with a-spectrin linked rHS and HPP using PCR-based single stranded conformational polymorphism (SSCP) analysis, followed by nucleotide sequence analysis cloning and structural analysis of the cDNA and genomic fragments of the a-spectrin gene relevant to its expression and regulation by the use of recombinant DNA technology; study of cis-acting sequences by gene manipulation followed b, 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 vitrc and in vivo analyses, and guanine-adenine ligation-mediated PCR (GA-LMPCR) dimethyl sulfate in vivo footprinting developmental and tissue-specific studies of the regulatory sequences of the a-spectrin gene promoter in transgenic mice; In vitro transduction of a-spectrin deficient MEL cells with a retrovirus containing the a-spectrin cDNA transduction of hematopoietic stem cells (HSCs) from a-spectrin deficient sph/sph mice with an ecotropic retrovirus containing the a-spectrin cDNA, followed by HSC-retroviral gene transplant into the sph/sph mice. These studies will provide important insights into our studies of the role of a-spectrin in normal and disease states.