MOLECULAR BIOLOGY OF HUMAN ERYTHROCYTE ALPHA-SPECTRIN

描述：（研究者摘要）本提案的长期目标是 以阐明正常和异常的 红细胞膜蛋白α-血影蛋白的表达。红细胞 α-血影蛋白是红细胞膜骨架的重要组分。的 该建议的第一个目的是鉴定患有以下疾病的患者中的α-血影蛋白突变： 遗传性球形红细胞增多症和遗传性 热变红细胞症，严重的溶血性贫血，并分析结构 和/或这些异常的功能意义。第二个目的是 建议是确定和表征启动子和其他关键调控 控制红细胞血影蛋白基因表达的因子。这些 结果将应用于α-血影蛋白基因的作用研究 转录在红细胞生成和膜生物发生中的作用， 对已发现有突变的溶血性贫血患者的研究 在假定的α-血影蛋白基因启动子中。本建议的第三个目的是 纠正α血影蛋白缺陷鼠红白血病细胞系中的缺陷 在sph/sph小鼠的红系细胞中， 遗传性溶血性贫血，通过逆转录病毒转导的α-血影蛋白cDNA。 本研究所采用的一般方法包括： 使用基于PCR的方法从患有α-血影蛋白连接的rHS和HPP的患者的基因组DNA 单链构象多态性（SSCP）分析，然后 cDNA的核苷酸序列分析、克隆和结构分析， 与其表达相关的α-血影蛋白基因的基因组片段， 利用重组DNA技术调控;顺式作用的研究 序列通过基因操作，然后B，基因转移/表达研究 组织培养细胞.电泳法研究反式作用因子 迁移率变动分析，DNAse-I足迹法，甲基化干扰 技术和定点诱变，然后在体外和体内 鸟嘌呤-腺嘌呤连接介导的PCR（GA-LMPCR）硫酸二甲酯 体内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; 用逆转录病毒体外转导α-血影蛋白缺陷MEL细胞 含有α-血影蛋白cDNA转导的造血干细胞（HSC） 从α-血影蛋白缺陷的SPH/SPH小鼠，用含有 a-血影蛋白cDNA，随后将HSC-逆转录病毒基因移植到 sph/sph小鼠。这些研究将为我们研究 α-血影蛋白在正常和疾病状态中的作用。

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.