MOLECULAR BIOLOGY OF HUMAN ERYTHROCYTE ALPHA-SPECTRIN

描述：（研究者的摘要）该提案的长期目标是 阐明正常和异常的分子机制 红细胞膜蛋白a-血影蛋白的表达。红细胞 a-血影蛋白是红细胞膜骨架的重要组成部分。的 该提案的首要目标是确定患有以下疾病的患者中的a-血影蛋白突变： 隐性遗传性遗传性球形红细胞增多症和遗传性 焦磷酸细胞增多症、严重溶血性贫血，并分析结构 和/或这些异常的功能意义。本次活动的第二个目的 建议的目的是确定发起人和其他关键监管机构并描述其特征 控制红细胞血影蛋白基因表达的因素。这些 结果将应用于a-血影蛋白基因作用的研究 红细胞生成和膜生物发生中的转录以及遗传 对已发现突变的溶血性贫血患者的研究 在假定的a-血影蛋白基因启动子中。该提案的第三个目标是 纠正血影蛋白缺陷型小鼠红白血病细胞系中的缺陷 在 sph/sph 小鼠的红细胞中，a-血影蛋白缺陷模型 遗传性溶血性贫血，通过逆转录病毒转导 a-血影蛋白 cDNA。 本研究中使用的一般方法包括： 使用基于 PCR 的方法从患有 a-血影蛋白关联的 rHS 和 HPP 的患者中获取基因组 DNA 单链构象多态性（SSCP）分析，然后 核苷酸序列分析 cDNA 的克隆和结构分析 a-血影蛋白基因与其表达相关的基因组片段 使用重组 DNA 技术进行监管；顺式作用研究 b、基因转移/表达研究 组织培养细胞；反式作用因子的电泳研究 迁移率变化分析、DNAse-I 足迹、甲基化干扰 技术和定点诱变，然后是体外和体内 分析和鸟嘌呤-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a-血影蛋白基因启动子的调控序列；在 用逆转录病毒体外转导a-血影蛋白缺陷的MEL细胞 含有 a-血影蛋白 cDNA 转导造血干细胞 (HSC) 来自a-血影蛋白缺陷的 sph/sph 小鼠，带有共向性逆转录病毒，其中含有 a-血影蛋白 cDNA，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.