MOLECULAR GENETICS OF THE RED CELL MEMBRANE SKELETON

这项重建建议的长远目标是 正常和异常的分子机制的阐明 表达编码膜骨架蛋白的基因， 特别是血影蛋白和锚蛋白的基因， 红细胞膜骨架的成分以及非- 红系细胞 对这些基因的研究具有科学意义， 有几个原因：I）几种人类疾病与 这些基因中的突变; II）与这些基因中的膜蛋白类似的膜蛋白， 红细胞，但有一些不同的结构表达在 非红系细胞，以及它们的多种分子来源， 以及它们的时间和空间的调节 表达是未知的;和III）的结构/功能关系 相互作用膜骨架蛋白 是 仍不 完全 理解 并保证 进一步调查 具体目标 旨在澄清这些问题包括：一）完成克隆 和染色体的选定区域的DNA序列分析 血影蛋白、胞衬蛋白和锚蛋白基因，重点是启动子区 和被认为参与选择性剪接的区域; II） 顺式作用元件和反式作用因子的表征 参与这些基因的组织特异性表达; III） 表征引起异常血影蛋白、锚蛋白和 带3表达，特别强调识别突变 负责遗传性球形红细胞增多症;和IV）分析 选择的重组血影蛋白的结构-功能关系， 胞衬蛋白结构域，例如α-血影蛋白的SH3结构域。 主要 实验方法包括：克隆和结构 分析相关的cDNA和基因组DNA片段， 利用重组DNA技术研究基因; 这些基因的不同遗传性疾病的个体通过使用 聚合酶链反应（PCR）技术;顺式作用的研究 通过基因操作随后进行基因转移/表达的序列 组织培养细胞的研究; 凝胶阻滞试验、足迹法和甲基化干扰 技术;通过生产研究结构/功能关系 重组蛋白在各种表达系统中的表达和分析 它们在生物化学和/或生物学测试系统中的功能。

This renewal proposal has as its long-term objective the elucidation of molecular mechanisms of normal and abnormal expression of genes encoding proteins of the membrane skeleton, in particular the genes for spectrin and ankyrin which are important constituents of the membrane skeleton of erythrocytes as well as non- erythroid cells. Study of these genes is of scientific interest for a number of reasons: I) several human diseases are associated with mutations in these genes; II) membrane proteins analogous to those in red cells but having a somewhat different structure are expressed in non-erythroid cells, and the molecular sources of their multiple isoforms as well as the regulation of their temporal and spatial expression is unknown; and III) the structure/function relationships of interacting membrane skeleton proteins is still not completely understood and warrants further investigation. Specific aims designed to clarify these issues include:I) completion of the cloning and DNA sequence analysis of selected regions of the chromosomal spectrin, fodrin and ankyrin genes, with emphasis on promoter regions and regions thought to be involved in alternative splicing; II) characterization of cis-acting elements and trans-acting factors involved in tissue-specific expression of these genes; III) characterization of mutations causing abnormal spectrin, ankyrin and band3 expression, with special emphasis on identifying mutations responsible for hereditary spherocytosis; and IV) analysis of structure-function relationships of selected recombinant spectrin and fodrin domains, e.g. the SH3 domain of alpha-spectrin. Major experimental methods to be employed include: cloning and structural analysis of the cDNAs and genomic DNA segments of the relevant genes by use of recombinant DNA technology; study of the DNA from individuals with different hereditary disorders of these genes by use of the polymerase chain reaction (PCR) technique; studies of cis-acting sequences by gene manipulation followed by gene transfer/expression studies in tissue culture cells; studies of trans- acting factors by gelretardation assays, footprinting and methylation interference techniques; studies of structure/function relationshipsby production of recombinant proteins in various expression systems and analysis of their function in biochemical and/or biological test systems.