RED CELL MEMBRANE PROTEIN 4.1--STRUCTURE AND FUNCTION

红细胞膜蛋白 4.1--结构与功能

• 批准号: 6301081
• 负责人: JOHN G CONBOY
• 金额: $ 21.52万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-15 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301081
• 关键词: artificial chromosomes; erythrocyte membrane; gene expression; gene rearrangement; gene targeting; genetic mapping; hereditary elliptocytosis; human genetic material tag; human tissue; membrane proteins; molecular cloning; nucleic acid sequence; protein 4.1; protein isoforms; protein structure function; protein transport

The about 80kD erythroid membrane skeletal component, protein 4.1, is the prototypical member of a complex family of structural protein isoforms encoded by a single genetic locus via multiple alternative pre- mRNA splicing pathways. Whereas the structure of the major red cell 4.1 isoforms has been defined, and some of its functional domains well characterized, a molecular genetic explanation for the diversity in 4.1 size (30-210kD) and intracellular localization (peripheral membranes, stress fibers, nuclei, centrosomes, Golgi) is lacking. In order to completely define the genetic repertoire of the 4.1 gene, and to characterize developmental switched in 4.1 expression mediated by regulated transcription and splicing events, the following aims are proposed. (1) Determine the DNA sequence of the entire about 250kb human 4.1 gene locus, to provide a wealth of primary data essential for analysis of gene function in normal individuals and in selected patients with hereditary elliptocytosis. (2) Explore the structural determinants of 4.1 isoform localization, by (a) characterizing transcription- and splicing-mediated changes in the complement of 4.1 mRNAs expressed in differentiating erythroid and epithelial cells, and (b) determining the subcellular compartmentalization of individual epitope-tagged isoforms of known primary sequence. (3) Develop genetic approaches toward manipulation of 4.1 expression in tissue-specific and isoform-specific fashion, by generating mice with 4.1 gene disruptions and rearrangements. 4.1 gene knockouts by homologous recombination in ES cells, and gene rescue experiments with normal or mutated YAC transgenes, are proposed to explore the biological significance of selected isoforms or functional domains. Studies will focus primarily on erythroid and epithelial cells, in which regulated switches in 4.1 expression have been demonstrated, and on fibroblasts, in which nuclear 4.1 isoforms have been identifies. The protein 4.1 gene will serve as a valuable model for exploring tissue-specific expression of a large and highly complex gene encoding a critical component of multiple intracellular skeletal structures. Successful accomplishment of these aims will facilitate elucidation of the biological functional significance of this diverse protein family, and may provide insight into the pathophysiologic consequences of disruptions in 4.1 gene expression.

约80kD的红细胞膜骨架成分，蛋白质4.1是 结构蛋白复杂家族中的典型成员 由单个遗传位点通过多个可供选择的前染色体编码的异构体 信使核糖核酸剪接途径。鉴于主红细胞4.1的结构 异构体已经被定义，它的一些功能域很好 特征，对4.1中多样性的分子遗传学解释 大小(30-210kD)和细胞内定位(外周膜， 应力纤维、核、中心体、高尔基体)缺乏。为了 完全定义了4.1基因的遗传谱，并 发育转换型4.1基因表达的特征 受调控的转录和剪接事件，目标如下 建议。(1)测定全长约250kb的DNA序列 人类4.1基因座，提供了丰富的原始数据 正常人和部分患者的基因功能分析 遗传性椭圆形红细胞增多症。(二)探索结构性决定因素 4.1亚型定位，通过(A)鉴定转录和 剪接介导的4.1mRNAs补体的变化 分化红系细胞和上皮细胞，以及(B)确定 单个表位标记异构体的亚细胞区划 已知的初级序列。(3)开发遗传方法以实现 4.1在组织特异性和异构体特异性表达中的操作 时尚，通过产生4.1基因中断和 重新安排。4.1 ES中同源重组的基因敲除 细胞，以及与正常或突变的YAC的基因拯救实验 转基因，是为了探索转基因的生物学意义。 选定的异构体或功能结构域。研究将主要集中在 在红系和上皮细胞上，在4.1中调节开关 已经证明，在成纤维细胞上有表达，其中核 4.1已鉴定出异构体。蛋白4.1基因将作为一种 有价值的模型探索组织特异性表达的大型和 高度复杂的基因编码多个 细胞内骨骼结构。成功实现这些目标 AIMS将有助于阐明生物功能 这个不同的蛋白质家族的意义，并可能提供洞察力 4.1基因突变的病理生理后果 表情。