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: 6564216
• 负责人: JOHN G CONBOY
• 金额: $ 14.33万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564216
• 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 的红细胞膜骨骼成分，蛋白质 4.1，是 结构蛋白复杂家族的典型成员 同种型由单个遗传位点通过多个替代预编码编码 mRNA 剪接途径。 而主要红细胞的结构 4.1 同工型已经被定义，并且它的一些功能域得到了很好的定义 4.1 多样性的分子遗传学解释 大小（30-210kD）和细胞内定位（外周膜， 缺乏应力纤维、细胞核、中心体、高尔基体）。 为了 完整定义了4.1基因的遗传库，并 4.1 表达介导的发育转换特征 调控转录和剪接事件，以下目标是 建议的。 (1)确定整个约250kb的DNA序列 人类4.1基因位点，提供丰富的重要原始数据 正常个体和选定患者的基因功能分析 患有遗传性椭圆形红细胞增多症。 (2)探索结构性决定因素 4.1 同工型定位，通过 (a) 表征转录和 4.1 mRNA 补体中剪接介导的变化 区分红细胞和上皮细胞，以及 (b) 确定 单个表位标记亚型的亚细胞区室化 已知的一级序列。 (3) 开发遗传方法 4.1 组织特异性和异构体特异性表达的操作 时尚，通过产生具有 4.1 基因破坏的小鼠和 重新安排。 4.1 ES同源重组基因敲除 使用正常或突变的 YAC 进行细胞和基因拯救实验 转基因，旨在探索其生物学意义 选定的亚型或功能域。 研究将主要集中于 对红细胞和上皮细胞的影响，其中 4.1 中的调节开关 表达已被证明，并且在成纤维细胞上，其中核 4.1 同种型已被鉴定。 蛋白质4.1基因将作为 探索大的组织特异性表达的有价值的模型 高度复杂的基因编码多个关键组成部分 细胞内骨骼结构。 成功实现这些 目标将有助于阐明生物功能 这种多样化蛋白质家族的重要性，并可能提供见解 4.1 基因破坏的病理生理后果 表达。