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: 6410295
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410295
• 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80 kD的红细胞膜骨架成分蛋白4.1， 一个复杂结构蛋白家族的原型成员 同种型由单个遗传位点通过多个替代前体编码， mRNA剪接途径。 而主要红细胞4.1的结构 已经定义了它的异构体，并且它的一些功能结构域很好地 特征，4.1中多样性的分子遗传学解释 大小（30- 210 kD）和细胞内定位（外周膜， 应力纤维、细胞核、中心体、高尔基体）缺乏。 为了 完全定义4.1基因的遗传库， 表征由以下介导的发育转换4.1表达： 调节转录和剪接事件，以下目的是 提出了 (1)确定整个约250 kb的DNA序列 人类4.1基因位点，提供了丰富的基本数据， 正常个体和选定患者的基因功能分析 遗传性椭圆形红细胞增多症 (2)探索结构决定因素 4.1同种型定位，通过（a）表征转录-和 剪接介导的4.1 mRNA补体的变化， 区分红系细胞和上皮细胞，和（B）确定 单个表位标记的同种型的亚细胞区室化 已知的原始序列。 (3)发展遗传学方法， 在组织特异性和同种型特异性中操纵4.1表达 通过产生具有4.1个基因破坏的小鼠， 重新安排 4.1 ES同源重组基因敲除 细胞，以及用正常或突变的YAC进行的基因拯救实验 转基因，被提议探索的生物学意义 选择的同种型或功能结构域。 研究将主要集中在 在红细胞和上皮细胞上，其中4.1中的调节开关 在成纤维细胞中，细胞核 4.1已鉴定出同种型。 蛋白质4.1基因将作为 有价值的模型，探索组织特异性表达的大， 一种高度复杂的基因，编码多种 细胞内骨架结构 成功完成这些 目的将有助于阐明生物功能 这一不同的蛋白质家族的意义，并可能提供洞察力 4.1基因破坏的病理生理后果 表情