EXPRESSION OF RENAL H+ ATPASE IN METANEPHROGENESIS

肾H-ATP酶在肾再生过程中的表达

• 批准号: 6105520
• 负责人: STEPHEN L GLUCK
• 金额: $ 12.29万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105520
• 关键词: adenosinetriphosphatase; developmental genetics; embryogenesis; gene expression; genetic promoter element; genetically modified animals; isozymes; kidney; laboratory mouse; mammalian embryology; tissue /cell culture; vesicle /vacuole

During metanephric the epithelial cells in different segments of the nephron and collecting duct begin to express protein markers characteristic of the mature, differentiated cell. We have established in prior work that amplified expression of the vacuolar H+ATPase, containing the B1 isoform of the B subunit, is a marker for intercalated cell differentiation, and is first detectable by antibody staining on gestational day 17. In studies using transgenic mice and cultured cells, we have established a region of the B1 subunit gene that confers intercalated cell-specific expression. We have recently discovered that a GAn ("GAGA") box in this region is essential for expression of the B1 isoform. The long term goals of this application are to understand the mechanisms controlling renal epithelial cell differentiation. The specific aims of this proposal are: l. To define the time course for expression of mRNA for vacuolar H+ATPase subunits during embryonic kidney development in vivo using in situ hybridization. 2. To isolate and characterize the GAn-binding protein(s) required for B1 subunit isoform expression. Oligonucleotide screening of a kidney bacterial expression library, or the one-hybrid method for screening a yeast GAL4 activation domain plasmid library, will be used to isolate clones for the GAn-binding proteins. The DNA binding properties and expression of the proteins in kidney will then be examined. 3. To define the promoter elements on the kidney isoform of the kidney vacuolar H+ATPase required for amplification of expression during development. Promoter-reporter constructs will be assayed in cultured cells, kidney tissue, and intact mice using several approaches, to identify cis-regulatory elements essential for B1 subunit expression. These studies should advance our understanding of developmental abnormalities of the collecting duct, and may provide information pertinent to mechanisms controlling renal epithelial cell differentiation that occurs following ischemic renal injury.

在跨载体期间 肾单位和收集管开始表达蛋白质标记 成熟的，分化的细胞的特征。我们已经建立了 在先前的工作中，放大了液泡H+ATPase的表达， 包含B亚基的B1同工型，是 插入的细胞分化，首先可通过抗体检测 妊娠第17天染色。在使用转基因小鼠和 培养的细胞，我们建立了B1亚基基因的区域 这赋予了互插入的细胞特异性表达。我们最近有 发现该区域中的一个gan（“ gaga”）盒对于 B1同工型的表达。 该应用程序的长期目标是了解 控制肾上皮细胞分化的机制。这 该提案的具体目的是：l。定义时间课程 胚胎期间的液泡H+ATPase亚基mRNA的mRNA表达 使用原位杂交在体内发育。 2。分离 并表征B1亚基所需的GAN结合蛋白 同工型表达。肾细菌的寡核苷酸筛查 表达式库，或用于筛选酵母gal4的单杂交方法 激活域质粒库将用于隔离克隆 GAN结合蛋白。 DNA结合特性和表达 然后将检查肾脏中的蛋白质。 3。定义发起人 需要肾脏液泡H+ATPase的肾同工型元素 用于扩大发育过程中的表达。启动子培养基 将在培养的细胞，肾脏组织和完整的构造中分析结构 使用多种方法的小鼠识别顺式调节元件 B1亚基表达必不可少的。 这些研究应该提高我们对发展的理解 收集管的异常，并可能提供信息 与控制肾上皮细胞的机制有关 缺血性肾脏损伤发生的分化。