EXPRESSION OF THE RENAL H+ ATPASE IN METANEPHROGENESIS

在后肾过程中，不同节段的上皮细胞 肾单位和集合管开始表达蛋白质标记物 成熟的，分化的成熟的、分化的细胞的特征我们已经确立了 在先前工作中，液泡H+ATPase的扩增表达， 含有B亚基的B1亚基，是嵌合的标志 细胞分化，并首次通过抗体染色检测到 妊娠第17天。在使用转基因小鼠和培养的 细胞，我们已经建立了B1亚单位基因的一个区域，它赋予 嵌入性细胞特异性表达。我们最近发现， 该区域的GA ETA(“Gaga”)框对于表达 B1亚型。这个应用程序的长期目标是理解 控制肾上皮细胞分化的机制。这个 这项建议的具体目标是：1.确定时间进程 空泡H+ATPase亚基在胚胎发育过程中的表达 用原位杂交法研究肾脏在体内的发育。2.隔离 并鉴定了B1亚基所需的GA eta结合蛋白(S) 同构表达式。一株肾脏细菌的寡核苷酸筛选 酵母GAL4表达文库或单杂交筛选方法 激活域质粒文库，将用于分离cdna克隆 对于GA ETA结合蛋白。DNA结合特性和 然后将检测这些蛋白在肾脏中的表达情况。3.至 确定肾脏亚型上的启动子元件 表达扩增所需的液泡H+-ATPase 发展。启动子-报告构建将在培养的 细胞、肾组织和完整的小鼠使用几种方法，以 确定B1亚基表达所必需的顺式调控元件。 这些研究应该促进我们对发展的理解 集合管的异常，并可提供信息 与控制肾上皮细胞的机制有关 在缺血性肾损伤后发生的分化。

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 GA eta ("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: 1. 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 GA eta-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 cDNA clones for the GA eta-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.