REGULATION OF CARBONIC ANHYDRASE II GENE EXPRESSION

胃酸的产生取决于酶的功能， H+ K+ ATP酶，其能够将质子泵入分泌的 小管对6-log浓度梯度。 哦-就是说 由该泵产生的二氧化碳通过第二个反应器的作用转化为HCO 3-。 碳酸酐酶II（CA II）。 先前的研究已经证明 编码这两种基因的胃粘膜表达 壁细胞酶在发育过程中经历重大变化。 稳定 H+ K+ ATP酶mRNA水平在出生时较低， 3周大。 这些变化伴随着平行发展 酸分泌能力的变化。 相反，CA II mRNA水平是 在新生儿中高，但在生命的接下来的3周内迅速下降。 CA Ⅱ和H+ K+ ATP酶之间的这种相互关系， 发育与编码基因的平行调节形成对比， 壁细胞中的两种酶。 的指示 这种差异的基础是由以前的研究提供的， CA II的表达，通过原位杂交到所有的细胞中， 新生儿胃粘膜，但主要是腺基部的细胞 和成熟动物的表面上皮。 这些数据表明， CA II在维持细胞pH的潜在重要性， 在细胞分裂过程中， 如在发育中的动物或在腺细胞的更新过程中 在胃表面上皮细胞分泌HCO 3的过程中， 对抗胃酸环境的第一道粘膜防线 流明 因此，了解CA II基因表达的调控 将揭示酶的许多重要功能， 胃粘膜 因此，本提案的目的是审查 在遗传水平上，CA II表达的机制是 监管. 为实现这一目标，提出了以下具体目标： 通过连接CA II基因的顺式调节区， 基因的启动子/增强子区的各个部分（有或没有 突变和/或缺失）转化为氯霉素乙酰转移酶（CAT） 表达系统;鉴定和表征特定的核蛋白 其可作为转因子调节启动子/增强子/沉默子 活性的顺式调节区;确定是否CA II基因是 在发育过程中受到转录调控，如果是顺式-反式 通过调节的凝胶阻滞试验确定的相互作用 体外转录;克隆编码转因子的cDNA， 调节CA II基因表达。 我们希望，这项建议中所包括的工作将有助于了解 在发育过程中调节CA II基因的重要机制 并认为所获得的信息可能具有广泛的意义， 尊重整个成长和发展的过程。

The production of gastric acid is dependent of the function of an enzyme, H+ K+ ATPase, that is capable of pumping protons into a secretory canaliculus against a 6-log concentration gradient. The OH- that is generated by this pump is converted to HCO3- by the action of a second enzyme, carbonic anhydrase II (CA II). previous studies have demonstrated that the gastric mucosal expression of the genes encoding these two parietal cell enzymes undergoes major changes during development. Steady state levels of H+ K+ ATPase mRNA are low at birth and increase rapidly at 3 weeks of age. These changes are accompanied by parallel developmental changes in acid secretory capacity. In contrast, CA II mRNA levels are high in the newborn but rapidly decline over the next 3 weeks of life. This reciprocal relationship between CA II and H+ K+ ATPase during development contrast with the parallel regulation of the genes encoding the two enzymes in parietal cells by acid secretagogues. An indication as to the basis of this disparity was provided by previous studies localizing the expression of CA II by in situ hybridization to all of the cells in the gastric mucosa of the newborn but primarily to cells at the glandular base and surface epithelium in mature animals. These data served to underscore the potential importance of CA II in maintaining cellular pH under circumstances other than during acid secretion: during cell division such as in developing animals or in the process of renewal of glandular elements and during HCO3-secretion by surface epithelial cells in the stomach as a first line of mucosal defense against the acid environment of the gastric lumen. Thus, an understanding of the regulation of CA II gene expression would shed light on a multitude of important functions of the enzyme in gastric mucosa. Accordingly, the objective of this proposal is to examine at a genetic level, the mechanism by which the expression of CA II is regulated . Toward this goal, the following specific aims are proposed: Characterized the cis-regulatory regions of the CA II gene by linking various portions of the gene's promoter/enhancer region (with or without mutation and/or deletions) to a chloramphenicol acetyl transferase (CAT) expression system; identify and characterize specific nuclear proteins which may serve as transfactors to regulate promoter/enhancer/silencer activity in the cis-regulatory regions; determine if the CA II gene is transcriptionally regulated during development and if cis-trans interactions thus identified by gel retardation assays regulated transcription in vitro; clone the cDNA(s) encoding transfactors that regulate CA II gene expression. We hope that the work included in this proposal will shed light on the important mechanism by which the CA II gene is regulated during development and believe that the information obtained may have broad significance with respect to the entire process of growth and development.