CA++ GENE REGULATION

胃酸的产生取决于一种酶的功能， H+K+ATPase，能够将质子泵入分泌物 与6对数浓度梯度相反的小管。OH-也就是 由该泵产生的气体在第二秒的作用下被转化为HCO3 碳酸氢酶II(CA II)。之前的研究已经证明 胃粘膜中编码这两种基因的表达 壁细胞酶在发育过程中经历了重大变化。稳稳 H+K+ATPase mRNA在出生时处于低水平，在出生后迅速增加 3周龄。这些变化伴随着平行的发展。 酸性分泌量的变化。相比之下，CA II的mRNA水平是 在新生儿中很高，但在接下来的3周内迅速下降。 CAⅡ与H+K+ATPase的这种相互关系 与编码基因的平行调节的发育形成对比 酸性促分泌剂在壁细胞中的两种酶。关于……的指示 这种差异的基础是以前的研究提供的本地化的 用原位杂交法将CA-II表达于所有细胞。 新生儿的胃粘膜，但主要是腺基底部的细胞 和成熟动物的表面上皮细胞。这些数据强调了 CA II在维持细胞pH中的潜在重要性 酸分泌期间以外的情况：在细胞分裂过程中 如在发育中的动物或在腺体成分更新的过程中 在胃表面上皮细胞分泌HCO3的过程中 胃粘膜抵御酸性环境的第一道防线 流明。因此，对CA II基因表达调控的理解 将阐明该酶在体内的一系列重要功能 胃粘膜。因此，这项提案的目标是审查 在遗传水平上，CA II表达的机制是 受监管的。为实现这一目标，提出了以下具体目标： 通过将CA II基因的顺式调控区 基因启动子/增强子区域的不同部分(有或没有 突变和/或缺失)导致氯霉素乙酰转移酶(CAT) 表达系统；鉴定和鉴定特定的核蛋白 它们可能作为转导因子来调节启动子/增强子/沉默因子 顺式调节区的活性；确定CA II基因是否 在发育过程中转录调控，如果顺式-反式 通过凝胶滞留分析确定的相互作用 体外转录；克隆编码转移因子的c DNA(S) 调节CAⅡ基因表达。 我们希望这项提案中所包括的工作将有助于 CA II基因在发育过程中的重要调控机制 并认为所获得的信息可能对 尊重成长和发展的整个过程。

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.