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CA++ GENE REGULATION

CA基因调控

基本信息

批准号：
2391515
负责人：
LUCYNDIA R MARINO
金额：
$ 18.37万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-04-15 至 1999-03-31
项目状态：
已结题

项目摘要

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.

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。