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.

胃酸的产生取决于酶的功能， H+ K+ ATPase，能够将质子泵入分泌物针对6型物体浓度梯度的管。哦，那是该泵产生酶，碳酸酐酶II（Ca II）。先前的研究表明编码这两个基因的胃粘膜表达顶细胞酶在发育过程中发生了重大变化。稳定的 H+ K+ ATPase mRNA的状态水平在出生时很低，并且在 3周龄。这些变化伴随着平行的发展酸分泌能力的变化。相反，Ca II mRNA水平为新生儿的较高，但在接下来的三个星期中迅速下降。 Ca II和H+ K+ ATPase之间的这种相互关系发展与编码基因的平行调节形成对比酸性促分子在顶细胞中的两种酶。表明以前的研究本地研究提供了这种差异的基础通过原位杂交与所有细胞中的所有细胞表达Ca II的表达新生儿的胃粘膜，但主要针对腺碱基的细胞和成熟动物的表面上皮。这些数据实现了 Ca II在维持细胞pH下的潜在重要性除酸分泌期间以外的情况：在细胞分裂期间就像在发育中的动物或腺元素更新过程中一样在胃中表面上皮细胞的HCO3分泌过程中粘膜防御抗胃酸环境的第一线流明。因此，了解Ca II基因表达的调节会阐明酶在中的多种重要功能胃粘膜。因此，该提议的目的是检查在遗传水平上，Ca II表达的机制为受监管。为了实现这一目标，提出了以下特定目标：通过联系来表征Ca II基因的顺式调节区域基因的启动子/增强子区域的各个部分（有或没有突变和/或缺失）至氯霉素乙酰基转移酶（CAT）表达系统；识别和表征特定的核蛋白可以用作调节启动子/增强子/消音器的转录器顺式调节区域的活性；确定Ca II基因是否为在开发过程中受到转录调节的调节以及if顺式传播通过调节的凝胶延迟测定法确定的相互作用体外转录；克隆cDNA（S）编码转录器的cDNA 调节Ca II基因表达。我们希望该提案中包含的工作能够阐明在发育过程中调节Ca II基因的重要机制并认为获得的信息可能具有广泛的意义尊重整个增长和发展过程。