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Detection and purification of the promoter region-binding protein involved in the tissue-specific gene expression

参与组织特异性基因表达的启动子区结合蛋白的检测和纯化

基本信息

批准号：
62570102
负责人：
OGAWA Hirofumi
金额：
$ 0.19万
依托单位：
Toyama Medical and Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

In thid laboratory, several genomic clones as well as cDNA clones of rat liver enzymes involved in amino acid metabolism have been isolated. Among them, serine dehydratase is the inducible enzyme, whose activity is subject to change by the treatment of various hormones or by feeding various composition of diet. To learn the molecular mechanim of enzyme induction/suppression, we attempoted to clone the serine dehydratase cDNA. One clone was screened from a gtll library carrying rat licer cDNAs. The cDNA was found to encode 363 amino acids and the expected molecular weight atrees well that obtained previously by sds/page of the purified protein. We also obtained the corresponding genomic clones and determined their DNA sequence. The serine dehydratase gene consisted of 9 exons and 8 introns, whose boundary sequences are obeyed by a gt/ag rule. The 5' flanking region of the gene contains a variety of sequences that are similar to the consensus elements which are proved to aggect the transcription of other genes. Two sequences similar to glucocorticoid response element are located at positions -2240 and -280. Since serine dehydratase is under control by glucagon and dexamethasone, the occurrence of such sequences is quiet reasonable. to prove the function of these sequences and to look for the specific factors which might regulate the gene transcription, we are developing the in vitro transcription system usin unclear extracts obtained from rat licers maintained on various diets, and the g-less cassette vector (gorski et al. 1987) having fragments of the 5' flanking region of the serine dehydratase gene.

在本实验室中，已分离出几个与氨基酸代谢有关的大鼠肝酶的基因组克隆和cDNA克隆。其中，丝氨酸蛋白酶是诱导酶，其活性受各种激素处理或喂养不同组成的饮食的影响。为了研究丝氨酸脱氢酶诱导/抑制的分子机制，我们克隆了丝氨酸脱氢酶的cDNA。从携带大鼠licer cDNA的gtll文库中筛选一个克隆。该cDNA编码363个氨基酸，预期分子量与纯化蛋白的SDS/PAGE结果一致。我们还获得了相应的基因组克隆并测定了它们的DNA序列。丝氨酸蛋白酶基因由9个外显子和8个内含子组成，其边界序列符合gt/ag规则。该基因的5'侧翼区含有多种与共有元件相似的序列，这些共有元件被证明可以聚集其他基因的转录。两个与糖皮质激素反应元件相似的序列位于-2240和-280位。由于丝氨酸脱氢酶受胰高血糖素和地塞米松控制，因此发生此类序列是完全合理的。为了证明这些序列的功能并寻找可能调节基因转录的特异性因子，我们正在开发体外转录系统，使用从维持在各种饮食中的大鼠地衣中获得的不透明提取物，和具有丝氨酸脱氢酶基因5'侧翼区片段的无G盒载体（Gorski等，1987）。