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

本实验室已分离到多个与氨基酸代谢相关的大鼠肝脏酶的基因组克隆和cDNA克隆。其中，丝氨酸脱水酶是一种诱导性酶，其活性会因多种激素处理或饲喂不同的饲料成分而发生变化。为了了解酶诱导/抑制的分子机制，我们尝试克隆了丝氨酸脱水酶基因。从携带大鼠LICER基因的GT11文库中筛选出一个克隆。该基因编码363个氨基酸，其预期相对分子质量与纯化蛋白的SDS/PAGE分析结果一致。我们还获得了相应的基因组克隆并测定了它们的DNA序列。丝氨酸脱水酶基因由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‘侧翼区包含一系列序列，这些序列与已被证明能整合其他基因转录的共同元件相似。两个类似糖皮质激素反应元件的序列位于-2240和-280位。由于丝氨酸脱水酶受胰升糖素和地塞米松的控制，因此这些序列的出现是非常合理的。为了证明这些序列的功能并寻找可能调控基因转录的特定因素，我们正在开发体外转录系统，使用从各种饮食维持的大鼠许可证中获得的不清楚提取物，以及无g盒载体(Gorski等人)。1987)具有丝氨酸脱水酶基因5‘侧翼区的片段。

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.