THE MOLECULAR BIOLOGY OF THE MAMMALIAN GRP GENE

哺乳动物前体蛋白的结构、功能和表达 胃泌素释放肽（GRP）基因的研究。 实验 进行a）表征结构和调节 B）过表达人GRP 激素原蛋白分析翻译后加工， 肽激素功能，c）测试自分泌生长 通过引入组成型表达的GRP基因的假说 转化为对外源性GRP激素有反应的细胞， 增加细胞生长。 a. 大鼠前原GRP基因。 结构和整个核苷酸 大鼠前原GRP基因的序列测定，使用 从大鼠脑cDNA分离的基因组克隆和cDNA克隆 图书馆 与人类基因相比， 在表达该基因的大鼠组织中观察到的RNA加工。 的 GRP及其相关肽的编码区是 进化上保守，与生物学功能一致， 两个领域。 大鼠GRP基因转录自一种组织， 在大脑中的特定启动子，创造一个替代途径， 调节基因表达。 B。 人前GRP原基因成功过表达 一种新型杆状病毒蛋白表达系统。 使用此 在模型系统中，可以产生足够的蛋白质， 翻译后加工事件的分析， 产生生物活性肽并纯化这些肽 建立功能。 C. 将组成型表达的人GRP基因导入 导入小鼠胚胎Swiss 3T3成纤维细胞 转染 这些成纤维细胞对增加的GRP作出反应， 细胞分裂，未能完全处理GRP激素原， 生物活性肽，因此没有显示生长 变化 本实验确定了玻璃钢的加工工艺 激素原仅限于特定的细胞类型。

The structure, function, and expression of mammalian prepro gastrin releasing peptide (GRP) genes was studied. Experiments were performed to a) characterize the structure and regulation of the rat prepro GRP gene, b) overexpress the human GRP prohormone protein to analyze post-translational processing and peptide hormone function, c) test the autocrine growth hypothesis by introducing a constitutively expressed GRP gene into cells which respond to exogeneous GRP hormone by increased cell growth. A. Rat prepro GRP gene. The structure and entire nucleotide sequence of the rat prepro GRP gene was determined, using genomic clones and cDNA clones isolated from a rat brain cDNA library. In contrast to the human gene, there is no alternative RNA processing observed in rat tissues expressing this gene. The coding regions for GRP and its associated peptide are evolutionarily conserved, consistent with a biologic function for both domains. The rat GRP gene is transcribed from a tissue specific promoter in the brain, creating an alternative route for regulating gene expression. B. The human prepro GRP gene was successfully overexpressed in a novel baculovirus protein expression system. Using this model system, sufficient protein can be produced to allow analysis of post-translational processing events needed to generate biologically active peptides, and purify those peptides to establish function. C. A constitutively expressed human GRP gene was introduced into mouse embryonic Swiss 3T3 fibroblasts using DNA transfection. These fibroblasts, which respond to added GRP by cell division, failed to fully process the GRP prohormone into biologically active peptides and consequently showed no growth changes. This experiment establishes that processing of GRP prohormone is restricted to specific cell types.