Construction of promoter-detection plasmid in animal cells by using the metapyrocatechase gene

利用偏焦儿茶酶基因构建动物细胞启动子检测质粒

• 批准号: 03557013
• 负责人: NAKAZAWA Atsushi
• 金额: $ 3.84万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557013/
• 关键词: Promoter; Vector; Cultured cells; Catechol; C230; ベペクター; プロモ-タ-; ベクタ-; カテュ-ル

To test the ability of expression of the metapyrocatechase (C230) gene in the animal cells, we first constructed two plasmids. In one plasmid, the C230 gene was put under the control of the promoter and enhancer of SV40 early genes, while in the other, it was controlled by the promoter and enhancer of the human elongation factor gene. Neither of these plasmid showed production of active C230 in HeLa and CHO cells. Next, we made a gene fusion in which the N-terminal two residues of chicken adenylate kinase was ligated to the N-terminal truncated C230. The C230 gene of the above two plasmids was replaced by the gene fusion, and the plasmid products were checked for their expression in the animal cells. However, the results were negative. Since the transcription of these plasmids were thought to be active in the animal cells, failure in the expression of the C230 gene is probably due to a barrier in the process after the translation.During the course of this study, we obtained a plasmid pKS230, in which the C230 gene is put under the control of the lac promoter. The restriction sites for HincII and EcoRV can be used as a cloning site for foreign DNA, since insertion to these sites make the plasmid inactive to produce the active C230. The inserted plasmid is easily detected by yellow color development after spraying a catechol solution. Therefore, this plasmid can be used a less expensive cloning vector comparing with the lac promoter vector that uses X-Gal as a detector.

为了检测间苯二酚(C230)基因在动物细胞中的表达能力，我们首先构建了两个表达载体。其中一个由SV40早期基因的启动子和增强子控制C230基因，另一个由人延长因子基因的启动子和增强子控制。两种载体均未在HeLa和CHO细胞中产生活性C230。接下来，我们将鸡腺苷酸激酶N端的两个残基连接到N端截短的C230上，进行了基因融合。用基因融合技术取代上述两种载体的C230基因，并检测其在动物细胞中的表达。然而，结果是否定的。由于这些质粒的转录在动物细胞中被认为是活跃的，C230基因的表达失败可能是由于翻译后的过程中的障碍。HincII和EcoRV的限制性内切酶切点可用作外源DNA的克隆切点，因为插入这些切点会使质粒失去产生活性C230的活性。在喷洒邻苯二酚溶液后，通过黄色显影很容易检测到插入的质粒。因此，与使用X-Gal作为检测器的Lac启动子载体相比，该质粒可以作为一种更便宜的克隆载体。

项目成果

M.Gomada: "Analysis of an upstream requlatory sequence required for activation of the requlatory gene xyls in xylene metabolism directed by the TOL plasmid" Molecular and General Genetics. 233. 419-426 (1992)

M.Gomada：“在 TOL 质粒指导的二甲苯代谢中激活调节基因 xyls 所需的上游调节序列的分析”《分子与通用遗传学》。

M.Gomada et al.: "Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida" Mol. Gen. Genet.233. 419-426 (1992)

M.Gomada 等人：“在恶臭假单胞菌的 TOL 质粒指导的二甲苯代谢中激活调节基因 xylS 所需的上游调节序列的分析”Mol。