以ilv-lac融合脱氧核糖核酸为模板体外合成β-半乳糖苷酶。
In vitro synthesis of beta-galactosidase with ilv-lac fusion deoxyribonucleic acid as template.
作者信息
Wild J, Smith J M, Umbarger H E
出版信息
J Bacteriol. 1977 Dec;132(3):876-83. doi: 10.1128/jb.132.3.876-883.1977.
Abstract
An in vitro protein-synthesizing system has been developed to study the mechanism of induction of ilvC gene in Escherichia coli strain K-12. Deoxyribonucleic acid (DNA) from a lambda phage carrying an ilvC-lac fusion was employed as a template for the in vitro synthesis of beta-galactosidase under the control of the ilvC promoter. The use of this template allowed an investigation of the components required for induction of the ilvC gene and the kinetics of the induction. The in vitro synthesis of beta-galactosidase under the control of the ilvC promoter was found to be DNA, acetohydroxy acid, and guanosine-3'-diphosphate-5'-diphosphate dependent, and sensitive to rifampin, actinomycin D, and chloramphenicol. Uncoupling experiments indicate that the inducer, acetohydroxybutyrate, acts at the transcriptional level. Investigation of a proposed noninducible ilvC regulatory mutant has shown normal induction in vitro. It was also observed that an intact ilvA gene is not required for the induction of the ilvC gene.
摘要
已开发出一种体外蛋白质合成系统，用于研究大肠杆菌K - 12菌株中ilvC基因的诱导机制。携带ilvC - lac融合基因的λ噬菌体的脱氧核糖核酸（DNA）被用作模板，在ilvC启动子的控制下体外合成β - 半乳糖苷酶。使用该模板可以研究ilvC基因诱导所需的成分以及诱导动力学。发现在ilvC启动子控制下β - 半乳糖苷酶的体外合成依赖于DNA、乙酰羟酸和鸟苷 - 3'-二磷酸 - 5'-二磷酸，并对利福平、放线菌素D和氯霉素敏感。解偶联实验表明，诱导剂乙酰羟丁酸在转录水平起作用。对一个假定的非诱导性ilvC调节突变体的研究表明其在体外诱导正常。还观察到ilvC基因的诱导不需要完整的ilvA基因。
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