化学合成乳糖操纵基因的克隆。II. 经EcoRI连接的操纵基因

Cloning of chemically synthesized lactose operators. II. EcoRI-linkered operators.

作者信息

Sadler J R, Betz J L, Tecklenburg M, Goeddel D V, Yansura D G, Caruthers M H

出版信息

Gene. 1978 May;3(3):211-32. doi: 10.1016/0378-1119(78)90033-1.

Abstract

A 40 base, mainly duplex DNA segment, with the following sequence pAATTCCACATGTGGAATTGTGAGCGGATAACAATTTGTT (3') GGTGTACACCTTAACACTCGCCTATTGTTAAACACCTTAAp (5') has been synthesized by combination of chemical and enzymatic methods. It consists of a wild-type lactose operator sequence (boxed) bracketed by "linker" sequences which permit excision of the segment from plasmid vehicles by the EcoRI restriction endonuclease. This segment has been ligated into the pMB9 plasmid and the resulting operator plasmids used to transform E. coli K-12. Among the transformant products were strains carrying plasmids with one, two, three, or four operator segments in tandem. Derepression of the lactose operon effected by these plasmids in vivo as well as the lifetimes of complexes formed between repressor and these plasmids in vitro increase with increasing numbers of operators per plasmid.

摘要

一段40个碱基的、主要为双链的DNA片段，其序列如下：pAATTCCACATGTGGAATTGTGAGCGGATAACAATTTGTT (3') GGTGTACACCTTAACACTCGCCTATTGTTAAACACCTTAAp (5')，已通过化学和酶促方法合成。它由一个野生型乳糖操纵序列（加框）组成，两侧为“接头”序列，这些序列允许通过EcoRI限制性内切酶从质粒载体中切除该片段。该片段已连接到pMB9质粒中，所得的操纵质粒用于转化大肠杆菌K-12。在转化产物中，有携带串联有一个、两个、三个或四个操纵片段的质粒的菌株。这些质粒在体内对乳糖操纵子的去阻遏作用以及阻遏物与这些质粒在体外形成的复合物的寿命随着每个质粒中操纵子数量的增加而增加。