ANALYSIS OF GENE-CONTROL SIGNALS BY DNA-FUSION AND CLONING IN ESCHERICHIA-COLI
ANALYSIS OF GENE-CONTROL SIGNALS BY DNA-FUSION AND CLONING IN ESCHERICHIA-COLI
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DOI：
10.1016/0022-2836(80)90283-1
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发表时间：
1980-01-01
影响因子：
5.6
通讯作者：
COHEN, SN
中科院分区：
文献类型：
--
作者：
CASADABAN, MJ;COHEN, SN
构建能够在诱导型ara（阿拉伯糖）启动子和lac（乳糖）结构基因之间插入DNA片段的质粒克隆载体，并用于检测和分析控制基因转录的信号。在缺乏诱导剂阿拉伯糖的情况下lac基因的表达表明转录起源于插入片段内;在存在阿拉伯糖的情况下lac基因的不表达表明转录被片段终止。使用不同的克隆载体，由多种限制性内切酶产生的DNA片段可以插入ara和lac之间。该方法用于鉴定和分离E.大肠杆菌染色体、各种R质粒、噬菌体T5和黑腹果蝇中含有能够在大肠杆菌中作为启动子的核苷酸序列。杆菌lac表达的特征水平由从位于每个片段内的启动子进行到lac基因的转录量决定。作用于启动子的遗传调节机制的作用可以通过改变乳糖表达水平来测定。这些克隆载体还用于将位于插入的DNA片段内的结构基因置于ara启动子的控制下。插入HindIII核酸内切酶产生的片段携带pSC 101的四环素抗性决定簇或R6-5质粒的磺酰胺抗性决定簇到这样的载体中导致阿拉伯糖诱导的对四环素或磺酰胺的抗性。
Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes were constructed and used for the detection and analysis of signals that control gene transcription. Expression of the lac genes in the absence of the inducer arabinose indicates that transcription originates within the inserted fragment; nonexpression of lac with arabinose present indicates that transcription is terminated by the fragment. Using different cloning vectors, DNA fragments generated by a wide variety of restriction endonucleases can be inserted between ara and lac. This procedure was used to identify and isolate endonuclease-generated DNA fragments from the E. coli chromosome, various R plasmids, bacteriophage T5, and Drosophila melanogaster that contain nucleotide sequences capable of functioning as promoters in E. coli. A characteristic level of lac expression is determined by the amount of transcription that proceeds to the lac genes from a promoter located within each fragment. The effects of genetic regulatory mechanisms acting on a promoter can be assayed by alterations in the level of lac expression. These cloning vectors were also used to bring structural genes located within an inserted DNA fragment under the control of the ara promoter. Insertion of HindIII endonuclease-generated fragments carrying the tetracycline-resistance determinant of pSC101 or the sulfonamide-resistance determinant of the R6-5 plasmid into such vectors resulted in arabinose-induced resistance to tetracycline or sulfonamide.