A LOW-COPY-NUMBER VECTOR UTILIZING BETA-GALACTOSIDASE FOR THE ANALYSIS OF GENE-CONTROL ELEMENTS
A LOW-COPY-NUMBER VECTOR UTILIZING BETA-GALACTOSIDASE FOR THE ANALYSIS OF GENE-CONTROL ELEMENTS
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DOI：
10.1016/0378-1119(87)90051-5
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发表时间：
1987-01-01
期刊：
影响因子：
3.5
通讯作者：
BOURGEOIS, S
中科院分区：
文献类型：
--
作者：
KOOP, AH;HARTLEY, ME;BOURGEOIS, S
构建了一个低拷贝数的载体pFZY1，其中M1 3mp18的多酶切位点连接子插入了编码一个无启动子的β-半乳糖苷酶(β-Gal)的lacZ基因的上游，为分析体内的调控元件提供了一个方便而准确的系统。该质粒含有没有PAR位点的ORIF复制起点，并以每个基因组一到两个拷贝的形式存在于细胞中。在氨苄西林存在的情况下，它被保留在宿主中，并且在所有测试的条件下，每个插入的启动子产生一致的β-半乳糖酶活性。比较了pFZY1和高拷贝数pKO载体中的一系列四环素抗性(TCR)启动子片段和lac启动子片段。含有相同TCR启动子的不同片段在所测试的几个构建体中的转录活性在6倍的范围内变化。野生型Lac启动子和突变体在pFZY1中的调控与在染色体中观察到的Lac启动子相似，而在PKO-1mp18中它们的调控受到高拷贝数的显著影响，正如预期的那样。
A low-copy-number vector, pFZY1, with the multiple restriction site linker of M1 3mp18 inserted upstream from a promoterless .beta.-galactosidase (.beta.Gal)-coding lacZ gene has been constructed to provide a convenient and accurate system to analyze regulatory elements in vivo. The plasmid contains the oriF replication origin without the par locus and is present in the cell in one to two copies per genome. It is retained in the host by the presence of ampicillin, and each inserted promoter yielded consistent values of .beta.Gal activity under all the conditions tested. A series of tetracycline resistance (TcR) promoter fragments and lac promoter fragments have been compared in pFZY1 and the high-copy-number pKO-vector series. The transcriptional activity measured for different fragments containing the same TcR promoter varied within a six-fold range among the several constructs tested. Regulation of the wild-type lac promoter and mutants in pFZY1 was similar to that observed for lac promoters in the chromosome while their regulation in pKO-1mp18 was significant affected by the high copy number, as expected.