New pSC101-derivative cloning vectors with elevated copy numbers
New pSC101-derivative cloning vectors with elevated copy numbers
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DOI：
10.1016/j.plasmid.2008.01.004
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发表时间：
2008-05-01
期刊：
影响因子：
2.6
通讯作者：
Phillips, Gregory J.
中科院分区：
文献类型：
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作者：
Peterson, James;Phillips, Gregory J.
增加pSC 101复制子拷贝数的突变已用于构建新的克隆载体。替换RepA中93位的谷氨酸产生以中等（27个拷贝/细胞）和高（类似于240个拷贝/细胞）拷贝数复制的质粒。基于RepE（来自F因子的结构相似的复制起始蛋白）的晶体结构，预测pSC 101 repA突变体在二聚化方面有缺陷。克隆载体允许增加基因产物的表达，沿着pSC 101衍生质粒的优点，包括稳定维持和与ColE 1质粒的相容性。质粒还允许对DNA插入物进行蓝/白色筛选，并赋予对氨苄青霉素、氯霉素和卡那霉素的抗性。该载体被用于基因测定，以抑制温度敏感的突变体ffh，编码的蛋白质组分的大肠杆菌信号识别颗粒，过量生产的4.5S RNA。虽然来自野生型pSC 101衍生质粒的4.5S RNA的表达不足以抑制，但使用新载体确实抑制了温度敏感性表型。(c)2008年爱思唯尔公司All rights reserved.
Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (similar to 240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the Escherichia coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype. (c) 2008 Elsevier Inc. All rights reserved.