SUPERPOLYLINKERS IN CLONING AND EXPRESSION VECTORS
SUPERPOLYLINKERS IN CLONING AND EXPRESSION VECTORS
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DOI：
10.1089/dna.1989.8.759
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发表时间：
1989-12-01
期刊：
影响因子：
--
通讯作者：
BROSIUS, J
中科院分区：
文献类型：
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作者：
BROSIUS, J
构建了超过300 bp的通用DNA多聚接头。它们包含所有限制性内切酶的识别序列--无论是已知的还是尚待发现的--这些酶都能识别回文六聚体。除了这64个不间断的六聚体识别位点之外，还存在一些含有中断的回文序列和非回文序列以及两个具有8bp的识别序列的位点。将多接头（在几种变体中）插入常用的大肠杆菌克隆载体中，例如pBlue-script（分别产生pSL 10、pSL 250、pSL 260、pSL 270和pSL 300）、pUC 18/pUC 19（分别产生pSL 180和pSL 190）或pUC 118/pUC 119（分别产生pSL 1180和pSL 1190）。在许多测试连接中观察到pSL 300中插入物的存在和不存在之间的细微颜色区分（中蓝色至浅蓝色或白色）。由pSL 300产生的中蓝色可能是由于翻译重新启动。在质粒pSL 251、pSL 261、pSL 271和pSL 301中使用不同的基因间区域进行翻译重新启动。还将多接头插入表达载体pUC 120中，产生pSE 1200，并插入表达载体pKK 233 -2中，产生pSE 220及其缩短形式pSE 280。最后，将多接头插入pTrc 99 A中，产生携带lac阻遏物基因的pSE 380。这将表达系统的使用从lacIq菌株扩展到其他细菌宿主。这些多功能载体在基因工程中有着广泛的应用。
Versatile DNA polylinkers of more than 300 bp were constructed. They contain the recognition sequences of all restriction enzymes-whether known or still to be discovered-that recognize palindromic hexamers. In addition to these 64 uninterrupted hexameric recognition sites, a number of sites containing interrupted palindromes and nonpalindromic sequences and two recognition sequences with 8 bp are present. Polylinkers (in several variants) were inserted into frequently utilized Escherichia coli cloning vectors such as pBlue-script (yielding pSLJ10, pSL250, pSL260, pSL270, and pSL300), pUC18/pUC19 (yielding pSL180 and pSL190, respectively), or pUC118/pUC119 (yielding pSL1180 and pSL1190, respectively). A subtle color discrimination between presence and absence of insert in pSL300 (mid-blue to light-blue or white) was seen in a number of test ligations. The mid-blue color that is generated by pSL300 is presumably due to translational restarts. A different intergenic region for translational restarts was used in plasmids pSL251, pSL261, pSL271, and pSL301. The polylinker was also inserted into expression vector pUC120, yielding pSE1200, and into expression vector pKK233-2, yielding pSE220 and a shortened version thereof, pSE280. Finally, the polylinker was inserted into pTrc99A, resulting in pSE380, which carries a lac repressor gene. This expands the use of the expression system beyond lacIq strains to other bacterial hosts. These versatile vectors have broad applications in genetic engineering.