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首页> 外文期刊>Molecular and Cellular Biology >Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.
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Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

机译：lac阻遏物转化为哺乳动物细胞的变构调节转录激活因子。

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摘要

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.
机译：通过使用大肠杆菌lac阻遏物创建了一种新型的哺乳动物调节系统。通过修饰lac阻遏物编码区使其包含simian病毒40（SV40）大肿瘤抗原的核定位信号和来自单纯疱疹病毒1型病毒粒子蛋白16的转录激活结构域，将lac阻遏物转化为哺乳动物转录激活因子。 lac激活蛋白（LAP）融合蛋白是几种启动子的有效激活剂，这些启动子包含位于转录单位上游或下游的lac操纵子序列。单个lac操纵子允许反式激活，而多个操纵子在相距一小段距离时会协同起作用。响应LAP，含有14或21个操纵子序列的启动子被诱导至少1,000倍，达到的活性水平比HeLa细胞中SV40早期启动子的活性高20至30倍。活化受到异丙基-β-D-硫代半乳糖苷（IPTG）的强烈抑制，表明LAP保留了变构调节所需的功能。 LAP是双功能的，还可以充当SV40启动子表达的阻遏物，该启动子在TATA盒的下游直接包含一个操纵子。最后，开发了遗传选择方案，使得可以从培养中的既定细胞或原代细胞中高频产生表达LAP的细胞系。

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