IMPROVED VECTOR SYSTEM FOR CONSTRUCTING TRANSCRIPTIONAL FUSIONS THAT ENSURES INDEPENDENT TRANSLATION OF LACZ
IMPROVED VECTOR SYSTEM FOR CONSTRUCTING TRANSCRIPTIONAL FUSIONS THAT ENSURES INDEPENDENT TRANSLATION OF LACZ
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DOI：
10.1128/jb.172.2.1077-1084.1990
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发表时间：
1990-02-01
影响因子：
3.2
通讯作者：
STPIERRE, R
中科院分区：
文献类型：
--
作者：
LINN, T;STPIERRE, R
已经开发了一种改进的载体系统，用于体外构建与lacZ的转录融合体。主要特征是在多接头克隆位点和无启动子lacZ基因之间插入RNaseIII切割位点。当这些载体用于构建转录融合体时，随后在RNase III位点处切割杂合mRNA产生lacZ mRNA的不变的5“末端。与早期的载体相比，该特征有助于确保lacZ mRNA的独立翻译，并因此确保β-lacZ mRNA的水平。所产生的半乳糖苷酶应准确地反映上游DNA序列的转录频率。载体的其它修饰包括去除克隆位点和lacZ之间的弱转录终止子，在lac下游插入终止子，以及改变限制性内切核酸酶切割位点以促进融合体的体外构建。组装了多拷贝质粒（pTL 61 T）和单拷贝λ（γ TL 61）载体。这些载体通常可用于扫描转录调节信号。
An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ. The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene. When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIII site generates an unchanging 5'' end for the lacZ mRNA. In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of .beta.-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences. Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions. Both multicopy plasmid (pTL61T) and single-copy lambda (.gamma.TL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.