Linn T, St Pierre R
Department of Microbiology and Immunology, Faculty of Medicine, University of Western Ontario, London, Canada.
J Bacteriol. 1990 Feb;172(2):1077-84. doi: 10.1128/jb.172.2.1077-1084.1990.
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 (lambda TL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.
已开发出一种改良的载体系统，用于体外构建与lacZ的转录融合体。其主要特点是在多克隆位点与无启动子的lacZ基因之间插入了一个RNaseIII切割位点。当这些载体用于构建转录融合体时，随后在RNaseIII位点对杂交mRNA的切割会为lacZ mRNA产生一个不变的5'端。与早期载体相比，这一特性有助于确保lacZ mRNA的独立翻译，因此，所产生的β-半乳糖苷酶水平应准确反映上游DNA序列的转录频率。载体的其他修饰包括去除克隆位点与lacZ之间的一个弱转录终止子，在lac下游插入一个终止子，以及改变限制性内切酶切割位点以促进体外融合体的构建。已组装了多拷贝质粒（pTL61T）和单拷贝λ（λTL61）载体。这些载体在扫描转录调控信号方面应具有普遍用途。
