Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ

Abstract

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.