Abstract
A low-copy-number vector, pFZY1, with the multiple restriction site linker of M13mpl8 inserted upstream from a promoterless β-galactosidase (βGal)-coding lacZ gene has been constructed to provide a convenient and accurate system to analyze regulatory elements in vivo. The plasmid contains the oriF replication origin without the par locus and is present in the cell in one to two copies per genome. It is retained in the host by the presence of ampicillin, and each inserted promoter yielded consistent values of β Gal activity under all the conditions tested. A series of tetracycline resistance (Tc R) promoter fragments and lac promoter fragments have been compared in pFZYl and the high-copy-number pKO-vector series. The transcriptional activity measured for different fragments containing the same Tc R promoter varied within a six-fold range among the several constructs tested. Regulation of the wild-type lac promoter and mutants in pFZYl was similar to that observed for lac promoters in the chromosome while their regulation in pKO-lmpl8 was significantly affected by the high copy number, as expected.
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