Figure 3

pvuII-lacZ transcriptional fusions. A. Sequence from the pvuIIC initiator codon to the pvuIIR-fused lacZ gene. The vector, and source of the polylinker (green) and lacZ gene (blue), is pLex3B (ATCC #87200) [58]. The primers indicated by black arrows and gray shading were used to PCR-amplify pvuIIC and part of pvuIIR; pvuIIR retains its native RBS. The pvuIIC gene includes two unique sites, ClaI and EspI (equivalent to BlpI), at which different null mutations in pvuIIC were generated as previously described [17]. The WT and three different mutants were cloned between the vector XmnI and EcoRI sites such that 'A' of the XmnI site blunt ligated to the 'TG' of the insert on the 5' end to regenerate the pvuIIC initiation codon (under the control of the vector's promoter and RBS); the 3' end ligation used the EcoRI site. In derivatives, synthetic oligonucleotides were inserted between the BglII and EcoRI sites (underlined) to fuse pvuIIC to lacZ or to introduce other changes. The pair of red arrows indicates primers used for mRNA quantitation. B. Oligonucleotides used to alter the pvuIIC-pvuIIR overlap region. A 30 bp oligonucleotide was cloned between the BglII and EcoRI sites (in the sequence shown in Figure 3) to put pvuIIC in-frame with the lacZ gene. In each case, the pvuIIR initiation codon is highlighted in green, and the pvuIIC-frame terminator in red. A unique XbaI site was included to help identify the desired clones and to facilitate shifting the fusion reading frame. A pvuIIC stop codon was introduced in some cases (bottom sequences), to restore it to its native location relative to the pvuIIR initiator. In lines 1 and 2, the pvuIIC-frame terminator is off to the right (not shown).