Figure 4.

In vivo promoter activity of the minus-strand origin sequence. (A) Nucleotide sequences inserted into pKK232–8 plasmid, which carries a promoter-less cat gene (12). Only the sequences of the nontemplate strand are shown. The insert in pKKminus-Ori carries the f1 wild-type sequence of nucleotides 5659–5707 on the strand shown, which corresponds to the top strand of the minus-strand origin (see Fig. 3A). pKKOri-10cons is identical to pKKminus-Ori except for three base substitutions (marked with asterisks), which yield the consensus sequence of the promoter −10 region. pKKlacUV5 carries a region of the lacUV5 promoter ranging from −46 to +3. (B) CAT activity in E. coli DH5 cells harboring pKK232–8, pKKminus-Ori, pKKOri-10cons, or pKKlacUV5 was determined with [¹⁴C]chloramphenicol as substrate. An autoradiograph of a chromatogram is shown. The products of the CAT reaction are indicated on the top. CAT activity was determined from intensity of the spots on the autoradiograph and shown on the right.