Figure 6.
Detection of transfer-independent cleavage activity at the oriTs of Hfr strains requires synthesis of F transfer proteins in vivo. (A) Forty microliters from ONCs of three F(−) strains (lanes 1–3) and four Hfr strains without (lanes 4, 6, 10, and 11) and with (+) the R1-16 plasmid (lanes 5, 7, 8, and 9) were prepared for a runoff nucleotide sequencing assay. (B) Forty and 80 μl of ONCs of 122-1[pGZ119] (lanes 1 and 2) and 122-1[pGZarcA] (lanes 3 and 4), or aliquots of cultures of 122-1[pGZ119] (lane 5) or 122-1[pGZarcA] (lanes 6 and 7), which were diluted first in fresh medium containing 10 mM isopropyl β-d-thiogalactoside and incubated with shaking at 37°C for 2.5 h, were prepared for the nicking assay. The numbers of viable cells analyzed in the reaction mixtures in lanes 1–7 were, respectively, 0.65, 1.3, 30.8, 61.5, 22.1, 0.09, and 0.15 (× 106 cfu). The nic-specific reaction products are indicated by arrows.