FIG. 3.

Location of the nic site within pC221. (A) Denaturing polyacrylamide gel electrophoresis of 5′-end-labeled restriction products. Negatively supercoiled pC221 previously digested with HpaII (lanes 1 to 5) or pC221 obtained from whole-cell lysates (lanes 6 to 10) was further digested with BstBI (lanes 1 and 6), BstEII (lanes 2 and 7), BstXI (lanes 3 and 8), ClaI (lanes 4 and 9), or XbaI (lanes 5 and 10). 5′-End-labeled fragments were detected by autoradiography. Fragment sizes are indicated in nucleotides; the doublet seen in lane 3 results from the uneven upper and lower strand lengths after digestion. (B) Location of restriction sites within the 692-bp AluI fragment previously shown to contain oriT (49). Larger fragments were not resolved on the gel shown. (C) Mapping of the labeled (★) fragments BE and BX locates the nic site 3′ to the HpaII site and in the sense strand with respect to cat and the mob genes; failure to generate similar fragments in lanes 6, 9, and 10 indicates the 5′ end at nic is blocked (•).