Genetic analysis of promoters on the insertion sequence IS21 of plasmid R68.45.

Tandem duplication of a 2.1-kb DNA sequence on R68 leads to the active insertion element IS21 on the enhanced chromosome mobilizing plasmid R68.45. The HindIII/SalI fragment which carries the single copy or the tandem duplication of IS21 was cloned from R68 and R68.45, respectively, into the multicopy plasmid pED815. Promoters on the two HindIII/SalI fragments were subsequently identified by cloning Sau3A fragments into the BglII site of the promoter cloning vector pGA46. Three promoters were identified on the HindIII/SalI fragment derived from R68 or R68.45, two of them mapped on Sau3A fragments of 214 bp and 82 bp, respectively, on IS21. The promoter on the 82 bp Sau3A fragment which maps at the SmaI site close to the left end of IS21 reads inward. The Sau3A fragment of 214 bp contains the left end of IS21 and transcription from its promoter proceeds outward. In R68.45, readthrough from this preexisting promoter located near the junction of the tandem copies of IS21 proceeds from the right-hand copy into the left, opposing the reading direction of the promoter mapped at the SmaI site of IS21. The expression of genes on one copy of IS21 by readthrough from a promoter on the other one is a possible explanation for the transpositional activity of the tandem configuration of IS21. The similarity of IS21 to other insertion sequences and especially to "mobile promoters" is discussed.