Atsushi Mori1, Koichi Ito1, K Mizobuchi2, Yoshikazu Nakamura1
1Department of Tumour Biology, The Institute of Medical Science, The University of Tokyo, PO Takanawa, Tokyo 108, Japan
2Department of Applied Physics and Chemistry, University of Electro-Communications, Chofu-shi, Tokyo 182, Japan
Tóm tắt
Replication of the Inclα plasmid CoIIb‐P9 requires the repZ gene, which encodes an essential, unstable initiator protein termed RepZ. Although many functional features of the CoIIb‐P9 replicon resemble those of structurally unrelated IncFII plasmids R1 and NR1, the role of transcription of repZ towards the replication origin is poorly understood. Using a series of deletion and substitution mutants of the CoIIb‐P9 replicon, we found that RepZ prefers to act in cis and that a spacer sequence between repZ and the origin is required for replication. This spacer element, referred to as CIS, retained strong transcription terminator activity. Efficient transcription terminators, whether Rho‐dependent or ‐independent, were capable of replacing CIS function for in vivo replication; CoIIb‐P9 replicated better as transcription terminated more efficiently within CIS. When the CIS element was substituted for by a strong Rho‐dependent terminator, such as λ tR1 or E. coli trp tt', in vivo replication of these recombinant replicons became dependent on the Rho factor, in contrast to the authentic CoIIb‐P9 replicon.
