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Nucleic Acids Research, 1984, Vol. 12, No. 13 5175-5191
© 1984

MOLECULAR BIOLOGY

Initiation of plasmid R1 replication in vitro is independent of transcription by host RNA polymerase

Ramon Diaz1,2 and Sagrario Ortega2

1Max-Planck-Institut für Molekulare Genetik Abt. Schuster, Ihnestrasse 63–73. D-1000 Berlin 33, FRG 2Instituto de Biologia Celular (C.I.B.) Velazquez 144, Madrid 6, Spain

Received May 23, 1984. Accepted June 19, 1984.

A Sau3A fragment containing most of the repA gene of plasmid Ri has been cloned in the BamH1 site of the expression vector pAS1. One of the recombinants, pSO1 codes for a fused RepA' protein which is efficiently synthesized both in vivo and in vitro from transcriptional and translational signals of the vector. It is shown that following pSO1 promoted accumulation of RepA' in cell-free extracts of E.coli, in vitro replication of the R1 miniplasmid pKN 182 can initiate and proceed uncoupled from further protein synthesis. Using this uncoupled system and also a M13mp9 based ori-R1 recombinant, pRD95, it is also shown that RepA' acts at the origin region of R1 to promote initiation of replication that is independent on transcription by host RNA polymerase. This is indicated by the insensitivity of pRD95 and pKN182 replication to rifampicin as well as to RNA polymerase antibodies. The properties of the uncoupled in vitro replication system are further described.


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