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Nucleic Acids Research, 1984, Vol. 12, No. 16 6443-6454
© 1984

MOLECULAR BIOLOGY

Gene A protein cleavage of recombinant plasmids containing the {varphi}X174 replication origin

A.C. Fluit*, P.D. Baas, J.H. Van Boom1, G.H. Veeneman1 and H.S. Jansz

Institute of Molecular Biology and Laboratory for Physiological Chemistry, State University of Utrecht 3584 CH Utrecht 1Department of Organic Chemistry, State University of Leiden Leiden, The Netherlands

*To whom correspondence should be addressed

Received May 14, 1984. Revised July 27, 1984. Accepted July 27, 1984.

Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to con struct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage {varphi}X174 (nucleotides 4299–4328 of the {varphi}X174 DNA sequence).

The double-stranded DNA fragments were cloned into the unique SmaI or R HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (Amp KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the {varphi}X174 origin region were thus obtained.

Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the {varphi}X174 origin region are nicked by the {varphi}X174 gene A protein. However, the other supercoiled plasmids are not nicked by the {varphi}X174 gene A protein. These results show that the first 27 b.p. of the {varphi}X174 origin region are sufficient as well as required for the initiation step in {varphi}X174 RF DNA replication, i.e. the cleavage by gene A protein.


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