Gene a Protein Interacting with Recombinant Plasmid DNAs Containing 25–30 b.p. of the ΦX174 Replication Origin
Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b. p. of the 30 b. p. origin region of bacteriophage ΦX174 (nucleotides 4299–4328 of the ΦX174 DNA sequence).
The double-stranded DNA fragments were cloned into the kanamycin resistance gene of pACYC177 (AmpR, KmR). Transformants were picked up by antibiotic selection and filter-hybridization using one of the oligodeoxyribonucleotides as a probe. Approximate lengths of the inserts were determined by restriction enzyme analysis. Exact length and orientation of each insert was determined by DNA sequencing.
Plasmid DNA with an insert homologous to the first 25 b. p. of the ΦX174 origin is not nicked by the gene A protein. However, plasmid DNA containing the 27 b. p. fragment in either orientation is nicked by the gene A protein, as well as plasmid DNAs containing the first 28 b. p. or the complete 30 b. p. conserved origin region of the isometric phages.
KeywordsReplication Origin Plasmid DNAs Restriction Enzyme Analysis Kanamycin Resistance Gene SmaI Site
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