Origin of replication of colicin E1 plasmid DNA.

Abstract
Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA [from Escherichia coli], were constructed by using [EcoRI, HaeII, HaeIII, HhaI, HinfI and HPaII] restriction endonuclease. The nucleotide sequence of a region that contains the origin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [.gamma.-32P]ATP and polynucleotide 5''-hydroxyl-kinase (ATP:5''-dephosphopolynucleotide 5''-phosphotransferase, EC 2.7.1.78) at the 5''-OH groups which were uncovered by alkali treatment. The site is 1 of 3 consecutive nucleotides, dA, dA and dC, located at a unique position. One or a few rA residues were attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of 5 A residues. Both sides of this segment are rich in G and C.