The mouse p53 gene, spreading over 16 kb, was divided into 10 segments of approximately 1,000 bp and subcloned into pUC19. The clones were transfected into mouse L cells together with an expression vector of the hygromycin B- or blasticidin S-resistance gene. The transfected cells were cultured in the drug-containing medium to establish cell lines resistant to the drug. Among the established lines, a high percentage of the cells originally transfected with pp53(H-R), possessing the HindIII-EcoRI fragment located downstream from the last coding exon of the p53 gene, harbored plasmid DNAs in an episomal state. The plasmid DNAs recovered from the 53HR cell-lines, transfected with pp53(H-R), were indistinguishable in structure from the original plasmids used for transfection, as assayed by Southern blotting and back-transformation to bacteria. The plasmids in episome of the cells replicated once per cell cycle in S phase in concert with chromosomal DNA. In the HindIII-EcoRI fragment (i.e., the p53(H-R) fragment) which showed a highly efficient replication activity, there exists a putative sequence for p53 recognition near the HindIII site. A DNA bending region with the clusters of AT tracts also exists near the EcoRI site of the same p53(H-R) fragment. In a transient replication system, pp53(H-R) autonomously replicated in episome of transfected cells, while mutant plasmids lacking either the p53 recognition sequence or the bending region did not. The results suggest that the HindIII-EcoRI region downstream from the p53 gene contains an activity of cellular DNA replication and both the p53 recognition sequence and the bending region are necessary for the replication activity.

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