AUTONOMOUSLY REPLICATING SEGMENT IDENTIFIED IN THE MURINE P53 GENE CONTAINS P53 RECOGNITION SEQUENCE AND BENDING REGION

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 t ransfected into mouse L cells together with an expression vector of th e hygromycin B- or blasticidin S-resistance gene. The transfected cell s 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 Hin dIII-EcoRI fragment located downstream from the last coding exon of th e p53 gene, harbored plasmid DNAs in an episomal state. The plasmid DN As recovered from the 53HR cell-lines, transfected with pp53(H-R), wer e indistinguishable in structure from the original plasmids used for t ransfection, as assayed by Southern blotting and back-transformation t o bacteria. The plasmids in episome of the cells replicated once per c ell cycle in S phase in concert with chromosomal DNA. In the HindIII-E coRI fragment (i.e., the p53(H-R) fragment) which showed a highly effi cient replication activity, there exists a putative sequence for p53 r ecognition near the HindIII site. A DNA bending region with the cluste rs of AT tracts also exists near the EcoRI site of the same p53(H-R) f ragment. In a transient replication system, pp53(H-R) autonomously rep licated in episome of transfected cells, while mutant plasmids lacking either the p53 recognition sequence or the bending region did not. Th e results suggest that the HindIII-EcoRI region downstream from the p5 3 gene contains an activity of cellular DNA replication and both the p 53 recognition sequence and the bending region are necessary for the r eplication activity.