Autonomously replicating sequences identified in transcriptional regulatory regions of human N-myc gene

The regions upstream from the human N-myc gene were examined for both transcription and replication. The plasmids containing several N-myc regions linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected to human neuroblastoma IMR32 cells. The results indicated that the region spanning from the PstI to the BamHI site (N-mycP-B; about 800 bp adjacent to the RNA start site), possessed enhancer activity. Various segments of the N-myc gene, overlapping the transcriptional regulatory regions, were subcloned into pUC19, and the plasmids were transfected to IMR32 cells together with a hygromycin B resistance-expression vector. The transfected cells were cultured in the presence of hygromycin B to establish cell lines resistant to the drug. Among the cell lines obtained, only the cells co-transfected with pN-mycP-B, the region of which contains enhancer activity described above, harboured the replicated pN-mycB-P in an episomal state. The results of BrdU labelling followed by CsCl isopycnic centrifugation suggested that the pN-mycP-B replicated once per cell cycle (in S phase) in concord with the replication of chromosomal DNA. In an in vitro DNA replication system using the nuclear extract from IMR32 cells, pN-mycP-B functioned as an template DNA of replication. Deletions up to 200 bp in the N-mycP-B region did not abolish the replicating activity, regardless of the positions of deletion, but deletions of longer than 300 bp did. These results suggest that the replicating activity of the fragment upstream of the human N-myc gene requires certain length, as well as the specific sequences not yet determined.