Cis-acting effects of sequences within 2.4-kb upstream of the human c-myc gene on autonomous plasmid replication in HeLa cells.

We have used density shift analysis to monitor the autonomous replicating sequence (ARS) activity of plasmids containing various DNA fragments from the 5'-flanking region of the human c-myc gene. The ARS activity of certain of these plasmids implied that structures in the c-myc DNA could be recognized for the initiation of replication in the absence of chromosomal integration. The plasmid pNeo.Myc-2.4 contains 2.4 contains 2.4 kb of c-myc 5'-flanking DNA, and replicated semiconservatively as a circular extrachromosomal element. Deletion derivatives of pNeo.Myc-2.4 containing either of two nonoverlapping regions of c-myc DNA semiconservatively incorporated bromodeoxyuridine into discrete populations of heavy-light supercoiled molecules to roughly the same extent as the chromosomal DNA in the same cultures. Some constructs displayed lower ARS activity, implying that distinct cis-acting sequences in the c-myc 5'-flanking DNA may independently affect DNA replication. The ARS activity of two separate c-myc sequences suggests that replication initiation signals are redundant in the c-myc origin. The smallest c-myc insert that displayed substantial ARS activity was 930 bp long and contained three 10/11 matches to the yeast ARS consensus and several additional features found in eukaryotic replication origins.