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 replicat ing sequence (ARS) activity of plasmids containing various DNA fragmen ts from the 5'-flanking region of the human c-myc gene. The ARS activi ty of certain of these plasmids implied that structures in the c-myc D NA could be recognized for the initiation of replication in the absenc e of chromosomal integration. The plasmid pNeo.Myc-24 contains 2.4 kh of c-myc 5'-flanking DNA, and replicated semiconservatively as a circu lar extrachromosomal element. Deletion derivatives of pNeo.Myc-2.4 con taining either of two nonoverlapping regions of c-myc DNA semiconserva tively incorporated bromodeoxyuridine into discrete populations of hea vy-light supercoiled molecules to roughly the same extent as the chrom osomal DNA in the same cultures. Some constructs displayed lower ARS a ctivity, implying that distinct cis-acting sequences in the c-myc 5'-f lanking 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 t hat displayed substantial ARS activity was 930 bp long and contained t hree 10/11 matches to the yeast ARS consensus and several additional f eatures found in eukaryotic replication origins.