LOCALIZATION OF A BIDIRECTIONAL DNA-REPLICATION ORIGIN IN THE NATIVE LOCUS AND IN EPISOMALLY AMPLIFIED MURINE ADENOSINE-DEAMINASE LOCI

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,0 00 extrachromosomal copies of the mouse adenosine deaminase (ADA) ampl icon in B-1/50 cells initiate their replication remarkably synchronous ly in early S phase and at approximately the same time as the single-c opy chromosomal locus from which they were derived. The abundance of A DA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA ep isomes within a preferred region and whether this region is the same a s that used at the corresponding chromosomal locus prior to amplificat ion. This study reports the detection and localization of a discrete s et of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template stran d complementarity of Okazaki fragments, indicative of the initiation o f bidirectional DNA replication, was found to lie within the same regi on. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a prefer red region and that the same region is used to initiate DNA synthesis within the native locus.