Replication of the dihydrofolate reductase genes on double minute chromosomes in a murine cell line

Abstract

The purpose of this study is to determine the kinetics of the replication of intrachromosomal versus extrachromosomal amplified dihydrofolate reductase (DHFR) genes. Previous studies reported that the DHFR gene, when carried intrachromosomally on a homogeneously staining region, replicates (as a unit) within the first 2 h of the S phase of the cell cycle. We wished to determine if the extrachromosomal location of the amplified genes carried on double minute chromosomes effects the timing of their replication. Equilibrium cesium chloride ultracentrifugation was used to separate newly replicated (BUdR-labeled) DNA from bulk DNA in a synchronized cell population. Hybridization with the cDNA for the DHFR gene allowed us to determine the period of time within the cell cycle in which the DHFR DNA sequences were replicated. We found that, in contrast to intrachromosomal dihydrofolate reductase genes that uniformly replicate as a unit at the beginning of the S phase of the cell cycle, dihydrofolate reductase genes carried on double minute chromosomes (DMs) replicate throughout the S phase of the cell cycle. These results suggest that control of replication of extrachromosomal DNA sequences may differ from intrachromosomal sequences.