DNA replication during amplification of the C3 puff of Rhynchosciara americana initiates at multiple sites in a 6 kb region.

Two independent two-dimensional agarose gel electrophoresis methods have been used to map the origin of replication that directs amplification of the C3 DNA puff of Rhynchosciara americana. The results of neutral/neutral two-dimensional gel electrophoresis show that DNA replication initiates at multiple sites in a zone of at least 6 kb situated immediately upstream from the promoter of the main transcription unit of this puff. The complementary neutral/alkaline two-dimensional gel electrophoresis technique shows that, within the initiation zone, forks move in both directions. In contrast, unidirectional fork movement away from the initiation zone is observed at the ends of the region, implying that it is the only place in the amplified region of the C3 puff where initiations occur. Since the initiation zone coincides with the region that is most highly amplified, amplification of the C3 puff probably occurs by an onion skin-type mechanism.