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 be en 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 mult iple sites in a zone of at least 6 kb situated immediately upstream from th e promoter of the main transcription unit of this puff. The complementary n eutral/alkaline two-dimensional gel electrophoresis technique shows that, w ithin the initiation zone, forks move in both directions. In contrast, unid irectional fork movement away from the initiation zone is observed at the e nds of the region, implying that it is the only place in the amplified regi on of the C3 puff where initiations occur. Since the initiation zone coinci des with the region that is most highly amplified, amplification of the C3 puff probably occurs by an onion skin-type mechanism.