Physical and genetic mapping of mammalian replication origins

The neutral/neutral and neutral/alkaline two-dimensional gel electrophoreti c techniques are sensitive physical mapping methods that have been used suc cessfully to identify replication initiation sites in genomes of widely var ying complexity. We present detailed methodology for the preparation of rep lication intermediates from mammalian cells and their analysis by both neut ral/neutral and neutral/alkaline two-dimensional gel approaches. The method s described allow characterization of the replication pattern of single-cop y loci, even in mammalian cells. When applied to metazoans, initiation is f ound to occur at multiple sites scattered throughout zones that can be as l ong as 50 kb, with some subregions being preferred. Although these observat ions do not rule out the possibility of genetically defined replicators, th ey offer the alternative or additional possibility that chromosomal context may play an important role in defining replication initiation sites in com plex genomes. We discuss novel recombination strategies that can be used to test for the presence of sequence elements critical for origin function if the origin lies in the vicinity of a selectable gene. Application of this strategy to the DHFR locus shows that loss of sequences more than 25 kb fro m the local initiation zone can markedly affect origin activity in the zone . (C) 1999 Academic Press.