Structural homologies and functional similarities between mammalian origins of replication and amplification promoting sequences

Abstract

MuNTS2, a 423 bp sequence isolated from the non-transcribed spacer of murine rDNA stimulates the amplification of cis-linked plasmid DNA in mouse cells under selective conditions. Here we demonstrate that a 180 bp subdomain of muNTS2 is highly homologous (approximately 70%) to three domains of the first well-characterized origin of replication of mammalian chromosomes, i.e. the origin of bidirectional replication (OBR) of the dihydrofolate reductase (DHFR) locus in Chinese hamster ovary (CHO) cells. When subcloned, the 180 bp homology region of muNTS2 was revealed to be essential for the amplification promoting activity of muNTS2. Fragments of the initiation zone of DNA replication from the DHFR locus of hamster cells containing the domains of homology to the mouse muNTS2 element proved also to promote DNA amplification. Thus, the screening system for amplification promoting elements turned out to detect an origin of bidirectional replication.

Activation of SV40 DNA replication in vivo by amplification-promoting sequences of the mouse ribosomal gene cluster

“…Substituting the aux-2 sequences by APS1 (lane 3) or APS2 (lane 4) at the late side increases replication efficiency to approximately 50% and 70%, respectively, as compared with the full-length origin (lane 1). On the other hand, insertion of a fragment of the DH-FR origin, which shares 73% homology with APS2 (Stolzenburg et al 1994) or of a fragment from the 28S rRNA coding region does not restore replication (lanes 5, 6, respectively). This result demonstrates that both APS elements specifically reactivate the core 1 origin of SV40 DNA replication.…”