FIG. 1.

(A) Features of the endogenous DHFR ori-β IR. Preferred start sites of DNA replication—the ori-β, ori-β′, and ori-γ sites; a 55-kb initiation zone (shaded area) between the DHFR- and 2BE2121-coding sequences; and matrix-attached regions (MARs, stippled boxes)—are indicated (5, 14, 42, 43, 44, 52, 55, 57, 71, 85, 86, 89). The 5.8-kb fragment of the DHFR ori-β region (pMCD), extending from the BamHI to the KpnI recognition sequences, is indicated. Arrows denote the positions of the primers used in the competitive PCR assay for origin function and correspond to primer pairs used in previous DHFR ori-β mapping studies (71). (B) Strategy for quantitating initiation occurring in exogenous DHFR ori-β fragments in ectopic chromosomal locations. A 5.8-kb wild-type or mutated DHFR ori-β fragment was coelectroporated with a neomycin resistance gene into the DR12 cell line, a CHO derivative containing a 150-kb deletion encompassing the entire DHFR locus (47). After selection with G418, total DNA was isolated, heat denatured, and size fractionated on a 5 to 30% linear neutral sucrose gradient. The fraction containing single-stranded DNA with a length of 1 to 2 kb, representing nascent DNA, was isolated, and the abundance of ori-β target sequences contained in the fraction was quantitated by competitive PCR.