LAGGING-STRAND, EARLY-LABELING, AND 2-DIMENSIONAL GEL ASSAYS SUGGEST MULTIPLE POTENTIAL INITIATION SITES IN THE CHINESE-HAMSTER DIHYDROFOLATE-REDUCTASE ORIGIN

There is general agreement that DNA synthesis in the single-copy and a mplified dihydrofolate reductase (DHFR) loci of CHO cells initiates so mewhere within the 55-kb spacer region between the DHFR and 2BE2121 ge nes, However, results of lagging-strand, early-labelling fragment hybr idization (ELFH), and PCR-based nascent-strand abundance assays have b een interpreted to suggest a very narrow zone of initiation centered a t a single locus known as ori-beta, while two-dimensional (2-D) gel an alyses suggest that initiation can occur at any of a large number of p otential sites scattered throughout the intergenic region, The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-beta and a second locus (ori-gamma) are somewhat preferred, To determine how these diff ering views are shaped by differences in experimental manipulations un related to the biology itself, we have applied the lagging-strand, ELF H, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way, In our experiments, the lagging-strand assay failed to identify a template s trand switch at ori-beta; rather, we observed a gradual, undulating ch ange in hybridization bias throughout the intergenic spacer, with hybr idization to the two templates being approximately equal near a center ed matrix attachment region, In the ELFH assay, all of the fragments i n the 55-kb intergenic region were labelled in the first few minutes o f the S phase, with the regions encompassing ori-beta and ori-gamma be ing somewhat preferred, Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multip le locations in the intergenic spacer, Thus, the results of all existi ng replicon-mapping methods that have been applied to the amplified DH FR locus in CHOC 400 cells are consistent with a model in which two so mewhat preferred subzones reside in a larger zone of multiple potentia l initiation sites in the intergenic region.