Lagging-strand, early-labelling, and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin.

Molecular and Cellular Biology
S WangJ L Hamlin

Abstract

There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-beta, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential 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 differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, 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 strand switch at ori-beta; rather, we observed a gradual, ...Continue Reading

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Citations

Jul 11, 1998·The Journal of Biological Chemistry·L Phi-vanW H Strätling
Jul 20, 1999·Molecular and Cellular Biology·M Malott, M Leffak
Aug 25, 1999·The Journal of Cell Biology·D S DimitrovaD M Gilbert
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Jan 21, 2015·The Journal of Cell Biology·Olivier Hyrien
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