Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin

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, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-beta and ori-gamma being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region.

c-Myc initiates illegitimate replication of the ribonucleotide reductase R2 gene

“…Genomic DNA from cell cycle synchronized Pre-B7 and Pre-B+ cells (Wang et al, 1998) was analysed by neutral-neutral two-dimensional (2D) gel electrophoresis at 0, 24, 48 and 72 h. The DNA samples were digested with HindIII, leaving the active R2 gene uncut (Thelander and Berg, 1986). We found a single replication fork in Pre-B7 cells at the active R2 gene 24 h after synchronization and seeding into complete medium ( Figure 2a (B,B'), arrow).…”

“…All bromodeoxyuridine (BrdU) incorporation assays were conducted in synchronized (Wang et al, 1998) Pre-B7 and Pre-B+ cells . BrdU incorporation and DNA replication was assessed during synchronization and at 0, 24, 48 and 72 h after seeding into complete medium and following mock-treatment (95% ethanol) or c-Myc activation (4-HT treatment).…”

“…Neutral ± neutral 2-dimensional (2D) gel electrophoresis was performed as follows: Genomic DNA was isolated from synchronized (Wang et al, 1998), non-activated and 4-HTactivated cells by standard procedures. One hundred mg of each DNA sample was digested overnight at 378C with HindIII (Roche Diagnostics, Laval, Que bec, Canada) and roto-evaporated to a 40 mL volume.…”

“…(b) Cyclin C replication is unaltered by c-Myc. 2D membranes of genomic DNA from synchronized Pre-B7 and Pre-B+ cells (Wang et al, 1998) were probed with 400 and 500 bp EcoRI cyclin C cDNA fragments (Li et al, 1996) Figure 3 c-Myc interaction with R2 E-boxes precedes initiation of de novo rereplication (a) The mouse ribonucleotide reductase R2 (R2) gene. This ®gure is a schematic representation of the mouse R2 gene (Thelander and Berg, 1986).…”

“…as the mass of DNA increases from n to 2n). (a) 2D gel electrophoresis and Southern analyses of non-activated (Pre-B7) (A ± D) and 4-HT-activated (Pre-B+) cells (E ± H) at 0, 24, 48 and 72 h after synchronizing and seeding into complete medium (Wang et al, 1998). The membranes were probed with a 1487 bp PstI R2 cDNA fragment (Thelander and Berg, 1986).…”