How a Replication Origin and Matrix Attachment Region Accelerate Gene Amplification under Replication Stress in Mammalian Cells
Figure 5
The breakage-fusion-bridge (BFB) cycle operates at chromosomal sites to generate and elongate fine-ladder homogeneously staining regions (HSRs).
Metaphase spreads (A, C) or paraformaldehyde (PFA)-fixed samples (B, D–I) from cells after BS selection (panels B and C) or cells grown in the presence of 5 nM methotrexate for 12 days (A, D to I) were analyzed. Panel A shows two representative images of symmetrical plasmid signals (green) arranged along the metaphase chromosome (red). Some cells contained anaphase bridges; (B) shows γ-H2AX signal (red) at the broken ends of a bridge, and (C) shows a bridge severed in the middle. Panels (D)–(H) show representative images of the segregation of fine-ladder HSRs (green) with anaphase chromosomes (gray). A chromatin bridge composed of a fine-ladder HSR could be severed at its end (D), at a site shifted from the middle of the HSR (E), or at the middle of the HSR (F). An HSR might also be delivered to one daughter nucleus by non-disjunction (yellow arrow in G), or evenly segregated without bridge formation (H). Frequencies of these events among the culture were counted and plotted in panel (H).