Figure 1.
Amplification of the initiation region/matrix attachment region (IR/MAR) plasmid.
The IR/MAR (+) plasmid generated double minutes (DMs) (A) or homogeneously staining regions (HSRs) (B) in human COLO 320 cells. In these images, plasmid sequence was detected by FISH (green; indicated by arrows), and chromosomes were counterstained with propidium iodide (PI; red). A previous study revealed the mechanism by which the IR/MAR plasmid generates DMs or HSRs in these cells; these findings are summarized in (C). In the drawing, the plasmid sequence is green, the chromatid is orange, and the centromeres are black circles. In (D)–(F), structures of the three plasmids used in this study are shown. The orientation and position of each PCR primer used for the detection of inverted repeats are depicted as black and red triangles with accompanying numbers. Black and red indicate hybridization to different DNA strands.
Figure 2.
The initiation region/matrix attachment region (IR/MAR) sequence increased transformation efficiency, initial multimerization, and chromosomal extension.
Three plasmids, shown in Figures 1D–F, were transfected to 1.5×106 CHO DG44 cells. The selection procedure is shown in (A). After selection with BS (stage “a”), total colony number from 8×105 tranfected cells and colony size in arbitrary units were scored at the stage “a” (B). At each selection stage, metaphase chromosome spreads were prepared, and plasmid sequences were detected by FISH. (C) Representative images for no signal (NO), paired single dots (SD), and homogeneously staining regions (HSRs) of various sizes (<1 to >7, expressed in units of chromosome width. (D) The frequency of each amplification status among cells receiving pSFV-V-Dhfr (dihydroflate reductase) (IR/MAR−) or pΔBN AR1-Dhfr (IR/MAR+) at each step was scored by examining 30–40 metaphases in triplicate. Values represent means; error bars indicate +/− standard deviation. ***; p<0.001, **; p<0.01, *; p<0.1.
Figure 3.
The initiation region/matrix attachment region (IR/MAR) increased the number of homogeneously staining regions (HSRs) per cell.
At stage “c” in Figure 2A, metaphase spreads were prepared from the IR/MAR (−) and (+) cultures, and these were analyzed by FISH. Representative images are shown in (A)–(D); HSRs are indicated by yellow arrows. The number of HSRs per cell was determined by examining 19–22 metaphases, which had at least one HSR, in three replicates, and means +/− standard deviation are plotted in (E). The result from Figures 2 and 3 are summarized in panel F, in which green and red represent IR/MAR (−) and (+) plasmids respectively, blue represents chromatin, and black circles represent centromeres.
Figure 4.
The initiation region/matrix attachment region (IR/MAR) increased the homogeneously staining region (HSR) elongation rate and decreased the frequency of micronuclei after elevation of methotrexate (Mtx) concentration.
The IR/MAR-bearing plasmid (A, E) or a plasmid lacking this sequence (B, F) was transfected into Chinese hamster ovary DG44 cells. Cells growing in 5 nM Mtx (stage “c” in Fig. 2A) were subsequently cultured in 50 nM Mtx. Then, metaphase spreads were prepared at the indicated times (in days), and plasmid sequences were detected by FISH. The frequency of cells bearing HSRs of various lengths was determined by observing 30–60 metaphase cells; the results are plotted in (A) and (B). Cells without (C) and with (D) plasmid HSRs contained micronuclei (arrowed). Frequencies were scored by observation of 200–500 cells in three replicates, and means +/− standard deviation are plotted in (E) and (F).
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).
Figure 6.
Clonal analysis of homogeneously staining region (HSR) elongation and inverted recombination.
After selection with blasticidine (BS) (stage “a” in Fig. 2A), cells were cloned by limiting dilution. Each clone was cultured in nucleotide-deficient medium containing 5 nM methotrexate (Mtx). The gene amplification status revealed by FISH before and after Mtx treatment is drawn schematically in each panel; plasmid signals are depicted in red. The inverted repeat in the plasmid sequence was detected by PCR, and images of ethidium bromide-stained agarose gels are shown in each panel. The numbers above each gel image correspond to the primer set used (Table S1). The results obtained with the initiation region/matrix attachment region (IR/MAR) (+) plasmid are shown in panels (A), (F)–(I), and results obtained with the IR/MAR (−) plasmid are shown in panels (B)–(E).
Figure 7.
H3K9 modification before and after the methotrexate (Mtx) treatment.
CHO DG44 cells were transfected with initiation region/matrix attachment region (IR/MAR) (−) pSFV-V-Dhfr (dihydroflate reductase) or IR/MAR (+) pΔBN AR1-Dhfr, as before. Chromatin was isolated from polyclonal transformants at stages “a” and “c” in Figure 2A (A–D). Alternatively, prior to Mtx treatment, chromatin was isolated from indicated clones classified as pattern 3 or 4. The chromatin was immunoprecipitated using anti-histone H3K9-trimethyl (H3K9me3) or anti-histone H3K9-acetyl (H3K9ac) antibody, and the precipitate was analyzed by real-time PCR.
Figure 8.
A model explaining how a circular molecule bearing the genomic initiation region/matrix attachment region (IR/MAR) sequence is amplified under replication stress.
For a detailed explanation, see the Discussion section. Green: IR/MAR sequence; orange: chromosome arm; black circle: centromere; blue chromosome fragile site.