Replication timing of the human 5q23/31.

<p><b>A.</b> Experimental strategy for determination of replication timing. Asynchronously replicating cells were labeled with BrdU and sorted by FACS into six fractions (G1, S1–4, G2/M) on the basis of DNA content. Genomic DNA from cells in each fraction was extracted, and newly replicated DNA was immunoprecipitated with anti-BrdU antibody. Semi-quantitative PCR was carried out using the newly replicated DNA as template. Relative band intensity was quantified. The values in each fraction were normalized by the levels of BrdU-labeled mitochondrial DNA (mtDNA; replicated equally throughout the cell cycle) used as an internal control for the recovery of DNA in each sample. <b>B.</b> 40,000 cells (Jurkat and HL-60) sorted (upper) and collected on the basis of DNA content (G1, S1–4, G2/M) were stained with PI, and analyzed by FACS (lower). <b>C.</b> Validation of cell cycle fractionation. The known early (PGK1) or late (F9) replicating region is enriched in appropriate fractions in comparison with the level of mtDNA in HL-60. <b>D.</b> DNA replication timing on the human chromosome 5q23/31 region (3.5 Mb) containing the cytokine cluster region in Jurkat (T cell) and HL-60 (non T cell). The 2.2 Mb segment containing the cytokine cluster (130.3–132.5) replicates in G1 or early in the S-phase (S1 and S2), whereas the 0.9 Mb segment distal to the cluster and proximal to the centromere replicates late in the S phase (S3, S4 and G2). The mean locations of the timing transition region (TTR) are located at around 130.15–130.25 (yellow box) in Jurkat (TTR-J) and at around 129.95–130.05 (green box) in HL-60 (TTR-H), and are offset by 180 kb in the two cell types. The left boundary of early replicating region coincided with the transition of chromosomal synteny (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042375#pone.0042375.s002" target="_blank">Fig. S2A</a></b>). <b>E.</b> The results of replication timing assays with fractionated cells are shown for each location along the 3.5 Mb human chromosome. The locations of the 9 primers used are indicated along the 5q23/31 region shown to the right of the panels. Small red solid boxes show the peak timing fraction for each probe, and large red dotted boxes show the maximum timing transition segments for Jurkat (129.98–130.22) and HL-60 (129.52–130.16).