Division of Diagnostic Imaging, Department of Radiology, Japan Labour Health and Safety Organization, Tokyo Rosai Hospital, Ohta-ku, Tokyo, Japan.
Methods Mol Biol. 2023;2519:117-126. doi: 10.1007/978-1-0716-2433-3_14.
A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.
细胞生物学的一个基本问题是 DNA 如何折叠成染色体。最近积累的大量证据表明，染色体的折叠与 DNA 复制的进展紧密相关。药物诱导的 PCC 是一种用于观察间期核的有用工具，特别是 S 期，因为 S 期过早浓缩染色体（S 期 PCC）。通过珠子加载方法，用 Cy3-dUTP 直接标记活性复制的 DNA，然后用 calyculin A 立即将 S 期核过早浓缩，以获得 S 期 PCC。在扫描共聚焦显微镜下观察 S-PCC 上的活性复制区域。用 Cy3-dUTP 标记的 S 期 PCC 清楚地揭示了染色体在 S 期通过“云状星云”到“串珠”到“带结构染色体的条纹阵列”（称为 G 带或 R 带模式）的形成的急剧转变。还在 S 期的各个亚相中测量了复制焦点的数量、分布和形状；在 S 期开始时可获得最大约 1400 个 0.35μm 平均半径大小的焦点，而在 S 期结束时数量减少到约 100 个。药物诱导的 PCC 清楚地提供了新的见解，即真核生物 DNA 复制与染色体浓缩/紧缩紧密相关，用于构建真核生物的高级染色体结构。
