Takanari H, Yamanaka H, Mitani H, Izutsu K
Department of Pathology, Mie University School of Medicine, Japan.
Biol Cell. 1994;82(1):23-31. doi: 10.1016/0248-4900(94)90062-0.
Double label immunofluorescence against PCNA and BrdU clearly revealed several characteristics of DNA replication sites in synchronized CHO cells. We observed that the distribution of replication sites changed from early to late S phase, particularly in the nucleolar and perinuclear regions, and the amount of PCNA in each replication site markedly decreased or disappeared with the progression of S phase. Although co-localization of PCNA and BrdU was usually seen, the intensity of fluorescence occasionally differed between the labeled PCNA and BrdU, particularly in late S phase. Based on the assumption that such a difference may reflect a different configuration of the chromatin, we propose that the brightly granular fluorescent staining of PCNA or BrdU in early S phase indicates a condensed segment of euchromatin. In the late S phase nucleus, we clearly observed a chromatin-like structure in the late replicating segments of heterochromatin in anti-PCNA stained material. In vincristine-induced multinucleate cells, a discrepancy between PCNA-distribution and BrdU-incorporation in sister nuclei was sometimes seen. Such observations indirectly support the following two mechanisms for premature chromosome condensation proposed by others: asynchronous initiation of DNA synthesis; and a difference in the rate of DNA synthesis. In addition, the finding that BrdU was not incorporated into sites that had PCNA deposits suggests a third mechanism: the local disturbance of replication sites. Furthermore, unusual distribution patterns for replication sites suggest that the nucleation process in multinucleate cells differs from that in normal cells.
针对增殖细胞核抗原（PCNA）和5-溴脱氧尿嘧啶核苷（BrdU）的双标记免疫荧光清晰地揭示了同步化的中国仓鼠卵巢（CHO）细胞中DNA复制位点的几个特征。我们观察到，复制位点的分布从S期早期到晚期发生了变化，特别是在核仁区和核周区，并且随着S期的进展，每个复制位点的PCNA量显著减少或消失。尽管通常可见PCNA和BrdU的共定位，但标记的PCNA和BrdU之间的荧光强度偶尔会有所不同，特别是在S期晚期。基于这种差异可能反映染色质不同构型的假设，我们提出S期早期PCNA或BrdU的明亮颗粒状荧光染色表明常染色质的浓缩片段。在S期晚期细胞核中，我们在抗PCNA染色材料中异染色质的晚期复制片段中清楚地观察到一种染色质样结构。在长春新碱诱导的多核细胞中，有时会在姐妹细胞核中看到PCNA分布与BrdU掺入之间的差异。这些观察结果间接支持了其他人提出的关于染色体早熟凝聚的以下两种机制：DNA合成的异步起始；以及DNA合成速率的差异。此外，BrdU未掺入有PCNA沉积的位点这一发现提示了第三种机制：复制位点的局部干扰。此外，复制位点的异常分布模式表明多核细胞中的成核过程与正常细胞中的不同。
