Mapping replicational sites in the eucaryotic cell nucleus.
Mapping replicational sites in the eucaryotic cell nucleus.
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DOI：
10.1083/jcb.108.1.1
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发表时间：
1989-01
期刊：
影响因子：
--
通讯作者：
Berezney R
中科院分区：
文献类型：
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作者：
Nakayasu H;Berezney R
我们用荧光显微镜研究了将生物素标记的dUTP掺入渗透的PTK-1袋鼠肾或3T3小鼠成纤维细胞后，间期细胞核中的DNA复制位点。散在的复制颗粒分布于核内及核周。发现了与细胞核染色质结构域和S期相关的三种不同的复制位点模式，分别命名为I型(S早期至中期)、II型(S中晚期)和III型(S晚期)。使用5-溴脱氧尿苷抗体在体内复制的DNA中也发现了类似的模式。用DNase I和0.2M硫酸铵抽提通透性细胞发现，尽管总DNA的去除率超过90%，但这些复制颗粒及其与剩余核基质结构的独特核内排列仍保持着显着的保持。原位制备的核基质结构还将生物素化的dUTP掺入复制颗粒中，与在完整核中检测到的复制颗粒没有区别。
We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus.