Vatolin S Y, Okhapkina E V, Matveeva N M, Shilov A G, Baiborodin S I, Philimonenko V V, Zhdanova N S, Serov O L
Institute of Cytology and Genetics, Academy of Sciences of Russia, Novosibirsk, Russia.
Mol Reprod Dev. 1997 May;47(1):1-10. doi: 10.1002/(SICI)1098-2795(199705)47:1<1::AID-MRD1>3.0.CO;2-R.
Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.
对姐妹染色单体交换（SCE）和某些小卫星DNA重组率的研究表明，它们在植入前阶段的水平明显高于胚胎发育后期。早期发育过程中的单链DNA断裂（SSB）可能与这两个事件都有关。基于此，我们估计了在体外视黄酸（RA）诱导的以及小鼠畸胎瘤（EC）和胚胎干细胞（ES）自发分化过程中的SSB。使用核仁沉降和单细胞DNA电泳方法，我们观察到在EC细胞开始分化后的最初2 - 4次有丝分裂期间，SSB急剧增加，随后逐渐恢复到未分化细胞的基础水平特征。SSB的增加表现为沉降速率缓慢的大量核仁的出现，以及大多数细胞DNA图谱中的低分子量片段。我们得出结论，在分化早期，不少于一半的基因组DNA已被切割。尽管根据胚胎抗原和形态学标准判断仍在持续分化，但观察到SSB水平下降。此外，SCE水平的增加与SSB的增加同时发生，可能是其结果。SSB的预定“激增”可能是在没有表型表现的阶段开始分化的最早事件。
