Dawson B A, Lough J
Department of Anatomy and Cellular Biology, Medical College of Wisconsin, Milwaukee 53226.
Dev Biol. 1988 Jun;127(2):362-7. doi: 10.1016/0012-1606(88)90322-3.
We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation ensues.
我们通过用生物素-11-dUTP原位修复固定细胞单层细胞核中的切口，随后用兔抗生物素抗体和异硫氰酸荧光素标记的山羊抗兔抗体进行免疫细胞化学检测，在体外鸡肌生成过程中定位了DNA链断裂。在分裂细胞的23小时培养物中未观察到足以进行免疫细胞化学检测的生物素积累。在33小时和43小时的培养物中，生物素首先仅在3%的细胞核中被检测到，所有这些细胞核似乎都处于融合的成肌细胞或小肌管中。相比之下，年轻的、高度融合的肌管（56小时）培养物中有18%的细胞核被生物素化；实际上所有这些细胞核，其中大多数聚集成团，都在肌管内。在较老的培养物（73小时和94小时）中，生物素掺入肌管细胞核的情况明显减少，而单核细胞核中的生物素掺入则有所增加。这些结果表明，年轻肌管的细胞核中发生了广泛的单链DNA切口，随后随着终末分化的发生而进行修复。
