Olins D E, Olins A L
J Cell Biol. 1987 May;104(5):1125-32. doi: 10.1083/jcb.104.5.1125.
Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.
用兔抗生物素蛋白抗体，随后用异硫氰酸荧光素偶联的羊抗兔IgG进行免疫荧光检测，发现从腹毛目纤毛原生动物宽口游仆虫分离出的大核将生物素化的dUTP特异性掺入复制带（RB）中。当在包埋前反应中使用金偶联的羊抗兔IgG时，随后的免疫电子显微镜分析表明，生物素化的核苷酸在RB的后部区域更集中，前部区域几乎没有标记。可以使用免疫荧光测定法确定，同时添加N-乙基马来酰亚胺或放线菌素D，以及省略任何一种其他未标记的dNTP，均可抑制生物素化dUTP的掺入。此外，在完整细胞裂解和体外测定之前，延长热休克时间会使掺入量明显降低。与已发表的关于[3H]胸腺嘧啶核苷体内掺入宽口游仆虫RB的数据进行比较，表明了体外反应的保真度。
