Strom C M, Dorfman A
Proc Natl Acad Sci U S A. 1976 Apr;73(4):1019-23. doi: 10.1073/pnas.73.4.1019.
In order to study the mechanism of the irreversible effects of BrdUrd on the differentiation of limb bud mesenchyme to cartilage, the reannealing behavior of DNA obtained from such cells was examined. Cells incubated with [3H]thymidine ([3H]dThd) during days 1 and 2 of culture incorporated label into repetitive, moderately repetitive, and unique classes of DNA. In contrast, when 5-bromo-2'-[3H]deoxyuridine ([3H]Brd Urd) was added during the first 48 hr (in the presence of 32 muM BrdUrd), the label was preferentially incorporated into a late moderately repetitive region. Simultaneous incubation of unlabeled BrdUrd and [3H]dThd revealed a selective inhibition of [3H]dThd incorporation into moderately repetitive regions. Cultures incubated during days 3 and 4 with [3H]dThd incorporated label into all three classes of DNA; however, when [3H]dThd was present during days 3 and 4 in cultures previously incubated with BrdUrd during days 1 and 2, the [3H]dThd was incorporated preferentially in the late moderately repetitive region. The melting behavior of this reannealed DNA was identical with that of the reannealed 1-2 day [3H]BrdUrd-labeled, late moderately repetitive DNA. Turnover experiments revealed that whereas there was no loss of [3H]deoxycytidine or [3H]dThd, 37% of [3H]BrdUrd activity was lost from the DNA in 2 days after removal of the isotopes.
为了研究5-溴脱氧尿苷(BrdUrd)对肢芽间充质细胞向软骨细胞分化产生不可逆影响的机制,对从这些细胞中提取的DNA的复性行为进行了检测。在培养的第1天和第2天用[³H]胸腺嘧啶核苷([³H]dThd)孵育的细胞,将放射性标记掺入到重复、中度重复和单拷贝的DNA类别中。相比之下,在最初的48小时内(在32μM BrdUrd存在的情况下)加入5-溴-2'-[³H]脱氧尿苷([³H]BrdUrd)时,放射性标记优先掺入到一个晚期中度重复区域。未标记的BrdUrd和[³H]dThd同时孵育显示,[³H]dThd掺入中度重复区域受到选择性抑制。在第3天和第4天用[³H]dThd孵育的培养物,将放射性标记掺入到所有三类DNA中;然而,当在第1天和第2天预先用BrdUrd孵育的培养物在第3天和第4天存在[³H]dThd时,[³H]dThd优先掺入晚期中度重复区域。这种复性DNA的解链行为与复性的1 - 2天[³H]BrdUrd标记的晚期中度重复DNA相同。周转实验表明,虽然[³H]脱氧胞苷或[³H]dThd没有损失,但在去除同位素后2天内,37%的[³H]BrdUrd活性从DNA中消失。