Mariani B D, Schimke R T
J Biol Chem. 1984 Feb 10;259(3):1901-10.
We have employed Chinese hamster ovary cells synchronized by mitotic selection to study the replication and amplification of the dihydrofolate reductase gene. Using bromodeoxyuridine to differentially label newly replicated DNA, we show that the dihydrofolate reductase gene is replicated during the first 2 h of S phase, a time when, at most, 10% of the total genome has been replicated. We find that a 6-h inhibition of DNA synthesis by hydroxyurea beginning 2 h after the initiation of S phase markedly increases the frequency with which cells become resistant to a 100-fold increment in methotrexate. When DNA synthesis resumes following removal of the hydroxyurea, virtually all of the DNA replicated prior to inhibition, including the dihydrofolate reductase gene, is rereplicated. Analysis of the dihydrofolate reductase enzyme content of cells 24 h after treatment with hydroxyurea using the fluorescence-activated cell sorter reveals a subset of cells with elevated dihydrofolate reductase. It is this subset that contains additional copies of the dihydrofolate reductase gene and from which emerge highly methotrexate-resistant cells. We propose that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome. As cells are subsequently placed under selection, a number of processes, including recombination events and loss of nonselected DNA sequences occur, resulting in what appears as differential gene amplification.
我们利用通过有丝分裂选择同步化的中国仓鼠卵巢细胞来研究二氢叶酸还原酶基因的复制和扩增。使用溴脱氧尿苷对新复制的DNA进行差异标记，我们发现二氢叶酸还原酶基因在S期的前2小时进行复制，而在这个时间段，整个基因组最多只有10%被复制。我们发现，在S期开始2小时后用羟基脲抑制DNA合成6小时，显著增加了细胞对甲氨蝶呤浓度增加100倍产生抗性的频率。当去除羟基脲后DNA合成恢复时，几乎所有在抑制前复制的DNA，包括二氢叶酸还原酶基因，都会再次复制。使用荧光激活细胞分选仪分析羟基脲处理24小时后细胞中二氢叶酸还原酶的含量，发现了一部分二氢叶酸还原酶含量升高的细胞。正是这部分细胞含有额外拷贝的二氢叶酸还原酶基因，并且从中产生高度抗甲氨蝶呤的细胞。我们提出，扩增的初始事件是基因组中可变但相对大量的DNA再次复制。随后当细胞处于选择压力下时，会发生许多过程，包括重组事件和非选择DNA序列的丢失，从而导致出现差异基因扩增。
