Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence-activated cell sorting.
Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence-activated cell sorting.
复制标题
荧光激活细胞分选显示快速自发二氢叶酸还原酶基因扩增。
DOI：
10.1073/pnas.80.12.3711
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发表时间：
1983
影响因子：
11.1
通讯作者：
Schimke,RT
中科院分区：
文献类型：
--
作者：
Johnston,RN;Beverley,SM;Schimke,RT
我们已经确定了编码二氢叶酸还原酶（5,6,7,8-四氢叶酸：NADP+氧化还原酶，EC 1.5.1.3）的基因在体外细胞中的基因拷贝数是否自发变化。细胞在非选择性条件下用荧光甲氨蝶呤染色，甲氨蝶呤定量结合二氢叶酸还原酶。用荧光激活的细胞分选器收集荧光最高的细胞，然后在没有甲氨蝶呤的情况下生长。在实验过程中，细胞没有处于代谢应激状态。经过连续10轮的生长和分选，衍生群体的荧光强度增加了50倍，对甲氨蝶呤具有高度抗性，二氢叶酸还原酶基因含量增加了40倍。我们还发现，已经扩增了基因的细胞，其荧光和基因拷贝数的增加或减少甚至比亲本细胞（每次分裂约10（-3）次）更快（每次细胞分裂高达3 × 10（-2）次扩增事件）。因此，我们得出结论，基因扩增可以在细胞中自发发生，其发生率随基因拷贝数的变化而变化。
We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell sorter and subsequently grown in the absence of methotrexate. At no time during the experiment were the cells placed under metabolic stress. After 10 successive rounds of growth and sorting, the derived population showed a 50-fold increase in fluorescence intensity, was highly resistant to methotrexate, and was amplified 40-fold in content of dihydrofolate reductase gene. We also found that cells already having amplified genes can undergo increases or decreases in their fluorescence and in gene copy number even more rapidly (at rates as high as 3 X 10(-2) amplification events per cell division) than do parental cells (ca. 10(-3) events per division). We therefore conclude that gene amplification can occur spontaneously in cells and that the rate of its occurrence varies with gene copy number.