AN AMPLIFIED CHROMOSOMAL SEQUENCE THAT INCLUDES THE GENE FOR DIHYDROFOLATE-REDUCTASE INITIATES REPLICATION WITHIN SPECIFIC RESTRICTION FRAGMENTS
AN AMPLIFIED CHROMOSOMAL SEQUENCE THAT INCLUDES THE GENE FOR DIHYDROFOLATE-REDUCTASE INITIATES REPLICATION WITHIN SPECIFIC RESTRICTION FRAGMENTS
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DOI：
10.1073/pnas.79.13.4083
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发表时间：
1982-01-01
期刊：
影响因子：
--
通讯作者：
HAMLIN, JL
中科院分区：
文献类型：
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作者：
HEINTZ, NH;HAMLIN, JL
建立了一株抗甲氨蝶呤的中国仓鼠卵巢细胞系（choc400），该细胞系含有135千碱基染色体DNA序列500倍扩增。该序列包括二氢叶酸还原酶基因（四氢叶酸脱氢酶，5,6,7,8-四氢叶酸：NADP+氧化还原酶，EC 1.5.1.3）。扩增序列的高拷贝数允许它被可视化为基因组消化中的一系列独特的限制性内切片段，分离在溴化乙啶染色琼脂糖凝胶上。在同步的CHOC 400细胞中，通过放射性标记S期开始时合成的DNA来研究扩增序列中DNA复制的起始。标记基因组消化的Southern印迹放射自显影显示，DNA合成始于一小部分来自扩增单位的EcoRI片段。这些早期标记的片段在S期后期不被合成，此时合成了不同的片段子集。无论在S期开始时使用何种药物收集细胞，观察到的复制模式都是相同的。扩增序列的复制显然是从每个重复单元的特定位置开始的，并在序列的其余部分以非随机顺序进行，也就是说，哺乳动物细胞染色体中DNA合成的起始是序列特异性的。
A methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) was developed containing a 500-fold amplification of a 135-kilobase chromosomal DNA sequence. This sequence includes the gene for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The high copy number of the amplified sequence permits it to be visualized as a distinct series of restriction fragments in genomic digests separated on ethidium bromide-stained agarose gels. Initiation of DNA replication in the amplified sequence was studied by radiolabeling DNA synthesized during the onset of S phase in synchronized CHOC 400 cells. Autoradiography of Southern blots of labeled genomic digests shows that DNA synthesis initiates in a small subset of the EcoRI fragments derived from the amplified units. These early labeled fragments are not synthesized at later times during S phase, when different subsets of fragments are synthesized. Regardless of the drug used to collect cells at the beginning of S phase, the replication pattern observed remains the same. Replication of the amplified sequence evidently initiates at specific sites within each repeated unit and proceeds in nonrandom order throughout the remainder of the sequence, i.e., that initiation of DNA synthesis in the chromosomes of mammalian cells is sequence specific.