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首页> 外文期刊>Molecular and Cellular Biology >Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.
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Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

机译：在独立衍生的耐甲氨蝶呤的中国仓鼠细胞中扩增了类似的150碱基碱基的DNA序列。

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摘要

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.
机译：我们从耐甲氨蝶呤的中国仓鼠卵巢细胞系（CHOC 400）的扩增的二氢叶酸还原酶结构域中分离出了代表150 KB连续DNA序列的重叠重组粘粒。该序列包括25碱基的二氢叶酸还原酶基因和DNA合成的起源。跨越该域的八个粘粒已被用作放射性杂交探针，以分析在四个独立衍生的耐甲氨蝶呤的中国仓鼠细胞系中二氢叶酸还原酶扩增子之间的相似性。我们已经观察到我们检查的150碱基碱基的DNA序列中的四个细胞系之间没有显着差异，除了多态性是由二氢叶酸还原酶结构域的两个可能的等位基因中的一个或另一个扩增引起的。我们还显示，在这四个抗性细胞系中扩增子的限制性酶切模式实际上与药物敏感性亲本细胞中相应的未扩增序列相同。此外，对来自扩增子的广泛分离区域的片段的相对拷贝数的测量表明，该150碱基对区域中的所有片段可以被一致地扩增。我们的数据表明，在耐甲氨蝶呤的中国仓鼠细胞中，相对于二氢叶酸还原酶基因本身而言，扩增的单位较大。此外，在我们已经研究的150碱基对扩增的共有序列中，在扩增过程中似乎没有发生重大的重排。

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