INDUCTION, BY THYMIDYLATE STRESS, OF GENETIC-RECOMBINATION AS EVIDENCED BY DELETION OF A TRANSFERRED GENETIC-MARKER IN MOUSE FM3A CELLS
INDUCTION, BY THYMIDYLATE STRESS, OF GENETIC-RECOMBINATION AS EVIDENCED BY DELETION OF A TRANSFERRED GENETIC-MARKER IN MOUSE FM3A CELLS
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DOI：
10.1128/mcb.6.10.3463
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发表时间：
1986-10-01
影响因子：
5.3
通讯作者：
SENO, T
中科院分区：
文献类型：
--
作者：
AYUSAWA, D;KOYAMA, H;SENO, T
对甲氨蝶呤指导的胸苷酸应激的遗传后果进行了研究，重点关注作为异源遗传标记引入小鼠胸苷酸脱氢酶阴性突变细胞的人胸苷酸合酶基因。胸苷酸应激诱导的胸苷酸合成酶阴性分离物伴随着人胸苷酸合成酶活性的丧失，其频率比一些但不是所有的胸苷酸系中未诱导的自发水平高1至2个数量级。 诱导的分离物几乎完全抑制放线菌酮和部分咖啡因。然而，通过测量对哇巴因或6-硫代鸟嘌呤的耐药性来确定，胸苷酸应激并没有诱导突变。胸苷酸脱氢酶阴性的分离子也诱导通过其他手段，如溴脱氧尿苷处理和X射线照射。在由胸苷酸胁迫诱导的每个腺苷酸酶阴性分离子中，包括转移的人胸苷酸合酶基因的几乎整个编码区的DNA片段以非常特异的方式缺失，如用人Alu序列和人胸苷酸合酶cDNA作为探针的Southern印迹分析所示。在低频率自发出现的分离体中，整个转移的遗传标记丢失。在X射线辐射诱导的分离体中，遗传标记的结构改变是随机的。这些结果表明，胸苷酸应激是一种生理因素，它以某种特定的方式引起这种外源掺入DNA的不稳定性，并在哺乳动物细胞中产生非随机遗传重组。
Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.