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首页> 外文期刊>Molecular and Cellular Biology >Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.
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Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

机译：在密切相关的人类对X射线的辐射反应不同的人类淋巴母细胞系中的重组能力不同。

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WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events.
机译：WIL2-NS和TK6是两个不同的人类淋巴母细胞细胞系，它们来自一个男性供体。 WIL2-NS细胞对X射线的细胞毒性作用具有更高的抵抗力，但对诱导的突变更为敏感。为了确定这些差异的机制基础，我们分析了对源自TK6和更易变的WIL2-NS的tk杂合子进行X射线处理后分离的胸苷激酶（tk）缺陷型突变体的物理结构。 Southern分析显示，虽然84％的TK6突变体是由于杂合性（LOH）的丧失而引起的，但来自WIL2-NS衍生物的所有106个突变体均在tk出现了LOH，除一个外，其余所有突变体均在17号染色​​体的其他连锁位点显示了LOH。运用荧光原位杂交技术区分了由于缺失导致仅保留一个tk等位基因的LOH和由于涉及同源染色体（例如重组）的机制而导致保留两个tk等位基因的LOH。在以这种方式分析的LOH突变体中，通过缺失产生了来自WIL2-NS的26个中的9个和来自TK6细胞系的17个中的11个。其余的突变体保留了tk基因的两个拷贝，因此通过涉及同源等位基因的机制产生。由于许多由同源机制引起的突变体保留了染色体17的部分杂合性，因此它们一定是通过重组或基因转化而不是染色体丢失和重复来产生的。最后，在具有质粒重组底物的转染测定中比较了WIL2-NS和TK6的重组能力。 WIL2-NS中的分子间重组频率高于TK6。这些数据与一个模型相符，该模型表明，WIL2-NS中的重组修复系统比TK6中的重组系统发挥更高的作用。分子间和分子内重组事件越频繁，WIL2-NS中tk基因座的变异性就越大。

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