| DNA Damage Sensitivity Assays with Arabidopsis Seedlings
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Author:
Date:
2014-04-05
[Abstract] We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The “true leaf assay” and the “UV resistance assay” are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, e.g. wild type, determining the most effective type of induced damage and the potential repair pathway affected.
[摘要] 我们描述快速和可重复的灵敏度测定以量化不同基因型的拟南芥幼苗对广泛的DNA损伤剂的反应。 我们应用（1）γ辐射，其产生DNA断裂，（2）bleocin，放射性模拟药物，（3）丝裂霉素C，DNA intrastrand交联剂，（4）羟基脲，DNA合成抑制剂， UV-C，其主要产生光产物。 "真叶试验"和"抗UV试验"基于容易确定的表型作为读数。 使用一组不同的损伤剂结合不同的读数允许相对于参考线（例如野生型）建立相对灵敏度/电阻，确定最有效类型的诱导损伤和潜在的修复途径受影响。
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| Measuring Homologous Recombination Frequency in Arabidopsis Seedlings
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Author:
Date:
2014-04-05
[Abstract] Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to increase in response to various chemical and physical agents that cause DNA damage and/or alter genome stability (reviewed in March-Díaz and Reyes, 2009). We monitor the frequency of SHR in transgenic Arabidopsis seedlings containing recombination substrates with two truncated but overlapping parts of the β-glucuronidase (GUS) reporter gene (Orel et al., 2003; Schuermann et al., 2005). Upon an SHR event, a functional ...
[摘要] 体细胞同源重组（SHR）是DNA双链断裂（DSB）修复的主要途径，其中完整的同源区域用作去除病变的模板。其在植物中的频率通常较低，因为大多数DSB通过非同源机制在高等真核生物中去除。然而，已显示SHR频率响应于引起DNA损伤和/或改变基因组稳定性的各种化学和物理试剂而增加（综述参见March-Díaz和Reyes，2009）。我们在包含具有β-葡糖醛酸糖苷酶（GUS）报道基因的两个截短但重叠部分的重组底物的转基因拟南芥幼苗中监测SHR的频率（Orel等人，2003 ; Schuermann等人，2005）。在SHR事件后，可以恢复转基因的功能版本（图1A）。适用于整个小植株的组织化学测定允许观察其中恢复报道分子的细胞，因为所编码的酶将无色底物转化为蓝色化合物。这种类型的报道基因已广泛用于鉴定调节植物中SHR水平所需的基因产物。我们通过用DNA损伤剂（bleocin，丝裂霉素C和UV-C）处理分析刺激SHR的植物，并将它们与未处理的植物进行比较。
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