Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae
Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae
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DOI：
10.1073/pnas.95.21.12438
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发表时间：
1998-10-13
影响因子：
11.1
通讯作者：
Lahue, RS
中科院分区：
文献类型：
--
作者：
Miret, JJ;Pessoa-Brandao, L;Lahue, RS
建立了一种定量和选择性的遗传分析方法来监测酵母中三核苷酸重复序列（TNR）的扩增。含有25个重复的启动子允许URA 3报告基因的表达，并产生对药物5-氟乳清酸的敏感性。将TNR扩增至30个或更多个重复会关闭URA 3并提供耐药性。当在两个染色体位点中的任一个上整合时，如果CTG重复在落后的子链上复制，则扩增速率为每代1 × 10（-5）至4 × 10（-5）。PCR分析表明，在95%的扩增等位基因中存在5-28个额外重复。在错配修复基因MSH 2或重组基因RAD 52缺陷的菌株中，CTG扩增率没有显著变化。CTG扩增的频繁性质表明，在该系统中，该重复的阈值数低于25。相比之下，互补重复CAG的扩增发生率低500至1,000倍，与随机（C，A，G）对照序列相似。当报告质粒在染色体内反转，切换复制的前导链和滞后链时，仅当CTG重复位于滞后子链上时才观察到频繁的扩增。在罕见的CAG扩增中，束大小的最大增益是38个重复。对照组重复CTA和TAG均未检测到扩增率。的方向依赖性和序列特异性数据支持的模型，CTG和CAG道的扩张导致通过发夹包含冈崎片段的DNA复制异常。
A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allow's expression of a URA3 reporter gene and yields sensitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 X 10(-5) to 4 X 10(-5) per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG shelved no detectable rate of expansions. The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.