Mechanistic features of CAG*CTG repeat contractions in cultured cells revealed by a novel genetic assay.
Mechanistic features of CAG*CTG repeat contractions in cultured cells revealed by a novel genetic assay.
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DOI：
10.1093/nar/gki880
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发表时间：
2005
影响因子：
14.9
通讯作者：
Lahue RS
中科院分区：
文献类型：
--
作者：
Pelletier R;Farrell BT;Miret JJ;Lahue RS
三核苷酸重复序列（tnr）经历高频突变导致至少15种神经退行性疾病。为了更好地了解培养细胞中TNR不稳定性的分子机制，利用穿梭载体建立了一种新的遗传检测方法。穿梭载体包含一个启动子-TNR报告基因结构，其表达依赖于TNR长度。该载体携带SV40基因和大T抗原基因，允许在灵长类细胞系之间携带。穿梭载体在培养细胞中繁殖，然后在酵母中回收和分析，使用选择报告基因表达。我们发现（CAG•CTG）25−33在293T和293人类细胞以及COS-1猴细胞中以高达1%的频率收缩，前提是质粒进行复制。发夹形成能力的重复序列刺激收缩。在COS-1细胞中，在25到33次重复之间观察到一个阈值的证据，在狭窄的重复长度范围内，收缩频率急剧增加（增加720%）。错配修复蛋白Mlh1的表达与重复不稳定性无关，这表明在我们的系统中，收缩与错配修复无关。总之，这些发现概括了人类遗传学的某些特征，因此建立了一种新的细胞培养系统，有助于为CAG•CTG重复不稳定性提供新的机制见解。
Trinucleotide repeats (TNRs) undergo high frequency mutagenesis to cause at least 15 neurodegenerative diseases. To understand better the molecular mechanisms of TNR instability in cultured cells, a new genetic assay was created using a shuttle vector. The shuttle vector contains a promoter-TNR-reporter gene construct whose expression is dependent on TNR length. The vector harbors the SV40 ori and large T antigen gene, allowing portability between primate cell lines. The shuttle vector is propagated in cultured cells, then recovered and analyzed in yeast using selection for reporter gene expression. We show that (CAG•CTG)25−33 contracts at frequencies as high as 1% in 293T and 293 human cells and in COS-1 monkey cells, provided that the plasmid undergoes replication. Hairpin-forming capacity of the repeat sequence stimulated contractions. Evidence for a threshold was observed between 25 and 33 repeats in COS-1 cells, where contraction frequencies increased sharply (up 720%) over a narrow range of repeat lengths. Expression of the mismatch repair protein Mlh1 does not correlate with repeat instability, suggesting contractions are independent of mismatch repair in our system. Together, these findings recapitulate certain features of human genetics and therefore establish a novel cell culture system to help provide new mechanistic insights into CAG•CTG repeat instability.