Miret J J, Pessoa-Brandão L, Lahue R S
The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12438-43. doi: 10.1073/pnas.95.21.12438.
A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows 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 showed 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.
开发了一种定量且具有选择性的遗传检测方法，用于监测酵母中三核苷酸重复序列（TNRs）的扩增情况。一个含有25个重复序列的启动子可使URA3报告基因表达，并产生对药物5-氟乳清酸的敏感性。TNR扩增至30个或更多重复序列会关闭URA3并赋予耐药性。当整合到两个染色体位点中的任何一个时，如果CTG重复序列在滞后子链上复制，每代的扩增率为1×10⁻⁵至4×10⁻⁵。PCR分析表明，95%的扩增等位基因中存在5 - 28个额外的重复序列。在错配修复基因MSH2或重组基因RAD52缺陷的菌株中，CTG扩增率没有显著变化。CTG扩增的频繁发生表明，在该系统中，这个重复序列的阈值数量低于25。相比之下，互补重复序列CAG的扩增率要低500至1000倍，类似于随机的（C、A、G）对照序列。当报告质粒在染色体内倒置，从而切换复制的前导链和滞后链时，只有当CTG重复序列位于滞后子链上时才会观察到频繁的扩增。在罕见的CAG扩增中，序列长度的最大增加为38个重复序列。对照重复序列CTA和TAG没有检测到扩增率。方向依赖性和序列特异性数据支持这样的模型，即CTG和CAG序列的扩增是由通过含发夹结构的冈崎片段的异常DNA复制导致的。
