Wu M J, Chow L W, Hsieh M
Department of Food Health, Chia-Nan College of Pharmacy and Science, Tainan 717, Taiwan.
Biochim Biophys Acta. 1998 Aug 14;1407(2):155-62. doi: 10.1016/s0925-4439(98)00033-7.
Several human hereditary neuromuscular and neurodegenerative diseases are caused by abnormal expansion of triplet repeat sequences (TRSs) CAG/CTG, CGG/CCG, or GAA/TTC on certain chromosomes. It is generally accepted that multiple slippage synthesis accounts for the instabilities of TRS. Earlier in vitro experiments by Behn-Krappa and Doerfler showed that TRS with high GC content can be expanded. In contrast, here we demonstrated that certain AT-rich TRSs, (TTC)17, (GAA)10/(TTC)10 and (GAA)17/(TTC)17, were also expansion-prone in PCR. With respect to the sequence of TRS, surprisingly, we found that the AT-rich (GAA)17/(TTC)17 extended more efficiently than the GC-rich (CAG)17/(CTG)17. This strongly suggested that the AT content of the repeat may influence TRS expansion. Furthermore, to examine the expansion of single-stranded TRS, we showed that only (TTC)17, but not the complementary (GAA)17, can be expanded. This suggested that a T-T mismatch may stabilize compatible secondary structures, most likely hairpins, for slippage synthesis. However, another poly-pyrimidine TRS, (CCT)17, is not amplification-prone in PCR. Due to the high C-content, this TRS is unlikely to adopt hairpin structures at the high pH used for PCR. Thus, the single-stranded PCR experiment may serve as an indirect assay for the ability of a sequence to adopt a hairpin conformation. When amplification was performed in reactions using Klenow DNA polymerase, only the double-stranded TRSs can be expanded. The reaction rate for (GAA)10/(TTC)10 was slower than for (GAA)17/(TTC)17, suggesting that the length of the repeat may be important for the amplification of TRS. The findings of these in vitro experiments may aid in understanding TRS expansion in vivo.
几种人类遗传性神经肌肉和神经退行性疾病是由某些染色体上的三联体重复序列（TRS）CAG/CTG、CGG/CCG或GAA/TTC异常扩增引起的。人们普遍认为，多次滑动合成导致了TRS的不稳定性。Behn-Krappa和Doerfler早期的体外实验表明，高GC含量的TRS可以扩增。相比之下，我们在此证明，某些富含AT的TRS，（TTC）17、（GAA）10/（TTC）10和（GAA）17/（TTC）17，在PCR中也易于扩增。关于TRS的序列，令人惊讶的是，我们发现富含AT的（GAA）17/（TTC）17比富含GC的（CAG）17/（CTG）17扩增效率更高。这强烈表明重复序列的AT含量可能影响TRS的扩增。此外，为了检测单链TRS的扩增，我们表明只有（TTC）17可以扩增，而互补链（GAA）17则不能。这表明T-T错配可能稳定了适合滑动合成的二级结构，很可能是发夹结构。然而，另一个聚嘧啶TRS，（CCT）17，在PCR中不易扩增。由于C含量高，该TRS在用于PCR的高pH条件下不太可能形成发夹结构。因此，单链PCR实验可作为序列形成发夹构象能力的间接检测方法。当使用Klenow DNA聚合酶在反应中进行扩增时，只有双链TRS可以扩增。（GAA）10/（TTC）10的反应速率比（GAA）17/（TTC）17慢，这表明重复序列的长度可能对TRS的扩增很重要。这些体外实验的结果可能有助于理解体内TRS的扩增。
