Yauk Carole L, Dubrova Yuri E, Grant Gemma R, Jeffreys Alec J
Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
Mutat Res. 2002 Mar 20;500(1-2):147-56. doi: 10.1016/s0027-5107(02)00005-2.
Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.
扩展简单串联重复序列（ESTR）位点包含小鼠基因组中一些最不稳定的DNA，并已广泛应用于种系突变的谱系研究。我们现在表明，小鼠ESTR位点Ms6-hm处的重复DNA不稳定性也可以通过对基因组DNA进行单分子PCR分析来监测。与不稳定的人类微卫星不同，后者几乎仅通过基于减数分裂重组的过程在种系中发生突变，而小鼠Ms6-hm在生殖（精子）DNA和体细胞（脾脏、大脑）DNA中均表现出重复序列不稳定性。同一近交系小鼠之间的突变频率没有显著差异。然而，不同组织之间存在显著差异，小鼠精子中的突变频率最高。体细胞和精子突变体的大小谱无法区分，且严重偏向于仅几个重复单元的增减，这表明通过在体细胞和种系中均起作用的有丝分裂复制滑移过程实现了重复序列更新。对急性减数分裂前暴露于X射线后的雄性小鼠进行分析发现，精子中的突变频率显著增加，但体细胞中的突变频率没有增加，尽管突变体的大小谱没有变化。Ms6-hm处辐射诱导的突变水平与父系照射后通过传统谱系分析确定的水平无法区分。这证实了小鼠ESTR位点对电离辐射非常敏感，并确定诱导的种系突变是由精子中存在的辐射诱导突变等位基因引起的，而不是由未修复的精子DNA损伤随后导致早期胚胎中出现突变体引起的。这种单分子监测系统有可能大幅减少种系突变监测所需的小鼠数量，并且不仅可用于研究种系突变，还可用于研究体内和细胞培养中的体细胞突变。
