GENOMIC INSTABILITY AND MOLECULAR MECHANISM OF DELAYED MUTAION IN Fl MICE BORN TO IRRADIATED SPERMATOZOA

已知辐射会引起基因组不稳定，其特征是诱导非靶向突变和延迟突变。在这项研究中，由于基因组不稳定的突变是测试F1小鼠出生的辐照父亲。发现小鼠Ms6hm高变小卫星位点母系遗传等位基因的长度变化突变在精子期受辐照的父亲所生的频率增加。同样，在辐射精子出生的Fl小鼠中，观察到母体遗传的粉红色眼睛不稳定等位基因的体细胞逆转频率更高。这些研究表明，受辐照精子引入受精卵的DNA损伤会引发受精卵和随后发育阶段胚胎的基因组不稳定，导致小卫星位点母系等位基因的非靶向突变，以及发育中的眼睛视网膜色素上皮细胞的延迟突变。上述研究表明，受损的父本基因组与未受损的母本基因组之间存在基因组串扰。为了研究这种基因组串扰的分子性质，我们研究了受辐照精子受精的小鼠受精卵的辐射反应。p53应答性报告基因在雌性原核中被有效激活。[3H]-Tdr标记实验表明，精子辐照受精卵不存在G1/S阻滞，但雄性和雌性原核DNA合成均受到抑制。P53 -/-合子缺乏这种抑制，通过微量注射GST-p53融合蛋白来纠正。6 Gy辐照受精卵中约有一半通过延长S期成功合成了完整的DNA，而另一半则没有。无论DNA含量如何，所有受精卵都分裂成2细胞期胚胎。这些结果揭示了p53依赖性原核串扰的存在以及p53在小鼠受精卵S期dna损伤检查点中的新功能。

Radiation is known to induce genomic instability Which is characterized by the induction of untargeted mutation and delayed mutation. In this investigation, mutation due to genomic instability was tested in F1 mice born to irradiated father. Length change mutation at the maternally inherited allele of the mouse Ms6hm hypervariable minisatellite locus was found to increase in frequencies when born to fathers irradiated at the spermatozoa stage. Similarly, a higher frequency of somatic reversion at the maternally inherited pink-eyed unstable allele was observed in Fl mice born to irradiated spermatozoa. These demonstrated that DNA damage introduced into zygote by irradiated sperm triggers genomic instability in zygotes and in embryos of subsequent developmental stages, resulting in induction of untargeted mutation at the maternal allele of the minisatellite locus, and delayed mutation in retinal pigment epithelial cells in developing eyes.The above study indicates that there exists a genomic crosstalk between damaged paternal genome and undamaged maternal genome. In order to investigate the molecular nature of this genomic crosstalk, we have studied radioresponses in mouse zygotes fertilized by irradiated sperm. A p53 responsive reporter was efficiently activated in female pronucleus. [3H]-Tdr labelingexperiments indicated that sperm irradiated zygotes was devoid of G1/S arrest, but pronuclear DNA synthesis was suppressed equally in male and female pronuclei. P53 -/- zygotes lacked this suppression which was corrected by microinjection of GST-p53 fusion protein. About a half of 6 Gy sperm irradiated zygotes managed to synthesize a full DNA content by prolonging S phase while the other half failed to do so. Regardless of the DNA content, all the zygotes cleaved to become 2 cell stage embryos. These results revealed the presence of p53 dependent pronuclear crosstalk and a novel function of p53 in S phase DNA-damage checkpoint of mouse zygotes.