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

受辐射精子所生 Fl 小鼠基因组不稳定性和延迟突变的分子机制

基本信息

批准号：
12480156
负责人：
NIWA Ohtsura
金额：
$ 10.37万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480156/
关键词：
radiation genomic instability untargeted mutation delayed mutation pink-eyed unstable allele minisatellite genomic crosstalk p53 dependent S checkpoint マウス初期胚 DNA合成抑制色素上皮細胞 pink-eyed unstabel allele p53-遺伝子

项目摘要

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.

辐射可诱导基因组不稳定性，其特征是诱导非靶向突变和延迟突变。在这项调查中，由于基因组不稳定性的突变进行了测试，在F1小鼠出生的辐射父亲。长度变化突变在母系遗传的小鼠Ms 6 hm高变小卫星位点的等位基因被发现增加的频率时，出生的父亲照射在精子阶段。同样，在辐照精子出生的F1小鼠中观察到母系遗传的粉红色眼睛不稳定等位基因的体细胞回复频率较高。这些结果表明，辐射精子对受精卵的DNA损伤，可引起受精卵及后续发育阶段胚胎的基因组不稳定，导致小卫星位点的母系等位基因发生非靶向突变，发育期眼的视网膜色素上皮细胞发生延迟突变，说明受损的父系基因组与未受损的母系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本质，我们研究了辐射精子受精的小鼠受精卵的辐射反应。在雌性原核中，p53应答报告基因被有效激活。[~ 3 H]-Tdr标记实验表明，精子辐照后的受精卵没有出现G1/S期阻滞，但雌雄原核DNA合成均受到抑制。P53 -/-合子缺乏这种抑制，这通过显微注射GST-p53融合蛋白来纠正。6戈伊照射的受精卵中，约有一半的精子能延长S期合成完整的DNA，而另一半则不能。无论DNA含量如何，所有的受精卵都裂解成2细胞期胚胎。这些结果揭示了p53依赖的原核串扰的存在和p53在小鼠受精卵S期DNA损伤检查点中的新功能。