Selection Mechanisms for Mutants As Studied by the Medaka Specific-Locus Test System.

通过青鳉特异性基因座测试系统研究的突变体选择机制。

• 批准号: 01440093
• 负责人: SHIMA Akihiro
• 金额: $ 11.39万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01440093/
• 关键词: Tester Medaka; Specific-locus mutation; Radiation; Developmental process; Selection; Dominant lethals; Malformation

1. The total cumulative number of the Medaka embryos examined at the end of this project reached 488, 890. This number of embryos corresponds to a total of 1, 198, 2-79 loci.2. The wild type male Medaka were exposed to gamma-rays, fission neutron or ethylnitrosourea. These treated males were allowed to mate with non-treated female tester Medaka which are homozygous recessive at the 3 loci, i. e. b/b ; gu/gu ; lf/lf.3. The gamma-irradiations were done using a 111-TBq ^<137>Cs gamma source located in the Research Center for Nuclear Science and Engineering, The University of Tokyo. The dose-rate used was 0.95 Gy/min.4. The irradiations with fission neutron were performed in the UTR-Kinki reactor (Atomic Energy Research Institute, Kinki University, in collaboration with Prof. S. Kondo and Dr. T. Ito) operated at 1 Watt with the following conditions : dDn/dt=16.9 cGy/hr, dDg/dt=24.8 cGy/hr.5. The treatments of the Medaka with ethylnitrosourea (ENU) were done by allowing the male Medaka to s … More wim in 1 mM aqueous solution of ENU (pH=5.8) at 27ﾟC for 2 hours, following by extensive rinse with tap water.6. The spontaneous total mutation 5 rate was 4.2x10^<-5>/locus, while the spontaneous viable mutation rate was 5.5x10^<-6>/locus, a value vdry close to that of the mouse (8.1x10^<-6>/locus, Ressell & Kelly (1982)).7. The induced rates of viable mutation per unit dose (rad) of gamma-rays for the Medaka sperm, spegmatids and spermatogonia were found to be respectively 176, 51 and 7.5 (x10^<-8>/locus).8. Based on the above-obtained values, the doubling gamma-ray dose for the induction of viable mutation was estimated to be 3.2, 12 and 98 rad (cGy) for sperm, spermatids and spermatogonia, respectively.9. Wg have established a method to obtain cell lines from individual Medaka embryos with malformation of various severities which is expected to result in dominant lethal mutation. Surprisingly, cell lines from moribund malformed : embryos tended to grow over a year more actively than those from the normal' controls without malformation.10. We also established an AP-PCR method to examine induced DNA damages in male getm cells, which is expected to give more direct clue to study the mechanisms of embryonic selection during development. Less

1. 在该项目结束时，Medaka胚胎的总累积数量达到488,890。这个数目的胚胎对应于1,198,2-79个位点。野生型雄鳉鱼分别暴露于γ射线、裂变中子和乙基亚硝基脲中。这些处理过的雄虫与未处理的雌虫交配，雌虫在3个位点（即b/b）上是纯合隐性的；顾/顾;低频/ lf.3。伽玛辐射是使用位于东京大学核科学与工程研究中心的111-TBq ^<137>Cs伽玛源进行的。使用的剂量率为0.95 Gy/min。裂变中子辐照在UTR-Kinki反应堆（近畿大学原子能研究所，与S. Kondo教授和T. Ito博士合作）中进行，操作条件为1瓦：dDn/dt=16.9 cGy/hr, dDg/dt=24.8 cGy/hr.5。5.采用乙基亚硝基脲（ENU）处理青鳉，将雄鳉在1 mM的ENU水溶液（pH=5.8）中浸泡2小时，温度为27ºC，然后用自来水冲洗。自发总突变率为4.2x10^<-5>/位点，而自发活突变率为5.5x10^<-6>/位点，与小鼠的数值（8.1x10^<-6>/位点，Ressell & Kelly(1982)）非常接近。单位剂量γ射线对Medaka精子、精母细胞和精原细胞的诱导活突变率分别为176、51和7.5 （x10^<-8>/位点）。根据上述数值，对精子、精细胞和精原细胞诱导活突变的双倍伽马射线剂量估计分别为3.2、12和98拉德（cGy）。我们已经建立了一种方法，从具有不同严重畸形的个体Medaka胚胎中获得细胞系，这些畸形有望导致显性致死突变。令人惊讶的是，来自垂死的畸形胚胎的细胞系往往比那些来自没有畸形的正常对照的细胞系生长得更活跃。我们还建立了一种AP-PCR方法来检测雄性生殖细胞诱导的DNA损伤，这有望为研究胚胎发育过程中的胚胎选择机制提供更直接的线索。少

项目成果

Shimada, A. and A. Shima.: "Some characteristics of radiation- or ENU-induced mutations at the b locus of the Medaka, Oryzias latipes." Zool. Sci.7. 1053 (1990)

Shimada, A. 和 A. Shima.：“辐射或 ENU 诱导的青鳉 (Oryzias latipes) b 基因座突变的一些特征。”

Shima,A.: "Induction of mutations in males the fish Oryzias latipes at a specific locus after gamma-irradiation." Mutation Res.198. 93-98 (1988)

Shima,A.：“伽马射线照射后，在雄性 Oryzias latipes 鱼的特定位点诱导突变。”

Shima,A.: "Development of a possible nonmammalian test system for radiation-induced germ-cell mutagenesis using a fish, the Japanese Medaka (Oryzias latipes)" Proc.Natl.Acad.Sci.,U.S.A.88. 2545-2549 (1991)

Shima,A.：“使用日本青鳉鱼（Oryzias latipes）开发一种可能的非哺乳动物测试系统，用于辐射诱导的生殖细胞突变”Proc.Natl.Acad.Sci.，U.S.A.88。

Kubota,Y.: "Anattempt to detect gammma-ray-induced DNA damages of matant embryos of the Medaka,Oryzias latipes." J.Radiat.Res.32. 82 (1991)

Kubota,Y.：“尝试检测伽马射线引起的青鳉、Oryzias latipes 休眠胚胎的 DNA 损伤。”

Komura,J: "Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells." Mutation Res.254. 191-198 (1991)

Komura,J：“培养金鱼细胞 c-ras 序列中嘧啶二聚体的优先切除修复和非优先光再激活。”