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.本项目结束时累计检查的Medaka胚胎总数达到488,890个。这个胚胎的数量相当于总共1,198个，2-79个。野生型雄性青竹受到伽马射线、裂变中子或乙基亚硝脲的照射。这些经过处理的雄性被允许与未处理的雌性测试者Medaka交配，这些雌性测试者在b/b、gu/gu和lf/lf.3个座位上是纯合子隐性的。伽马辐射是使用东京大学核科学与工程研究中心的111-Tbq^&lt；137&gt；Cs伽马源进行的。所用剂量率为0.95Gymin。裂变中子辐照是在UTR-KINKI反应堆(近藤大学原子能研究所，与近藤教授和T.Ito博士合作)中进行的，功率为1瓦，条件如下：DDN/dt=16.9cGy/hr，DDG/dt=24.8cGy/hr.5。用乙基亚硝脲(Enu)对青竹的治疗是通过让男性青竹给S…来完成的将更多的WIM放在1 mm的ENU水溶液中(pH=5.8)，在27ﾟC下放置2小时，然后用自来水大量冲洗。自发总突变率为4.2×10~(-5)/个基因座，自发存活突变率为5.5×10~(-6)个/基因座，与小鼠(8.1x10^；~(-6))/基因座(Ressell&Kelly(1982))相近。单位剂量伽马射线对青竹精子、精原细胞和精原细胞的诱变率分别为176、51和7.5(x10^~(-8))。根据上述数值，估算了精子、精子细胞和精原细胞诱变活性的伽马射线倍增剂量分别为3.2、12和98rad(CGy)。工作组已经建立了一种方法，从具有不同严重程度的畸形的单个Medaka胚胎中获得细胞系，预计这将导致显性致命突变。令人惊讶的是，来自垂死畸形的细胞系：胚胎往往比那些没有畸形的正常对照的胚胎在一年内生长得更活跃。我们还建立了AP-PCR检测雄性Getm细胞DNA损伤的方法，以期为研究胚胎发育过程中的选择机制提供更直接的线索。较少

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。”