Mechanism for control of mutagenesis in mammals

哺乳动物诱变控制机制

• 批准号: 09440255
• 负责人: SEKIGUCHI Mutsuo
• 金额: $ 8.96万
• 依托单位: Fukuoka Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09440255/
• 关键词: Reactive oxygen; spontaneous mutation; mutation rate; 8-oxoguanine; DNA precursor; DNA replication; replication error; MTH1 gene; 遺伝子障害; 酸化塩基; 突然変異; 変異タンパク; DNA修復; 遺伝子; 塩基置換

Reactive oxygen species produced during normal cellular matabolism damage DNA and its precursors. An oxidized form of guanine base, 8-oxoguanine, is regarded as most critical in terms of mutagenesis as well as carcinogenesis. An enzyme 8-oxo-dGTPase hydrolyzes 8-oxo-dGTP, an oxidized form of dGTP, to 8-oxo-dGMP, thereby preventing the occurrence of A : T to C : G transversion, caused by rnisincorporation of 8-oxo-dGTP into DNA.To elucidate the role of 8-oxo-dGTPase in carcinogenesis, it is necessary to construct cell or mouse lines with altered levels of the enzyme activity. We isolated the genomic sequence for mouse 8-oxo-dGTPase protein, identified the exon / intron region of the gene MTH1, and characterized the promoter in relation to the regulation of expression of the gene. Based on these data, we performed an gene targeting experiment using ES cells. The ES cells defective in both alleles of the MTH1 gene are viable but yield a moderately higher frequency of spontaneous mutation. By sing MTH1-defective ES cells, we constructed mouse lines defective in both alleles of the MTH1 gene. The mice are viable and apparently normal in appearance but produced a significantly large number of tumores in their organs, especially in stomach.The mouse MTH1 gene has 5 exons, among which the coding sequence resides on the third through the fifth exon, and spans approximately 9 kb. Part of the human and rat genes for 8-oxo-dGTPase has been isolated, and the human gene is composed of five exons while the rat gene has three exons. A comparison of the gross structures of the genes revealed that the overall structures of the human and rat genes are similar to that of the mouse gene.

正常细胞基质中产生的活性氧损伤DNA及其前体。鸟嘌呤碱基的氧化形式，8-氧代鸟嘌呤，被认为是诱变和致癌方面最关键的。一种酶8-oxo-dGTP水解8-oxo-dGTP（dGTP的一种氧化形式）为8-oxo-dGMP，从而防止由8-oxo-dGTP掺入DNA引起的A：T到C：G颠换的发生。我们分离了小鼠8-oxo-dGTdR蛋白的基因组序列，鉴定了基因MTH 1的外显子/内含子区域，并表征了与基因表达调控相关的启动子。基于这些数据，我们使用ES细胞进行了基因靶向实验。MTH 1基因的两个等位基因都有缺陷的ES细胞是可行的，但产生适度较高的自发突变频率。通过单一的MTH 1缺陷的ES细胞，我们构建了MTH 1基因的两个等位基因都有缺陷的小鼠品系。这些小鼠可以存活，外表明显正常，但在其器官中产生了大量肿瘤，尤其是在胃中。小鼠MTH 1基因有5个外显子，其中编码序列位于第三至第五个外显子上，跨度约为9 kb。已分离出人和大鼠的部分8-氧代-dGT β基因，人基因由5个外显子组成，而大鼠基因有3个外显子。对基因总体结构的比较显示，人类和大鼠基因的总体结构与小鼠基因的结构相似。

项目成果

Y.Tominaga: "Alkylation-indued apoptosis of embryonic stem cells in which the gene for DNA-repair methy 1 transferase been disrupted by gene targeting." Carcinogenesis. 18. 889-896 (1997)

Y.Tominaga：“烷基化诱导的胚胎干细胞凋亡，其中 DNA 修复甲基 1 转移酶基因被基因靶向破坏。”

P.J.Cai: "Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity." Nucl.Acids Res.25. 1170-1176 (1997)

P.J.Cai：“具有抗突变活性的人类 MTH1 蛋白的保守氨基酸序列的意义。”

D.W.Porter et al.: "Sensitivity of E.coli(Mut T)and human(MTH1)8-oxo-dGTPase to in vitro inhibition by carcinogenic metals" Carcinogenesis. 18. 1785-1791 (1997)

D.W.Porter 等人：“大肠杆菌 (Mut T) 和人 (MTH1)8-oxo-dGTPase 对致癌金属体外抑制的敏感性”致癌作用。

H.Igarashi: "Organization and expression of the mouse MTH1 gene for preventing transversion mutation." J.Biol.Chem.272. 3766-3772 (1997)

H.Igarashi：“用于预防颠换突变的小鼠 MTH1 基因的组织和表达。”

T.Iwakuma: "High incidence of nitrosamine-induced tumorigenesis in mice lacking DNA repair methyltransferase." Carcinogenesis. 180. 1631-1635 (1997)

T.Iwakuma：“缺乏 DNA 修复甲基转移酶的小鼠中亚硝胺诱导的肿瘤发生率很高。”