权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular analysis of induced mutation in radioadaptive response by low dose of radiation in mouse cells

小鼠细胞低剂量辐射诱导的放射适应性反应突变的分子分析

基本信息

批准号：
07680577
负责人：
TACHIBANA Akira
金额：
$ 1.47万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680577/
关键词：
radioadaptive response radiation-induced mutation DNA double strand break repair mouse cells mouse Hprt gene deletion mutation HPRT遺伝子

项目摘要

The DNA double strand break (dsb) has been implicated as the critical lesion induced by ionizing radiation leading to chromosome aberrations, mutations and cell death. We have attempted to refine the measurement and analysis of the rejoining of dsbs with the use of nuclear extracts applied to defined plasmid molecules carrying specific enzymatically-induced dsb. The plasmid pZEr0-2 containing the ccdB gene under the control of the lac gene promotor was used as a substrate. The ccdB gene is lethal to E.coli on being overexpressed, which makes it easy to select plasmids with the mutated ccdB gene caused by mis-rejoining. We have compared the activities of extracts from an ataxia-telangiectasia cell line (AT2KYSV) with those from a normal cell line (N2KYSV).The extract from AT2KYSV cells showed much higher frequency of mis-rejoining than the N2KYSV extract. Sequence analysis of the mutant plasmids revealed deletion mutations which occurred mostly between short direct repeats (3-6 basepairs). This feature resembles breakpoint junctions in deletion mutations, indicating that this in vitro system reflects the deletion formation mechanism in vivo. We are currently investigating the radioadaptive response using this in vitro system.To analyze mutations at the mouse Hprt locus in radioadaptive response, we first tried to determine the sequence of the gene. We have determined the sequences of exons 2,3,6,7,8, and 9 and the surrounding sequences of each exon, but not yet for exons 4 and 5.

DNA双链断裂（dsb）被认为是电离辐射引起染色体畸变、突变和细胞死亡的关键性损伤。我们试图通过将核提取物应用于携带特异性酶诱导dsb的确定质粒分子来改进dsb重连接的测量和分析。将含有在lac基因启动子控制下的ccdB基因的质粒pZEr 0 -2用作底物。ccdB基因在过表达时对大肠杆菌是致命的，这使得容易选择具有由错误重新连接引起的突变的ccdB基因的质粒。我们比较了共济失调-毛细血管扩张症细胞系（AT 2KYSV）和正常细胞系（N2 KYSV）的提取物的活性，AT 2KYSV细胞提取物显示出比N2 KYSV提取物高得多的错接频率。突变质粒的序列分析显示缺失突变主要发生在短的直接重复序列（3-6个碱基对）之间。该特征类似于缺失突变中的断点连接，表明该体外系统反映了体内缺失形成机制。我们目前正在使用这种体外系统研究辐射适应性反应。为了分析辐射适应性反应中小鼠Hprt基因座的突变，我们首先试图确定该基因的序列。我们已经确定了外显子2、3、6、7、8和9的序列以及每个外显子的周围序列，但尚未确定外显子4和5的序列。