ISOLATION AND STRUCTURE OF A CDNA EXPRESSING A MAMMALIAN 3-METHYLADENINE-DNA GLYCOSYLASE
ISOLATION AND STRUCTURE OF A CDNA EXPRESSING A MAMMALIAN 3-METHYLADENINE-DNA GLYCOSYLASE
复制标题
DOI：
10.1002/j.1460-2075.1990.tb07534.x
复制
发表时间：
1990-10-01
期刊：
影响因子：
11.4
通讯作者：
LAVAL, F
中科院分区：
文献类型：
--
作者：
OCONNOR, TR;LAVAL, F
利用大鼠肝癌细胞系H4细胞的poly(A)+ mRNA构建cDNA质粒表达文库。该文库被引入DNA中3-甲基腺嘌呤（3-meAde）残基修复缺陷的大肠杆菌BH290菌株中。这种DNA修复缺陷使得染色对烷基化剂的处理具有表型敏感性。筛选甲基甲烷磺酸存活的cDNA文库。携带来自存活细胞的质粒pAPDG10（烷基化n -嘌呤- dna糖基化酶）的BH290细胞对MMS的敏感性与野生型菌株相当。含有pAPDG10质粒的BH290细胞粗提物释放出3-meAde和7-甲基鸟嘌呤残基，这些残基来自于[甲基- 3h)二甲基硫酸盐甲基化的DNA。插入到pAPDG10中的993 bp的cDNA序列有一个长度大于85个氨基酸的开放阅读框。衍生的253个氨基酸的APDG蛋白序列和由alkA基因编码的大肠杆菌3-meAde-DNA糖基酶II均有保守序列区域。利用Southern杂交对基因组DNA进行分析表明，APDG基因的最小大小为6.5- 12kb。Northern blot分析显示，H4细胞中产生的转录本也存在于正常大鼠肝细胞中。
A cDNA plasmid expression library was constructed from the poly(A)+ mRNA of H4 cells, a rat hepatoma cell line. The library was introduced into Escherichia coli strain BH290 deficient in the repair of 3-methyladenine (3-meAde) residues in DNA. This DNA repair deficiency renders the stain phenotypically sensitive to treatment with alkylating agents. The cDNA library was screened for survivors to methylmethane sulfonate. BH290 cells hosting one of the plasmids, pAPDG10 (Alkylated N-Purine-DNA Glycosylase), from surviving cells had a sensitivity to MMS equivalent to that of the wild type strain. Crude extracts of BH290 cells harboring the pAPDG10 plasmid released 3-meAde and 7-methylguanine residues from DNA methylated with [methyl-3H)dimethylsulfate. The cDNA sequence of 993 bp inserted in pAPDG10 has a single open reading frame greater than 85 amino acids in length. The derived APDG protein sequence of 253 amino acids and the 3-meAde-DNA glycosylase II of E. coli coded for by the alkA gene have regions of conserved sequences. Analysis of the genomic DNA using Southern hybridization suggests that the APDG gene has a minimal size of 6.5-12 kb. Northern blot analysis shows that the transcript produced in H4 cells is also present in normal rat liver cells.