Kawate H, Ihara K, Kohda K, Sakumi K, Sekiguchi M
Department of Biochemistry, Kyushu University, Fukuoka, Japan.
Carcinogenesis. 1995 Jul;16(7):1595-602. doi: 10.1093/carcin/16.7.1595.
cDNA for mouse O6-methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state. Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells. A single cell of NIH3T3 contained 1.8 x 10(4) molecules of the methyltransferase protein. When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus. Using double-stranded oligomers containing a single O6-methylguanine or O4-methylthymine at predetermined sites, the mouse enzyme repaired O6-methylguanine and O4-methylthymine, at an almost equal efficiency. In the LacZ reversion assay, MNNG-induced A:T to G:C as well as G:C to A:T transition mutations were efficiently suppressed by the function of mouse methyltransferase, in vivo.
小鼠O6-甲基鸟嘌呤-DNA甲基转移酶的cDNA在缺乏甲基转移酶的大肠杆菌突变细胞中表达，过量产生的小鼠酶被纯化至同质状态。使用该纯化产物制备多克隆抗体，并用于估计细胞中甲基转移酶蛋白的量。一个NIH3T3细胞含有1.8×10⁴个甲基转移酶蛋白分子。当对小鼠成纤维细胞进行免疫染色时，结果显示大部分甲基转移酶蛋白存在于细胞质而非细胞核中。使用在预定位点含有单个O6-甲基鸟嘌呤或O4-甲基胸腺嘧啶的双链寡聚物，小鼠酶以几乎相等的效率修复O6-甲基鸟嘌呤和O4-甲基胸腺嘧啶。在LacZ回复试验中，MNNG诱导的A:T到G:C以及G:C到A:T的转换突变在体内被小鼠甲基转移酶的功能有效抑制。
