Tano K, Shiota S, Remack J S, Brent T P, Bigner D D, Mitra S
University of Tennessee-Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory 37831.
Mutat Res. 1991 Sep;255(2):175-82. doi: 10.1016/0921-8777(91)90051-p.
Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.
几个实验室已证明，用人类DNA转染中国仓鼠卵巢（CHO）细胞可产生稳定表达DNA修复蛋白O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的克隆，而亲本细胞系中缺乏该蛋白（Mex-表型）。我们使用人类MGMT cDNA和抗人类MGMT抗体作为探针，研究了许多此类MGMT阳性（Mex+）克隆中MGMT的遗传起源。五个独立分离的Mex+细胞系均无人MGMT基因序列。免疫印迹分析证实这些细胞提取物中不存在人类蛋白。在表达低水平MGMT（0.6 - 1.4×10⁴分子/细胞）的细胞系中，MGMT mRNA的大小（1.1 kb）与仓鼠肝脏中的相同。一个细胞系GC-1，其MGMT水平高得多（4×10⁴分子/细胞），有两种MGMT mRNA，主要的一种为1.3 kb，次要的一种为1.8 kb。它还有两种MGMT多肽（32 kDa和28 kDa），两者都比仓鼠肝脏和其他Mex+转染细胞中存在的25 kDa MGMT大。这些结果表明，所有Mex+ CHO细胞克隆中的MGMT均由内源基因编码。虽然不能排除Mex+细胞克隆中MGMT基因的自发激活，但人类DNA序列的干预可能是GC-1细胞系中内源基因激活的原因。
