Camakaris J, Petris M J, Bailey L, Shen P, Lockhart P, Glover T W, Barcroft C, Patton J, Mercer J F
Department of Genetics, University of Melbourne, Parkville, Victoria, Australia.
Hum Mol Genet. 1995 Nov;4(11):2117-23. doi: 10.1093/hmg/4.11.2117.
Three copper-resistant variants of cultured Chinese hamster ovary (CHO) cells were isolated and each was shown to accumulate less intracellular copper than the parental cells when grown in copper-supplemented media. The reduced copper accumulation was related to enhanced copper efflux. As cultured cells from patients with Menkes disease (mutations in MNK; ATP7A gene) accumulate copper, probably due to defective copper efflux, we investigated the possible role of the MNK gene in the molecular basis of copper resistance. We found increased MNK mRNA and MNK protein in all three resistant variants. The MNK protein, which has not been previously demonstrated experimentally in mammalian cells, was observed to have an apparent molecular weight of 178 kDa on SDS gels. The degree of increase in MNK mRNA and protein correlated well with the level of copper resistance and extent of copper efflux. By Southern blot and FISH analysis we determined that the molecular basis for overexpression of MNK was genomic amplification of the MNK gene. These data, combined with the clinical and cellular phenotype in Menkes disease, provide strong evidence that the MNK protein is involved in transmembrane copper efflux, and demonstrate a new system of gene amplification in mammalian cells.
我们分离出了三种对铜具有抗性的中国仓鼠卵巢（CHO）细胞培养变体，并且当在添加了铜的培养基中生长时，相较于亲代细胞，每种变体细胞内积累的铜都更少。铜积累的减少与铜外流增强有关。由于患有门克斯病（MNK；ATP7A基因突变）患者的培养细胞会积累铜，这可能是由于铜外流存在缺陷，因此我们研究了MNK基因在铜抗性分子基础中的可能作用。我们发现，在所有三种抗性变体中，MNK mRNA和MNK蛋白均有所增加。在SDS凝胶上观察到，MNK蛋白的表观分子量为178 kDa，此前尚未在哺乳动物细胞中通过实验证实过该蛋白。MNK mRNA和蛋白的增加程度与铜抗性水平及铜外流程度密切相关。通过Southern印迹和FISH分析，我们确定MNK过表达的分子基础是MNK基因的基因组扩增。这些数据，结合门克斯病的临床和细胞表型，有力地证明了MNK蛋白参与跨膜铜外流，并证明了哺乳动物细胞中的一种新的基因扩增系统。
