D'Urso M, Mareni C, Toniolo D, Piscopo M, Schlessinger D, Luzzatto L
Somatic Cell Genet. 1983 Jul;9(4):429-43. doi: 10.1007/BF01543044.
Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.
通过将次黄嘌呤鸟嘌呤磷酸核糖基转移酶和葡萄糖6-磷酸脱氢酶（G6PD）双缺陷的CHO细胞系（NA31）与正常的G6PD（+）人成纤维细胞融合，获得了人-仓鼠体细胞杂种。对NA31提取物的分析表明，尽管几乎没有G6PD活性，但以6-磷酸-2-脱氧葡萄糖作为底物时可检测到显著活性，因此该酶的突变型和正常型都能很容易地被检测到。所获得的细胞杂种表达人G6PD。人G6PD亚基分布于同二聚体分子以及人-仓鼠异二聚体分子中。然而，尽管杂种中存在的仓鼠G6PD亚基的量与仓鼠亲本细胞中的量相似，但与人亲本细胞相比，人G6PD亚基的量减少了3至10倍。这些结果表明，在杂种中G6PD基因的表达或基因产物的稳定性发生改变。通过在二酰胺（一种氧化细胞内谷胱甘肽的物质）中进行诱变和筛选，我们分离出了一个人G6PD活性增加3至5倍的克隆。该衍生物可能具有人G6PD结构基因表达速率的增加。
