Cloning mammalian genes by expression selection of genetic suppressor elements: association of kinesin with drug resistance and cell immortalization.

We describe a general strategy for cloning mammalian genes whose downregulation results in a selectable phenotype. This strategy is based on expression selection of genetic suppressor elements (GSEs), cDNA fragments encoding either specific peptides that act as dominant inhibitors of protein function or antisense RNA segments that efficiently inhibit gene expression. Since GSEs counteract the gene from which they are derived, they can be used as dominant selectable markers for the phenotype associated with downregulation of the corresponding gene. A retroviral library containing random fragments of normalized (uniform abundance) cDNA expressed in mouse NIH 3T3 cells was used to select for GSEs inducing resistance to the anticancer drug etoposide. Three GSEs were isolated, two of which are derived from unknown genes and the third encodes antisense RNA for the heavy chain of a motor protein kinesin. The kinesin-derived GSE induces resistance to several DNA-damaging drugs and immortalizes senescent mouse embryo fibroblasts, indicating that kinesin is involved in the mechanisms of drug sensitivity and in vitro senescence. Expression of the human kinesin heavy-chain gene was decreased in four of four etoposide-resistant HeLa cell lines, derived by conventional drug selection, indicating that downregulation of kinesin represents a natural mechanism of drug resistance in mammalian cells. IMAGES:

我们报道了一种通用的克隆策略，用于克隆那些下调后可产生可选择表型的哺乳动物基因。该策略基于遗传抑制元件（genetic suppressor elements, GSEs）的表达筛选，这类cDNA片段可编码两类物质：一类是作为蛋白质功能显性抑制因子的特异性肽段，另一类是可高效抑制基因表达的反义RNA片段。由于GSEs可靶向拮抗其来源基因，因此可作为对应基因下调相关表型的显性选择标记。我们使用构建于小鼠NIH 3T3细胞中的、携带丰度均一的随机cDNA片段的逆转录病毒文库，筛选可诱导细胞产生抗癌药物依托泊苷（etoposide）抗性的GSEs。最终分离得到3种GSEs：其中2种来源于功能未知的基因，第3种则编码针对动力蛋白驱动蛋白（kinesin）重链的反义RNA。该源自驱动蛋白的GSE可诱导细胞对多种DNA损伤类药物产生抗性，还可使衰老的小鼠胚胎成纤维细胞发生永生化，这表明驱动蛋白参与了细胞的药物敏感性及体外衰老调控机制。通过传统药物筛选方法获得的4株依托泊苷抗性海拉（HeLa）细胞系中，人类驱动蛋白重链基因的表达均出现下调，这表明驱动蛋白下调是哺乳动物细胞产生天然耐药性的一种机制。图像资料：