CLONING MAMMALIAN GENES BY EXPRESSION SELECTION OF GENETIC SUPPRESSOR ELEMENTS - ASSOCIATION OF KINESIN WITH DRUG-RESISTANCE AND CELL IMMORTALIZATION
CLONING MAMMALIAN GENES BY EXPRESSION SELECTION OF GENETIC SUPPRESSOR ELEMENTS - ASSOCIATION OF KINESIN WITH DRUG-RESISTANCE AND CELL IMMORTALIZATION
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DOI：
10.1073/pnas.91.9.3744
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发表时间：
1994-04-26
影响因子：
11.1
通讯作者：
RONINSON, IB
中科院分区：
文献类型：
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作者：
GUDKOV, AV;KAZAROV, AR;RONINSON, IB
我们描述了克隆哺乳动物基因的一般策略，其下调导致可选择的表型。该策略基于基因抑制元件（GSEs）的表达选择，基因抑制元件是编码特异性肽的cDNA片段，作为蛋白质功能的显性抑制剂或有效抑制基因表达的反义RNA片段。由于gse抵消了它们产生的基因，因此它们可以作为与相应基因下调相关的表型的显性选择标记。利用小鼠NIH 3T3细胞中表达的归一化（均匀丰度）cDNA随机片段逆转录病毒文库，筛选诱导对抗癌药物依托泊苷耐药的GSEs。分离到三个gse，其中两个来自未知基因，第三个编码运动蛋白激酶重链的反义RNA。运动蛋白衍生的GSE诱导对几种dna损伤药物的抗性，并使衰老的小鼠胚胎成纤维细胞永生，表明运动蛋白参与了药物敏感性和体外衰老的机制。通过常规药物选择获得的四种依托泊苷耐药HeLa细胞系中，有四种人激酶重链基因的表达降低，表明激酶的下调是哺乳动物细胞耐药的自然机制。
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.