Cloning Mammalian Genes by Expression Selection of Genetic Suppressor Elements: Association of Kinesin with Drug Resistance and Cell Immortalization

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 9; pp. 3744 - 3748
• Main Authors: Gudkov, Andrei V., Kazarov, Alexander R., Thimmapaya, Rama, Axenovich, Sergey A., Mazo, Ilya A., Roninson, Igor B.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 26-04-1994; National Acad Sciences; National Academy of Sciences
• Subjects: 3T3 Cells; Animals; Base Sequence; Biological and medical sciences; Biotechnology; Cancer; cDNA; CDNA libraries; Cell lines; Cells; Cellular Senescence; Cloning, Molecular - methods; Complementary DNA; Deoxyribonucleic acid; DNA; DNA Primers - chemistry; DNA, Complementary - genetics; down-regulation; Drug Resistance; Drugs; etoposide; Etoposide - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Library; Genes, Suppressor; Genetic engineering; Genetic technics; Genetic Vectors; Genetics; HeLa Cells; Humans; immortalization; kinesin; Kinesin - physiology; Libraries; Mammals; man; Methods. Procedures. Technologies; Mice; Molecular cloning; Molecular Sequence Data; NIH 3T3 cells; Polymerase chain reaction; RNA, Messenger - genetics; selection; senescence; Viruses; Antineoplastic agent; Resistance; Vertebrata; Phenotype; Mammalia; Selection; Microtubule associated protein; Inhibition; Method; Molecular cloning; Gene expression; Kinesin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.9.3744

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.