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Cited by 122Microtubule and mitosis dynamicsTelomeres, Telomerase, and SenescenceCancer-related Molecular Pathways

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

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Gudkov, A V, Kazarov, A R, Thimmapaya, R, Axenovich, S A, Mazo, I A, Roninson, I B·1994·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.91.9.3744PMC PMC43658
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Abstract

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.

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Authors
Gudkov, A. V., Kazarov, A. R., Thimmapaya, R., Axenovich, S. A., Mazo, I. A., & Roninson, I. B.
Affiliations
University of Illinois Chicago (US)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
1994-04-26
Volume
91
Issue
9
Pages
3744-3748
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.91.9.3744
PMC
PMC43658
OpenAlex
W1982745217
Cited by
122
References
25
Citations by year
2023: 1 · 2021: 1 · 2019: 1 · 2018: 1 · 2016: 1 · 2014: 1 · 2013: 1
FWCI
2.77
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • American Cancer Society
  • National Institutes of Health (5r01ca039365-08, 5r01ca056736-05)
  • National Cancer Institute (CA39365, CA40333, CA56736)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
Keywords
gene cloning, dominant selectable marker, kinesin heavy chain, drug resistance, etoposide resistance, DNA-damaging agents, cell immortalization, cellular senescence, antisense RNA, NIH3T3 cells, HeLa cells, gene downregulation
MeSH terms
Animals, Base Sequence, Cloning, Molecular, Drug Resistance, Etoposide, Genetic Vectors, HeLa Cells, Humans, Molecular Sequence Data, RNA, Messenger, Gene Library, Gene Expression, Genes, Suppressor, 3T3 Cells, Kinesins, Cellular Senescence, DNA Primers, DNA, Complementary, Mice

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