Related Experiment Videos
alpha-Amanithin-resistant mutants of Chinese hamster ovary (CHO) cells
Abstract:
Spontaneous and EMS-induced alpha-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent RNA polymerase II in cell-free extracts from a mutant (ARM-1) was partially resistant to alpha-amanitin. Growing mutants for several generations in the presence or absence of alpha-amanitin did not change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without alpha-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.
Insights
Researchers isolated alpha-amanitin-resistant Chinese hamster ovary (CHO) cells. These mutants possess altered DNA-dependent RNA polymerase II, suggesting potential changes in cell membrane properties.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Alpha-amanitin is a potent toxin that inhibits DNA-dependent RNA polymerase II.
- Understanding drug resistance mechanisms is crucial for developing new therapies.
Purpose of the Study:
- To isolate and characterize alpha-amanitin-resistant Chinese hamster ovary (CHO) cells.
- To investigate the molecular basis of alpha-amanitin resistance in these mutants.
Main Methods:
- Induction of mutations using Ethyl methanesulfonate (EMS) and isolation of resistant clones.
- Characterization of DNA-dependent RNA polymerase II activity in cell-free extracts.
- Growth studies of mutant cells in the presence or absence of alpha-amanitin.
Main Results:
- Spontaneous and EMS-induced alpha-amanitin-resistant CHO cell lines were successfully isolated.
- Mutant ARM-1 exhibited partial resistance of DNA-dependent RNA polymerase II to alpha-amanitin.
- Mutant cell growth patterns were unaffected by prolonged exposure to alpha-amanitin, indicating stable resistance.
Conclusions:
- The isolated mutants possess an altered DNA-dependent RNA polymerase II, conferring resistance to alpha-amanitin.
- Potential alterations in the cell membrane may also contribute to the observed resistance phenotype.