Related Experiment Videos
Isolation and characterization of tunicamycin resistant mutants from Chinese hamster ovary cells
Abstract:
Stable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross-resistance for ConA but are slightly cross-resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.
Insights
Researchers developed tunicamycin-resistant (TMR) Chinese Hamster Ovary (CHO) cell mutants. These novel membrane mutants show reduced glucosamine incorporation, indicating altered glycoprotein synthesis.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Tunicamycin (TM) is an antibiotic that inhibits N-linked glycosylation.
- Studying drug-resistant mutants can reveal cellular mechanisms of drug action and resistance.
- Chinese Hamster Ovary (CHO) cells are widely used for cell biology research and biopharmaceutical production.
Purpose of the Study:
- To isolate and characterize Chinese Hamster Ovary (CHO) cell mutants resistant to tunicamycin (TM).
- To investigate the biochemical and phenotypic properties of these novel tunicamycin-resistant (TMR) mutants.
- To determine if TMR mutants represent a new class of membrane mutants.
Main Methods:
- Selection of stable clones from CHO cells exhibiting resistance to tunicamycin.
- Assessment of TMR phenotype stability over time in the absence of the drug.
- Morphological characterization of TMR mutants.
- Testing for cross-resistance to other lectins like Concanavalin A (ConA) and Phytohemagglutinin (PHA).
- Biochemical labeling of membrane proteins and glycoproteins from Vesicular stomatitis virus (VSV) grown in TMR mutants using radioactive glucosamine.
Main Results:
- Stable tunicamycin-resistant (TMR) CHO cell clones were successfully isolated.
- The TMR phenotype remained stable for over nine months without drug pressure.
- TMR mutants exhibited varied morphologies, from epitheloid to abnormally elongated.
- Mutants showed no cross-resistance to ConA but slight cross-resistance to PHA.
- A marked reduction in radioactive glucosamine incorporation into viral glycoproteins was observed in TMR mutants.
Conclusions:
- Tunicamycin-resistant (TMR) CHO cell mutants represent a novel class of membrane mutants.
- These mutants are characterized by defects in glycoprotein synthesis, specifically reduced glucosamine incorporation.
- The findings provide insights into the cellular pathways involved in tunicamycin sensitivity and resistance.