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Characteristics of concanavalin A-resistant Chinese hamster ovary cells and certain revertants
Abstract:
Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant difference in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild typed, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/(3H) borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein closses were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitivity phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutation in different genes.
Insights
Chinese hamster ovary (CHO) cells resistant to Concanavalin A (ConA) showed altered cell properties and glycoprotein mobility. Revertants revealed complex genetic interactions influencing ConA resistance and temperature sensitivity.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Concanavalin A (ConA) is a lectin used to select for cell surface alterations.
- Chinese hamster ovary (CHO) cells are a widely used mammalian cell line in research.
- Understanding ConA resistance mechanisms can reveal insights into cell membrane and growth regulation.
Purpose of the Study:
- To isolate and characterize ConA-resistant CHO cell clones.
- To investigate the pleiotropic effects of ConA resistance on cellular properties.
- To analyze changes in cell surface glycoprotein mobility and their genetic basis.
Main Methods:
- Single-step selection of ConA-resistant CHO cell clones.
- Assessment of temperature sensitivity, morphology, adherence, and colchicine susceptibility.
- Lactoperoxidase/125I and galactose oxidase/(3H) borohydride labeling to analyze cell surface glycoproteins.
- Cell hybridization experiments to determine genetic complementation of temperature sensitivity.
Main Results:
- ConA-resistant clones exhibited 2-fold resistance to ConA but showed pleiotropic temperature sensitivity, altered morphology, and adherence.
- Increased mobility of three classes of cell surface glycoproteins (225,000, 200,000, and 130,000 daltons) was observed in ConA-resistant cells.
- Temperature revertants showed differential restoration of ConA sensitivity and glycoprotein mobility, indicating complex genetic regulation.
- Temperature sensitivity phenotypes were recessive and noncomplementing, suggesting a single gene locus.
- Reversions to temperature resistance appeared to be recessive suppressor mutations in different genes.
Conclusions:
- ConA resistance in CHO cells is associated with significant alterations in cell surface glycoproteins and pleiotropic cellular phenotypes.
- The genetic analysis suggests a complex interplay between genes controlling ConA resistance, temperature sensitivity, and cell surface protein modification.
- These findings provide a basis for understanding the genetic control of cell surface properties and their impact on cell physiology.