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Isolation and characterization of puromycin-resistant clones from cultured mosquito cells
Abstract:
We have isolated from an established Aedes albopictus (mosquito) cell line clones which are resistant to the antibiotic puromycin. On the basis of growth and plating efficiency, clones Pur-8026 and Pur-8612 were five- and seven-fold more resistant, respectively, to puromycin than wild-type cells. In vitro protein synthesis was resistant to puromycin only in extracts prepared from Pur-8612 cells. Measurements of puromycin transport, cross-resistance to colchicine, and sensitivity to Tween-80 indicating that resistance in Pur-8026 cells was due to membrane alteration(s) affecting permeability to puromycin. This is the first description of puromycin resistant in insect cells and also the first report of puromycin resistance in an animal cell variant associated with an alteration at the level of protein synthesis.
Insights
Researchers developed puromycin-resistant Aedes albopictus (mosquito) cell lines. One line showed altered protein synthesis, while the other had membrane changes affecting puromycin permeability.
Area of Science:
- Molecular Biology
- Cell Biology
- Insect Science
Background:
- Puromycin is an antibiotic that inhibits protein synthesis.
- Antibiotic resistance in insect cell lines is not well-characterized.
- Aedes albopictus is a significant vector for various diseases.
Purpose of the Study:
- To isolate and characterize puromycin-resistant mutants in Aedes albopictus cells.
- To investigate the mechanisms underlying puromycin resistance in insect cells.
- To establish a novel tool for genetic studies in mosquitoes.
Main Methods:
- Isolation of puromycin-resistant clones from an Aedes albopictus cell line using puromycin selection.
- Assessment of puromycin resistance levels based on growth and plating efficiency.
- In vitro protein synthesis assays using cell extracts.
- Analysis of puromycin transport, cross-resistance to colchicine, and sensitivity to Tween-80.
Main Results:
- Two puromycin-resistant clones, Pur-8026 and Pur-8612, were isolated, exhibiting 5- and 7-fold increased resistance, respectively.
- Pur-8612 cells showed resistance in in vitro protein synthesis, suggesting a defect at the translational level.
- Pur-8026 cells displayed altered membrane permeability to puromycin, indicated by transport measurements and cross-resistance patterns.
- This study represents the first description of puromycin resistance in insect cells.
Conclusions:
- Puromycin resistance in Aedes albopictus can arise from distinct mechanisms, including alterations in protein synthesis or membrane permeability.
- These resistant cell lines provide valuable models for studying drug resistance and fundamental cellular processes in insects.
- The findings open new avenues for genetic manipulation and functional studies in mosquito vectors.