Related Experiment Videos
Reversion in thymidine kinase deficient variants of mouse lymphoma P388
Abstract:
The ability of 5 independently isolated thymidine kinase-deficient clones of mouse lymphoma P388 to revert has been examined. We were unable to detect spontaneous revertants in any of the 5 clones. Treatment with the hypomethylating agent 5-azacytidine induced reversion in 4 of the clones, but the frequency of revertants was very low (less than 10(-6). The response was not dose-dependent. The mutagen EMS was capable of inducing reversion in 3 of the clones with a variable level of response. The activity of thymidine kinase in 16 revertants was determined. In half of these the level of enzyme activity was considerably greater than the original P388 cell line. The high frequency loss of thymidine kinase that occurs in these cells may represent a stable inactivation of gene activity rather than an alteration in the DNA base sequence.
Insights
Spontaneous reversion of thymidine kinase deficiency in mouse lymphoma cells was not observed. However, low-frequency reversion was induced by 5-azacytidine and EMS, suggesting stable gene inactivation.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Mouse lymphoma P388 cells were used to study gene reversion.
- Thymidine kinase deficiency was observed in 5 independently isolated clones.
Purpose of the Study:
- To investigate the reversion frequency of thymidine kinase-deficient mouse lymphoma P388 clones.
- To determine the effect of 5-azacytidine and EMS on reversion.
- To analyze thymidine kinase activity in revertant cells.
Main Methods:
- Isolation of 5 thymidine kinase-deficient clones from mouse lymphoma P388.
- Treatment of clones with 5-azacytidine (a hypomethylating agent) and EMS (a mutagen).
- Measurement of thymidine kinase activity in revertant cells.
Main Results:
- No spontaneous revertants were detected in any of the 5 clones.
- 5-azacytidine induced low-frequency reversion (<10^-6) in 4 clones, independent of dose.
- EMS induced reversion in 3 clones with variable response.
- 16 revertants showed thymidine kinase activity, with half exhibiting levels higher than the original P388 cell line.
Conclusions:
- Spontaneous reversion is rare in these thymidine kinase-deficient clones.
- Chemical agents can induce reversion, but at low frequencies.
- High-frequency loss of thymidine kinase may indicate stable gene inactivation rather than DNA sequence alteration.