Related Experiment Videos
Induction and isolation of frameshift mutants in cultured Chinese hamster DON cells
Abstract:
Induction, isolation and characterization of frameshift mutants were studied by using a Chinese hamster Don (CHD) cell line. ICR-191, known to be a potent frameshift mutagen, was used for the induction of frameshift mutations. The drug (10(-5) M), as well as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ethyl methanesulfonate (EMS), increased significantly the frequency of forward mutations from 8-azaguanine (8-AG) sensitivity (8-AGs) to resistance (8-AGr) over the untreated control to an extent of about 100-fold. 21 8-AGr mutants were isolated from the AP-01 (a sub-line of CHD) cells after treatment with appropriate concentrations of ICR-191 (10(-5) and 1.36 X 10(-5) M), and subsequently several reclonal mutants were tested for their ability to revert to 8-AG susceptibility after treatment with the three mutagens and a carcinogen, 2-nitrofluorene (2-NF), known to be a frameshift mutagen. Among the 8-AGr mutants tested, clone ICR-014 or ICR-172 showed a significant increase in reversion frequency over the control level only after treatment with ICR-191 or 2-NF, respectively; but not with the other two mutagens. These results suggest that each of these two kinds of mutant has a different frameshift mutation in one of the loci controlling 8-AG resistibility. It was also found that the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) activities in clones ICR-014 and ICR-172 were 1.9 and 34% of that of the original AP-01 cells, respectively.
Insights
This study investigated frameshift mutations in Chinese hamster cells using ICR-191 and other mutagens. Specific mutants showed differential reversion frequencies, suggesting distinct genetic alterations affecting 8-azaguanine resistance.
Area of Science:
- * Molecular Biology
- * Genetics
- * Toxicology
Background:
- * Frameshift mutations are critical in genetic toxicology and cancer research.
- * Chinese hamster cells are a standard model for mutagenicity testing.
- * Understanding mutagenic mechanisms is essential for risk assessment.
Purpose of the Study:
- * To induce, isolate, and characterize frameshift mutants in a Chinese hamster cell line.
- * To investigate the mutagenic and mutagenic potential of ICR-191, MNNG, EMS, and 2-NF.
- * To differentiate frameshift mutations based on reversion analysis and enzyme activity.
Main Methods:
- * Induction of mutations using ICR-191, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and ethyl methanesulfonate (EMS) in Chinese hamster Don (CHD) cells.
- * Isolation of 8-azaguanine-resistant (8-AGr) mutants.
- * Reversion analysis of isolated mutants using mutagens and 2-nitrofluorene (2-NF); measurement of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity.
Main Results:
- * ICR-191, MNNG, and EMS significantly increased 8-azaguanine sensitivity (8-AGs) to resistance (8-AGr) frequencies approximately 100-fold.
- * Two distinct 8-AGr mutants (ICR-014 and ICR-172) exhibited differential reversion frequencies when treated with ICR-191 or 2-NF.
- * HGPRT activities in ICR-014 and ICR-172 were significantly reduced (1.9% and 34%, respectively) compared to control cells.
Conclusions:
- * The study successfully induced and characterized frameshift mutants in Chinese hamster cells.
- * Distinct frameshift mutations in loci controlling 8-azaguanine resistance were identified.
- * Differential reversion patterns and HGPRT activity provide insights into the nature of specific frameshift mutations.