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Induction of frameshift mutations in cultured mammalian cells within a transfected sequence containing a
K L Riedinger1, M G Hanford, R A Farber
1Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill 27599-7525, USA.
Abstract:
A cultured mouse cell line with an integrated copy of a plasmid that contains a short dinucleotide repeat sequence (microsatellite) has been used to determine the frequencies and types of mutation induced by two frameshift mutagens. The presence of the microsatellite, which consists of 17 repeats of a poly(dC-dA).poly(dT-dG) sequence, disrupts the reading frame of a gene coding for neomycin resistance. Revertants were selected in G418, and mutations were analyzed by PCR. ICR-170 was found to increase the reversion frequency by ten- to 15-fold at its LD50, although most of the frameshifts that it induced were single-base insertions outside the microsatellite sequence. NA-AAF brought about a more modest increase in mutation frequency, but nearly all of the revertants in the NA-AAF-treated cultures had insertions or deletions of multiples of two base pairs within the DNA segment that included the microsatellite. This system can be modified to include different short tandem repeat sequences as targets for testing of compounds that are suspected of having frameshift-inducing activities.
Insights
This study used a mouse cell line with a microsatellite sequence to test frameshift mutagens. ICR-170 and NA-AAF induced mutations, with NA-AAF showing specific insertions/deletions within the microsatellite.
Area of Science:
- Molecular biology
- Genetics
- Toxicology
Background:
- Microsatellites are repetitive DNA sequences prone to mutation.
- Frameshift mutagens can cause insertions or deletions in DNA.
- Assessing mutagenicity is crucial for understanding genotoxicity.
Purpose of the Study:
- To evaluate the mutagenic potential of ICR-170 and N-acetoxy-2-acetylaminofluorene (NA-AAF) using a mouse cell line.
- To characterize the types and frequencies of mutations induced by these frameshift mutagens.
- To establish a cell-based system for detecting frameshift mutagenicity.
Main Methods:
- Utilized a cultured mouse cell line containing a disrupted neomycin resistance gene due to an integrated microsatellite.
- Selected revertants resistant to G418 (neomycin analogue).
- Analyzed mutations within the microsatellite sequence using Polymerase Chain Reaction (PCR).
Main Results:
- ICR-170 increased reversion frequency 10-15 fold but induced primarily single-base insertions outside the microsatellite.
- NA-AAF caused a modest increase in mutation frequency, with most revertants exhibiting insertions/deletions of two base pairs within the microsatellite.
- The study demonstrated differential mutagenic mechanisms of the tested compounds.
Conclusions:
- The developed cell line system effectively detects frameshift mutagenicity.
- NA-AAF specifically targets microsatellite sequences, inducing frameshift mutations.
- The system is adaptable for testing other suspected frameshift mutagens with varying repeat sequences.