Increased Transcription Levels Induce Higher Mutation Rates in a Hypermutating Cell Line

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green flu...

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Bibliographic Details
Published in:The Journal of immunology (1950) 2001-04, Vol.166 (8), p.5051-5057
Main Authors: Bachl, Jurgen, Carlson, Chris, Gray-Schopfer, Vanessa, Dessing, Mark, Olsson, Carina
Format: Article
Language:English
Subjects:
Animals
B-Lymphocytes - drug effects
B-Lymphocytes - immunology
B-Lymphocytes - metabolism
Clone Cells
Codon, Terminator - genetics
Codon, Terminator - immunology
Cytidine Deaminase - genetics
Doxycycline - pharmacology
Enhancer Elements, Genetic - drug effects
Enhancer Elements, Genetic - immunology
Flow Cytometry
Genes, Reporter - drug effects
Genes, Reporter - immunology
Genetic Vectors - immunology
Green Fluorescent Proteins
Hydroxamic Acids - pharmacology
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Heavy Chains - metabolism
Introns - genetics
Introns - immunology
Luminescent Proteins - biosynthesis
Luminescent Proteins - genetics
Lymphocyte Activation - genetics
Mice
Mutagenesis, Site-Directed - drug effects
Mutagens - pharmacology
somatic hypermutation
Transcription, Genetic - drug effects
Transcription, Genetic - immunology
Transfection
Tumor Cells, Cultured - drug effects
Tumor Cells, Cultured - immunology
Tumor Cells, Cultured - metabolism
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Summary:Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.
ISSN:0022-1767
1550-6606
DOI:10.4049/jimmunol.166.8.5051