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 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.
Bachl
J
J
Basel Institute for Immunology, Basel, Switzerland. bachl@gsf.de
Carlson
C
C
Gray-Schopfer
V
V
Dessing
M
M
Olsson
C
C
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D002999:Clone Cells; D018388:Codon, Terminator / Q000235:genetics / Q000276:immunology; D003564:Cytidine Deaminase / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000187:drug effects / Q000276:immunology; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000187:drug effects / Q000276:immunology; D005822:Genetic Vectors / Q000276:immunology; D049452:Green Fluorescent Proteins; D006877:Hydroxamic Acids / Q000494:pharmacology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics / Q000276:immunology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000187:drug effects; D009153:Mutagens / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology / Q000378:metabolism
D018388:Codon, Terminator; D006877:Hydroxamic Acids; D007143:Immunoglobulin Heavy Chains; D008164:Luminescent Proteins; D009153:Mutagens; D049452:Green Fluorescent Proteins; C012589:trichostatin A; D003564:Cytidine Deaminase; D004318:Doxycycline