Dual reporter system to dissect cis- and trans-effects influencing the mutation rate in a hypermutating cell line.
Activation induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching in the immunoglobulin genes of B-lymphocytes. AID expression by itself is sufficient to induce a GC-basepair biased mutator phenotype in lymphoid and non-lymphoid cell lines. Nevertheless a network of cis-regulatory elements and additional trans-factor proteins seems to govern the molecular mechanism of somatic hypermutation. To address the nature of mutation rate changes observed in the hypermutating pre-B cell line 18-81, we extended our previously described green fluorescent protein (GFP) reversion-system. Introducing an additional mutation reporter transgene enables us to discriminate between cis- and trans-factor caused alterations in the mutator phenotype. We show here that in cell line 18-81 the mutation rate declines upon prolonged periods of cell culture. The gradual loss of the mutator phenotype in cell line 18-81 is due to the downregulation of endogenous AID expression and can be reconstituted by overexpression of human AID protein. A correlation between AID mRNA levels and mutation rates is evident and even small changes in AID expression levels cause a significant effect on the mutability of the reporter transgenes.
Ruckerl
Florian
F
GSF-National Research Centre for Environment and Health, Institute of Clinical Molecular Biology and Tumour Genetics, Marchioninistrasse 25, 81377 Munich, Germany.
Mailhammer
Reinhard
R
Bachl
Jurgen
J
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology*; D002460:Cell Line; D003564:Cytidine Deaminase / Q000235:genetics; D015536:Down-Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D007700:Kinetics; D051379:Mice; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D027041:Somatic Hypermutation, Immunoglobulin; D014157:Transcription Factors; D014162:Transfection; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D012333:RNA, Messenger; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase