AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization.
Somatic hypermutation generates variants of antibody genes and underpins the affinity maturation of antibodies. It is restricted to the V-gene segments, and although it decays exponentially toward the 3'end, it includes recognizable hot spots. Although the detailed mechanism of hypermutation remains elusive, the process may take place in two separate stages, preferentially targeting G/Cs in the first and A/Ts in the second stage. It seems that MSH2 is involved in the second stage, and that activation induced deaminase (AID) is implicated in the control of hypermutation. The constitutively hypermutating cell line Ramos expresses AID, and we have prepared transfectants that express a chimeric AID-green fluorescent protein. The fluorescence is strongly detected in the cytoplasm but not in the nucleus. Yet, the chimeric protein increases the hypermutation rate either directly or, more likely, indirectly, by favoring the transport of AID into the nucleus. Thus, in Ramos, AID seems to be rate limiting. Unexpectedly, the proportion of deletions also is increased. The increase in mutation rate detected by a fast cytofluorimetric method based on the accumulation of sIgM-loss mutants correlates with the increase measured by mutations defined by sequence analysis. The higher mutation rate is largely explained by the higher proportion of mutated clones, indicating that AID controls the number of cells that undergo hypermutation but not the number of mutations that are incorporated in each mutation round.
Rada
Cristina
C
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom. car@mrc-lmb.cam.ac.uk
Jarvis
John M
JM
Milstein
César
C
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D049452:Green Fluorescent Proteins; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007075:Immunoglobulin M / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase