Roles for Transcription in Antibody Diversity

National Institute On Aging

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Abstract

Activation-induced deaminase (AID) is a cytosine DNA deaminase. AID introduces mutations to support increased antibody affinity (somatic hypermutation) and changed antibody effector function (class switch recombination). For somatic hypermutation, AID targeting to the variable region is poorly understood. AID is associated with transcription and starts at the promoter preceding each rearranged V gene. To firmly delineate the map of mutation, we mapped the mutations from AID from the VH exon to the Emu enhancer. We did this for each V gene segment rearranged to either JH1, JH2, JH3, and JH4 gene segments. Rearranged genes from Peyers patch B cells in mice were amplified by PCR using 5 primers for the ubiquitous VH1 (J558) family, and 3 primers located within the Emu intronic enhancer, positioned 1 kb downstream of JH4. Thus, rearranged V genes using JH1 generated a 2.5 kb fragment, genes using JH2 produced a 2.2 kb fragment, genes using JH3 had a 2.0 fragment, and genes using JH4 had a 1.5 kb fragment. These fragments were separated on an agarose gel, and the bands were cloned and sequenced to produce mutational maps. Preliminary results indicate that mutations were most abundant within a 1 kilobase distance downstream of the promoter, regardless of which JH gene segment was used. We will next determine why mutations stop after 1 kilobase.