Critical test of hot spot motifs for immunoglobulin hypermutation

In hypermutation at the immunoglobulin loci, some bases are much more mutable than others. The increased mutability of the hot spots has been attributed to their being embedded in short sequence motifs. Among the suggested motifs are palindromes, TAA and RGYW (i.e. A/G G C/T A/T). We have tested these proposed motifs in a transfection system in vitro, which ordinarily uses the hypermutable stop codon TAG. The stop codon TAA is not hypermutable in our system, even when embedded in the pentamer and hexamer palindromes TAATA and ATTAAT; in fact, the revertants isolated were due to deletions. Single or double base changes in an RGYW motif containing a hypermutable stop codon result in a reduction of one order of magnitude or more in point mutation frequency. When the nonamer GACTAGTAT, which includes the same RGYW motif, was moved over hundred base pairs upstream, hypermutability was reduced by an order of magnitude. Thus, while RGYW apparently is a hypermutability motif, it cannot be the sole determinant of mutability.