RAPID METHODS FOR THE ANALYSIS OF IMMUNOGLOBULIN GENE HYPERMUTATION -APPLICATION TO TRANSGENIC AND GENE TARGETED MICE

Hypermutation of immunoglobulin genes is a key process in antibody div ersification. Little is known about the mechanism, but the availabilit y of rapid facile assays for monitoring immunoglobulin hypermutation w ould greatly aid the development of culture systems for hypermutating B cells as well as the screening for individuals deficient in the proc ess, Here we describe two such assays, The first exploits the non-rand omness of hypermutation, The existence of a mutational hotspot in the Ser31 codon of a transgenic immunoglobulin V gene allowed us to use PC R to detect transgene hypermutation and identify cell populations in w hich this mutation had occurred, For animals that do not carry immunog lobulin transgenes, we exploited the fact that hypermutation extends i nto the region flanking the 3'-side of the rearranged J segments, We s how that PCR amplification of the 3'-flank of VDJ(H) rearrangements th at involve members of the abundantly-used V(H)J558 family provides a l arge database of mutations where the germline counterpart is unequivoc ally known, This assay was particularly useful for analysing endogenou s immunoglobulin gene hypermutation in several mouse strains, As a rap id assay for monitoring mutation in the J(H) flanking region, we show that one can exploit the fact that, following denaturation/renaturatio n, the PCR amplified J(H) flanking region DNA from germinal centre B c ells yields mismatched heteroduplexes which can be quantified in a fil ter binding assay using the bacterial mismatch repair protein MutS [Wa gner et al, (1995) Nucleic Acids Res, 23, 3944-3948], Such assays enab led us, by example, to show that antibody hypermutation proceeds in th e absence of the p53 tumour suppressor gene product.