V-H MUTANT RABBITS LACKING THE V(H)1A2 GENE DEVELOP A2(-CELLS IN THE APPENDIX BY GENE CONVERSION-LIKE ALTERATION OF A REARRANGED V(H)4 GENE() B)

We investigated the molecular basis for the appearance of V(H)a2 allot ype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of V-H, genes because o f a small deletion encompassing V(H)1a2, the 3'-most gene in the V-H l ocus, The V(H)1 gene is the major source of V(H)a allotype because thi s gene is preferentially. rearranged in normal rabbits, In young homoz ygous ali/ali animals, the levels of a2 molecules found in the serum i ncrease with age, In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Precious studies suggested th at positive selection results in expansion of a2 allotype-bearing B ce lls in the appendix of young mutant ali/ali rabbits, We separated appe ndix cells from a 6-wk-old Alicia rabbit by FAGS based on the expressi on of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM(+) a2(+) and IgM(+) a2(-) populations by reverse transcriptase-PCR, The cDNAs from both populations were cl oned and sequenced. Analysis of these sequences suggested that, in a2( +) B cells, the first D proximal functional gene in Alicia rabbits, V( H)4a2, rearranged and was altered further by a gene conversion-like me chanism, Upstream V-H genes were identified as potential gene sequence donors: V(H)9 was found to be the most frequently used gene donor, Am ong the a2(-) B cells, y33 was the most frequently rearranged gene.