(A) Alignment of anti-Rh(D) heavy chains to their nearest germline V, D, and J genes. Also shown are the putative intermediate heavy chain sequences (Ca, Cb, Da, Db, and Dc; see text and Fig 3). The number of nucleotide differences from a germline V_H is tabulated to the right of each sequence. In general, D segments showed poor homology with known D genes, so mutations were not scored in these regions. Key: Replacement mutations indicated with letters, silent mutations as “*”, identities as “.”, and insertions as “-”. Sequences derived from the 5′ V_H primers used in library construction22 are marked as “>”. CDR region designations are determined as per Kabat⁵⁹; numbering and H region designations per Chothia et al.31(B) Alignment of the four VH3 genes used by anti-Rh(D) heavy chains and (C) dendrogram of all human VH3 family germline genes shows relatedness of VH3-21, VH3-30, VH3-33, and VH3-30.3 and the surprising restriction in V_H gene usage. Note that the VH3-30.5 gene is present in only certain haplotypes and is identical to VH3-30.60Genbank accession numbers for anti-Rh(D) heavy chains are listed in the Appendix.