Antibody repertoire development in fetal and neonatal piglets.: XIII.: Hybrid VH genes and the preimmune repertoire revisited

The expressed porcine V-H genes belong to the V(H)3 family (clan), four of which, V(H)A, VHB, VHC, and VHE, alone comprise similar to 80% of the preimmune repertoire. However, so-called "hybrid" V-H genes that use CDR1 of one V-H gene and the CDR2 of another are frequently encountered. We studied > 3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (V(H)Z and VHY), together with VHF and the four genes mentioned above, constitute the major V-H genes and these account for > 95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major V-H genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of V-H repertoire diversification and 2) establish a library of 29 putative porcine V-H genes. One-third of these genes are chimeric genes and their sequences. suggest that the porcine V-H genome developed by duplication and splicing from a small number of prototypic genes.