Germline complexity, restriction fragment length polymorphism, and coding region sequences of the human VH7 gene family identified with family-specific FR3 segment oligonucleotides

Abstract

We have used the most family-specific gene segment, the 5'-end of framework 3 (FR3) to study the germline complexity and coding region sequences of the recently described human VH7 gene family. Because of the high degree of 5' sequence homology between members of the VH7 and VH1 families, full-length coding region probes are unable to distinguish between the two groups. Hybridization with a VH7 coding region probe to EcoRI digested genomic DNA revealed 12 fragments. Many of these hybridizing fragments were also identified with a full length VH1 coding sequence probe. However, examination of the same DNA samples with a VH7 family specific oligonucleotide, encompassing the 5'-end of the FR3 segment, greatly reduced hybridization complexity yielding only four fragments which included one polymorphic band of molecular weight 7.4 kb. The VH7 specific FR3 oligonucleotide was also used under conditions of moderate stringency to isolate VH7 clones from PCR-amplified genomic DNA libraries derived from six unrelated individuals. All clones isolated contained members of the VH7 family. Six sequences were obtained. Gene 7A.4, seen in all individuals, is identical to the previously described germline V 1–4.1b gene other than G-C substitutions at nucleotides 253 and 254. Five distinct pseudogenes were also identified. Stop codons were confined to frameworks 2 and 3. Previously described expressed VH7 genes from cord blood, normal adults and two rheumatoid factors are > 96% homologous to gene 7A.4.