Rearrangement of Exogenous Immunoglobulin VH and DJH Gene Segments in Immature Lymphoid Cell Lines

Abstract

A model substrate for the joining of immunoglobulin V🇭 and DJ🇭 elements has been constructed in a retroviral vector. This vector carries a selectable marker whose expression is independent of the arrangement of the resident immunoglobulin gene segments. The substrate was introduced into lymphoid and non-lymphoid cells, and recombination between the V🇭 and DJ🇭 elements was detected by a direct hybridization assay. Joining of the exogenous gene segments was observed in cell lines representative of three distinct stages in early B-cell differentiation. Rearrangement was not observed in three cell lines derived from mature B cells, or in a fibroblastoid cell line. The V🇭 and DJ🇭 gene segments were arranged in the substrate, so that the rearranged coding and non-coding sequences could be recovered from infected cell lines. By molecular cloning and nucleotide sequence determination, V-DJ🇭 junctions formed by rearrangement of the substrate were found to resemble similar junctions in functional heavy chain genes. The joining of V🇭 and DJ🇭 elements was observed to be asymmetric; loss of nucleotides and insertion of N regions occurred at the coding joints. At the noncoding joints, the joining was seen to be a precise fusion of the heptamer segments. These observations provide further evidence that the rearrangement of immunoglobulin gene segments occurs by a site specific mechanism, nonreciprocal in terms of coding and noncoding regions. The model substrate described here is potentially useful for defining the nucleotide sequences that mediate rearrangement and in examining the developmental specificity of this process.