PREFERENTIAL USAGE OF JH2 IN D-J JOININGS WITH DQ52 IS DETERMINED BY THE PRIMARY DNA-SEQUENCE AND IS LARGELY DEPENDENT ON RECOMBINATION SIGNAL SEQUENCES

Rearrangement of D(Q52) With J(H) in IgH genes takes place in a large part of lymphocytes at a very early stage of differentiation although usage of D(Q52) seems to be limited in the functional IgH genes of mature B cells. A possible and likely explanation for this, is secondary D-J(H) joinings occurring after the initial D(Q52)-J(H) joinings, which becomes possible only when D(Q52) segments join 5'-located J(H) segments. Previously we reported the non-random use of J(H) in joinings with D(Q52), in which D(Q52)-J(H2) joinings are dominant. We questioned how the preferential usage Of J(H2) in D(Q52)-J(H) joinings is determined by utilizing extrachromosomal DNA substrates. Extrachromosomal DNA substrates with a combination of D(Q52) and multiple J(H) segments were prepared. These plasmids with a polyoma-derived replication system were transfected into pre-B cell lines and recombination profiles of recovered plasmids were analyzed semi-quantitatively. Experiments with plasmids containing all four J(H) segments in normal configuration showed an apparent high frequency Of J(H2) usage, similar to the rearrangements previously observed in thymocyte genomes. In plasmids containing two J(H) segments, when one was J(H2), it was rearranged more frequently than the other, independent of its proximity to D(q52). The J(H3) with recombination signal sequences (RSS) substituted for J(H2) was rearranged more frequently than the naive J(H2) itself indicating the importance of RSS. A J(H3) hybrid with the 200 bp upstream sequence Of J(H2) but excluding RSS, however, failed to increase J(H3) use. The addition of one nucleotide to the 22-bp spacer Of J(H1) RSS increased the frequency Of J(H1) usage. These observations suggest that the preferential usage Of J(H2) is determined by the primary DNA sequence and is largely dependent on RSS.