Toshiyasu Hirama

Excision products of immunoglobulin gene rearrangements

We have isolated circular DNAs from splenocytes of euthymic and athymic mice, and prepared the DNA libraries of 1.5 X 10(6) clones. Hundreds of clones homologous to immunoglobulin (Ig) heavy chain segments (DSP2 and DQ52-JH) or light chain segments (J kappa and J lambda) have been identified. Southern hybridization predicted that three of 12 euthymic mouse clones homologous to DQ52-JH and four of 10 athymic mouse clones homologous to DSP2 contained reciprocal recombination products of D-J joining. Some of these clones were characterized by sequencing: two clones contained the precise excision product of the recombination of a DSP2 segment with either JH2 or JH3 segment; two clones showed imprecise ligation of the DSP2-JH2 coding joint and precise ligation of the DFL16.1-JH3 reciprocal joint in the same molecule. They seem to represent a replacement of the pre-existing DSP2-JH2 rearrangement by joining an upstream DFL16 segment to a downstream JH3 segment. The presence in extrachromosomal DNA of a reciprocal recombination product of DH-JH joining is consistent with the view that immunoglobulin genes, like T cell receptor (TCR) genes, can be rearranged in B cell lineage by the looping-out and excision of chromosomal DNA.

STRUCTURE OF EXTRACHROMOSOMAL CIRCULAR DNAS GENERATED BY IMMUNOGLOBULIN LIGHT CHAIN GENE REARRANGEMENTS

Recombination at the immunoglobulin kappa or lambda light chain locus generates extrachromosomal circular DNAs. We have isolated circular DNAs from adult mouse spleen cells and prepared a circular DNA clone library. We characterized four J kappa-positive and one J lambda 1-positive clones. The J kappa-clones contained both coding and signal joints of V kappa-J kappa joining, and the J lambda 1-clone contained a signal joint of V lambda 1-J lambda 1 joining. Genomic organization of the V kappa gene families used in these joints suggested the excision of circular DNA preceded by inversion. A specific dinucleotide (P) insertion in the coding joint was observed in two clones. Three coding joints were out of frame and one clone had an in-frame coding joint, although possibly combined with a pseudo-V kappa gene. These kappa-positive circular DNAs are possibly excised from the chromosome by secondary recombinations which replace non-productive primary rearrangements.

Myelodysplastic syndrome (MDS)-associated inhibitory activity on haematopoietic progenitor cells : contribution of monocyte-derived lipid containing macrophages (MDLM)

We studied myelodysplastic syndrome‐associated inhibitory activity (MDS‐IA), which inhibited colony formation in vitro of normal granulocyte‐macrophage progenitors (CFU‐GM). When adherent marrow cells were incubated with fetal calf serum for 21–24 d. monocyte‐derived lipid containing huge macrophages (MDLM) developed. MDLM from MDS marrow (MDS‐MDLMs) and their conditioned medium (MDLM‐CM) consistently suppressed the growth of normal CFU‐GM colony formation. MDS‐IA was active on CFU‐GM during the S‐phase and relatively resistant to heating. Monoclonal antibody against H subunit (acidic) ferritin and polyclonal antibody against placental ferritin neutralized the inhibitory activity of MDS‐MDLMs. In addition, cell lysates of MDS‐MDLMs reacted to both monoclonal anti‐H subunit ferritin and polyclonal anti‐placental ferritin in Western blotting analysis, indicating that the inhibitory activity was predominantly acidic isoferritin.