The generation of sequencing data for the hemiascomycetous yeast random sequence tag project was performed using the procedures established at GENOSCOPE. These procedures include a series of protocols for the sequencing reactions, using infra-red labelled primers, performed on both ends of the plasmid inserts in the same reaction tube, and their analysis on automated DNA sequencers. They also include a package of computer programs aimed at detecting potential assignation errors, selecting good quality sequences and estimating their useful length. ß

Four monoclonal antibodies (mAb) derived from an (NZB x NZW)F1 mouse bound to nucleosomes, total histones and to the H2A-H2B dimers but not to individual histones or DNA. Sequencing of their heavy (H)- and light (L)-chain variable region genes showed that they derived by somatic mutations from the same B cell precursor. The distribution of negatively and positively charged amino acids in the H-chain complementarity-determining regions was very similar to that observed not only in anti-H2A-H2B mAb derived from different lupus-prone mouse strains but also in anti-DNA mAb. Combined analysis of the mAb structures and their interactions with immobilized H2A-H2B dimer or total histones by plasmon resonance allowed us to assign the H-chain mutations a major role in the binding profiles of these anti-nucleosome mAb. Interestingly, four of the five H-chain mutations that distinguished mAb 3F6 from 2E1 generated negatively or positively charged amino acid residues, and two of them occurred at positions 56 and 76, which are frequently involved in the maturation process of anti-DNA antibodies. A modeling study of the 3F6 variable fragment (Fv) predicted that acidic residues occupy the cleft of the Ab combining site and have the potential to participate in electrostatic interactions. Thus, the demonstration that (NZB x NZW)F1-derived anti-H2A-H2B antibodies share certain structural features and mutation patterns with anti-DNA mAb suggest that common selection and maturation processes account for the production of these lupus-related autoantibodies.

In systemic lupus erythematosus, the nucleosome assumes a central role in the autoimmune response to self antigens. To gain insight into the etiology and pathogenesis of anti-nucleosome antibodies (Ab), we analyzed a panel of six IgG-secreting hybridomas derived from a single young MRL +/+ mouse at the onset of the autoimmune response. All monoclonal antibodies (mAb) bound exclusively the native nucleosome, and represented five different clonotypes that recognized diverse nucleosomal epitopes, typical of a polyclonal response. The VH-complementarity-determining region (CDR)3 regions exhibited unique stretches of charged amino acids with different polarity that may be important for the interaction with the nucleosome. These early anti-nucleosome mAb displayed striking structural differences with not only anti-DNA, but also with anti-nucleosome Ab, that appear later in disease. Two of the mAb deposited in kidney glomeruli after in vivo administration to RAG-1-deficient mice, suggesting that diverse B cell clones, possibly selected by the nucleosome itself, may play a role in the initiation of kidney damage.

No abstract

SUMMARYTwo IgM, k anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB × NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb.