It has been proposed that the anti-double-stranded DNA (dsDNA) response in patients with systemic lupus erythematosus (SLE) is antigen driven and that DNA or nucleosomes select anti-DNA reactive, somatically mutated B cells. We have used site-directed mutagenesis to systematically revert the somatic mutations of two human anti-dsDNA antibodies from SLE patients to analyze the resulting changes in DNA binding as well as binding to other autoantigens. Our data demonstrate that high-affinity binding to dsDNA and nucleosomes is acquired by somatic replacement mutations in a stepwise manner. Reactivity to surface structures of apoptotic cells is acquired by the same somatic mutations that generate high-affinity dsDNA binding. Importantly, revertant antibodies with germ-line V regions did not show any measurable DNA reactivity. We propose that anti-DNA autoantibodies are generated from nonautoreactive B cells during a normal immune response. B cells may acquire autoreactivity de novo during the process of somatic hypermutation. Nucleosomes, if available in lupus patients because of defects in clearing of apoptotic debris, might subsequently positively select high affinity anti-DNA B cells.somatic hypermutation ͉ systemic lupus erythematosus

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca 2+ signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca 2+ signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca 2+ responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium. Strains carrying mutations located downstream of nipB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations. DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB. The gene products of modB and modC are homologous to ChU and ChID of Escherichia coil and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively.ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein. The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other. Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum. Compared with that for the parental nijfDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants. No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants. The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase. Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake.The process of N2 fixation has been studied for many years in a variety of different diazotrophs, and the conventional molybdenum-containing nitrogenase has been well characterized. However, it was not realized until recently that some organisms harbor, in addition to the molybdenum nitrogenase, other genetically distinct nitrogenase enzyme complexes. These alternative nitrogenases have been best characterized in the obligate aerobic soil bacterium Azotobacter vinelandii, which contains three distinct nitrogenase systems. The conventional nitrogenase (nitrogenase 1) includes a molybdenum cofactor, nitrogenase 2 is a vanadium enzyme, and nitrogenase 3 is a heterometal-free enzyme complex (for a review, see reference 5). The phototrophic purple bacterium Rhodobacter capsulatus harbors two nitrogenase systems, corresponding to nitrogenase 1 and nitrogenase 3, whereas a vanadium-containing enzyme is apparently not present (36). The expression of alternative nitrogenases in both A. vinelandii and R capsulatus is repressed by extremely low molybdenum concentrations, indicating that high-affinity systems are involved in gene regulation.

The alternative nitrogenase of Rhodobacter capsulatus is expressed only under conditions of nitrogen and molybdenum depletion. The analysis of anfA-lacZ fusions demonstrated that this dual control occurred at the level of transcription of anfA, which encodes a transcriptional activator specific for the alternative nitrogenase. The anfA promoter was found to be activated under nitrogen-limiting conditions by NtrC in a 54 -independent manner. In addition, anfA transcription was repressed by traces of molybdenum. This molybdenum-dependent repression of anfA was released in R. capsulatus mutants carrying either lesions in the high-affinity molybdenum uptake system (modABCD) or a double deletion of mopA and mopB, two genes encoding molybdenumpterin-binding proteins. The expression of the molybdenum transport system itself was shown to be negatively regulated by molybdenum and, unexpectedly, to be also regulated by NtrC. This finding is in line with the presence of two tandemly arranged DNA motifs located in front of the R. capsulatus mopA-modABCD operon, which are homologous to R. capsulatus NtrC binding sites. Mapping of the transcriptional initiation sites of mopA and anfA revealed promoter sequences exhibiting significant homology to each other but no homology to known prokaryotic promoters. In addition, a conserved DNA sequence of dyad symmetry overlapping the transcriptional initiation sites of mopA and anfA was found. Deletions within this element resulted in molybdenumindependent expression of anfA, indicating that this DNA sequence may be the target of MopA/MopB-mediated repression.Molybdenum is an essential trace element required for the activities of several enzymes. These molybdenum enzymes contain either molybdopterin cofactors or the iron-molybdenum cofactor which is present only in nitrogenase. In addition to the conventional molybdenum nitrogenase, the phototrophic purple bacterium Rhodobacter capsulatus harbors an alternative nitrogenase, which is devoid of heterometals (24,39,40). The alternative nitrogenase of R. capsulatus is repressed by traces of molybdenum (39). Repression of alternative nitrogenases by molybdenum was also found for Azotobacter vinelandii, an obligate aerobic soil bacterium which is able to fix nitrogen via the molybdenum, the vanadium, or the iron-only nitrogenase (for a review, see reference 2). The molybdenum repression of the alternative nitrogenase in R. capsulatus is released in mutants unable to import molybdate by a high-affinity molybdenum uptake system (modABCD) or in a mutant strain devoid of mopA and mopB, which code for molybdenum-pterin-binding proteins (46). As found for most members of the superfamily of ABC transporters (11), the high-affinity molybdate transport system of R. capsulatus consists of a periplasmic substrate-binding protein (ModA), a transmembrane protein forming the entry pathway (ModB), an ATP-binding protein which couples ATP hydrolysis to translocation of the substrate into the cytoplasm (ModC), and a fourth protein (ModD) of unknown function (...

Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and Tcell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by`error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.