Immunoglobulin (Ig) and T cell receptor (TCR) genes are assembled during lymphocyte maturation through site-specific V(D)J recombination events. Here we show that E2A proteins act in concert with RAG1 and RAG2 to activate Ig VK1J but not Iglambda VlambdaIII-Jlambda1 rearrangement in an embryonic kidney cell line. In contrast, EBF, but not E2A, promotes VlambdaIII-Jlambda1 recombination. Either E2A or EBF activate IgH DH4J recombination but not V(D)J rearrangement. The Ig coding joints are diverse, contain nucleotide deletions, and lack N nucleotide additions. IgK VJ recombination requires the presence of the E2A transactivation domains. These observations indicate that in nonlymphoid cells a diverse Ig repertoire can be generated by the mere expression of the V(D)J recombinase and a transcriptional regulator.

Monoclonal antibodies directed toward metal chelating agents or metal-chelate complexes have many potential uses both in medicine and in environmental analysis. In medicine, chelator-linked antibodies have been used in vivo to transport and deliver radioisotopes to specific target sites, such as tumors (1-3). The availability of monoclonal antibodies to the chelating agent has permitted researchers to investigate the biodistribution of these "magic bullets" in cancer chemotherapy (4). In environmental analysis, the availability of monoclonal antibodies that can distinguish among different metals provides a basis for rapid, sensitive immunoassays that can be used onsite to assess heavy metal contamination (5, 6). Monoclonal antibodies that bind to chelators such as diethylenetriaminepentaacetic acid and 1,4,7,10-tetraazacyclododecane-N, NЈ, NЉ, Nٞ-tetraacetic acid have been reported (7, 8); however, these antibodies were directed primarily toward the chelate portion of the molecule and did not demonstrate the ability to differentiate among various metals bound in the chelate complex. Two hybridomas that synthesized monoclonal antibodies showing metal ion specificity have also been reported. The first, CHA255, was the result of immunization with a derivative of In(III)-EDTA coupled to a carrier protein by a thioureido-L-benzyl group (4). The monoclonal antibody synthesized by CHA255 bound to a variety of chelate complexes but bound to indium-chelate complexes more tightly than it did to other chelated metals (4, 9). Monoclonal antibodies directed toward mercuric ions have also been elicited by immunization of animals with a glutathione-Hg derivative (10). These antibodies, which bind to mercury with high affinity, have been used to detect mercuric ions in aqueous samples (6) and are the basis for a commercially available immunoassay to monitor mercury contamination in environmental samples.The molecular features responsible for an antibody's ability to differentiate among metals have been explored in some detail with CHA255 (11). Binding studies have shown that CHA255 was highly specific for the indium chelate; the antibody's affinity decreased from 24-to 20,000-fold when other metals were substituted for In(III) in the chelate complex. The structural basis for this fine discrimination was investigated by x-ray crystallographic analyses of the antigen-binding fragment complexed with either In(III) or Fe(III) chelates of thioureido-L-benzyl-EDTA. A notable feature of the antibodyIn(III)-EDTA complex was the additional coordination of the chelated metal by a histidine residue from the heavy chain's third complementarity determining region. This histidine coordination did not occur in the corresponding Fe(III) complex, due to a slightly different hapten coordination that reduced access to the metal. It was concluded that the absence of the histidine ligation was largely responsible for the 24-fold lower binding of the iron hapten to CHA255 relative to that of the indium hapten. The larger differences in the binding a...

Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VκI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vκ locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the DH, Vκ, and Vλ loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

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A wild-type strain of Staphylococcus aureus , which inactivates a wide variety of aminoglycosides (except the gentamicin components), has been found to harbor a plasmid (RAp01) that mediates the biosynthesis of a nucleotidyltransferase. This enzyme modifies the 4′-hydroxy function of these antibiotics. The plasmid has been studied, the enzyme responsible for this resistance pattern has been isolated by affinity chromatography, and its kinetics and physicochemistry have been characterized. The target of this enzyme has also been located by demonstrating the structure of one inactivated compound, 4′-( O )-adenylyltobramycin.