Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence.

We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].     

Purification and characterization of a protein that binds to the recombination signal sequence of the immunoglobulin J kappa segment.

A protein that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified almost to homogeneity from the nuclear extract of a murine pre-B cell line 38B9. A similar binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The binding activity was associated with a polypeptide with a molecular weight of 60,000. DNase I footprinting analysis demonstrated that this binding protein interacted with the heptamer and several 3' bases close to the heptamer. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. One base substitution in the heptamer of the J kappa RS greatly reduced the affinity of the J kappa RS binding protein. The high specificity of the binding site of the J kappa RS binding protein suggests that this protein may be involved in V-J recombination.     

Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.

DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.     

The V(D)J recombination signal sequence and kappa B binding protein Rc binds DNA as dimers and forms multimeric structures with its DNA ligands.

The murine DNA binding protein Rc binds to the heptamer motif of the V(D)J recombination signal sequences and to the kappa B motif of the immunoglobulin enhancer. Bacterial fusion proteins for Rc and DNA ligands of Rc form multiple protein-DNA complexes in electrophoretic mobility shift assays (EMSA). Large complexes formation is favored by an increased Rc concentration. In order to determine the architecture of these complexes, the apparent molecular weights of the protein-DNA complexes were first determined by their gel mobilities. The data suggest that Rc binds to its DNA ligands as dimers, tetramers, and multiples of tetramers. The inference that Rc binds DNA as dimers was substantiated by the formation of chimeric complexes when two electrophoretically distinguishable Rc proteins were employed in EMSA. Methylation interference experiments show that there are no contiguous protein binding sites evident in the DNA of the larger complexes. Apparently, multimerization occurs via protein-protein interactions. Such interaction was demonstrated by the formation of Rc dimers and tetramers in a chemical crosslinking experiment. Significantly, the multimerization of DNA-bound Rc could be involved in bringing the variable region gene segments together for the somatic V(D)J recombination.     

The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product

Mutations in the single-minded (sim) gene of Drosophila result in the loss of the precursor cells giving rise to the midline cells of the embryonic central nervous system. We have examined the structure of the sim product by sequencing a sim cDNA clone, and have also determined the subcellular localization of the protein and its developmental expression by staining embryos with an antiserum against a sim fusion protein. The results indicate that sim is a nuclear protein specifically expressed along the midline of the neuroepithelium, the same subset of cells that are missing in the mutant. No similarity is observed between sim and any known nuclear protein, but, surprisingly, it is similar to the Drosophila period (per) locus gene product, which controls the periodicity of biological rhythms.     

The Drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system development.

The J kappa RBP binds to the immunoglobulin recombination signal sequence flanking the kappa-type J segment. We previously isolated the highly conserved homolog of the J kappa RBP gene from D. melanogaster, which is not thought to have immunoglobulin molecules. Using many deficiency mutants and in situ hybridization, we mapped the Drosophila J kappa RBP gene in a region containing two recessive lethal mutations, i.e., br26 and br7, which shows the dominant Suppressor of Hairless (Su(H)) phenotype in heterozygotes. All six Su(H) alleles analyzed at the DNA level contained mutations in the Drosophila J kappa RBP gene. Since the Su(H) mutation affects peripheral nervous system development, the Drosophila J kappa RBP gene product is involved in gene regulation of peripheral nervous system development. The results also imply that the immunoglobulin recombination signal sequence and the target sequence of the Drosophila J kappa RBP protein might have a common evolutionary origin.     

DNA sequence of the constant gene region of the mouse immunoglobulin kappa chain.

The constant (C; ref. 3) gene segment of the immunoglobulin kappa light chain and about 1 kb upstream as well as downstream of the segment have been sequenced. The sequences of the C gene segment itself and parts of the upstream region were determined both in liver and in myeloma T DNA clones derived from the same mouse inbred strain. The sequences were identical, i.e. no somatic mutations were detected. Two sites in the region not coding for protein are discussed as possible targets of aberrant variable (V) gene translocations. Doublet frequencies were calculated in the approx. 2500 bp of the C region sequence reported in this paper and in the approx. 3400 bp of two rearranged V gene regions.     

The Drosophila gastrulation gene concertina encodes a G alpha-like protein

Gastrulation is a complex process requiring the coordination of cell shape changes and cell movements. In Drosophila, gastrulation begins immediately upon cellularization of the blastoderm stage embryo with the formation of the ventral furrow and posterior midgut. Cells that form both of these invaginations change their shape via apical constriction. Embryos from mothers homozygous for mutations in the concertina (cta) gene begin furrow formation by forming a zone of tightly apposed cells, constrict some cells, and then fail to constrict enough cells to form an organized groove. The cta gene has been cloned, and sequence analysis suggests that it encodes an alpha subunit of a G protein. G proteins have a role in cell-cell communication as mediators of signals between membrane-bound receptors and intracellular effectors. The phenotype of embryos from homozygous cta mothers suggests that the cta gene plays a role in a signal transduction pathway used during gastrulation.     

Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.