Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase zeta.

To examine a role of DNA polymerase zeta in somatic hypermutation, we generated transgenic mice that express antisense RNA to a portion of mouse REV3, the gene encoding this polymerase. These mice express high levels of antisense RNA, significantly reducing the levels of endogenous mouse REV3 transcript. Following immunization to a hapten-protein complex, transgenic mice mounted vigorous Ab responses, accomplished the switch to IgG, and formed numerous germinal centers. However, in most transgenic animals, the generation of high affinity Abs was delayed. In addition, accumulation of somatic mutations in the V(H) genes of memory B cells from transgenic mice was decreased, particularly among those that generate amino acid replacements that enhance affinity of the B cell receptor to the hapten. These data implicate DNA polymerase zeta, a nonreplicative polymerase, in the process of affinity maturation, possibly through a role in somatic hypermutation, clonal selection, or both.

Characterization of the human B cell RAG-associated gene, hBRAG, as a B cell receptor signal-enhancing glycoprotein dimer that associates with phosphorylated proteins in resting B cells.

Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG-associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane-integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated isoforms of hBRAG are detectable with these reagents. Additionally, their use in cell surface biotinylation and flow cytometry reveals subcellular hBRAG pools both at cell surface and intracellular locations. Co-immunoprecipitation experiments with hBRAG antisera detected the association of hBRAG with phosphorylated proteins in resting B cells, including the protein tyrosine kinase Hck, which is subsequently dephosphorylated upon B cell receptor (BCR) ligation. Consistent with its cell surface expression and possible link to BCR signaling, experiments in which alpha-hBRAG antibodies were used to generate early activation signals suggest a modest but specific element of tyrosine phosphorylation occurring through a putative hBRAG receptor. Additional experiments also suggest that hBRAG may be involved in positively enhancing BCR ligation-mediated early activation events. Collectively, these results are consistent with a function for hBRAG as a B cell surface signaling receptor molecule. Coupled with the earlier observation that hBRAG expression correlates with early and late B cell-specific RAG expression, we submit that hBRAG may mediate regulatory signals key to B cell development and/or regulation of B cell-specific RAG expression.

hBRAG, a novel B cell lineage cDNA encoding a type II transmembrane glycoprotein potentially involved in the regulation of recombination activating gene 1 (RAG1).

The different display reverse transcription-PCR (DD RT-PCR) technique was used to identify novel cDNA detecting mRNA transcripts co-expressed with human recombination activating gene-1 (RAG1). A 5.0-kb transcript detected by the differential display amplicon 3G1 was found to correlate strongly with RAG1 mRNA expression in various human cell lines. Subsequent screenings of a pre-B cDNA library with 3G1 led to the identification of a complete cDNA we have termed hBRAG (human B-cell RAG-Associated Gene). The hBRAG cDNA encodes a 503-amino acid (aa) protein with no known homology to any nucleotide or protein sequence. The predicted molecular mass of 55 kDa was confirmed by in vitro translation. Based on sequence analysis, the predicted open reading frame encodes for a type II transmembrane spanning glycoprotein with the N-terminal 81 -aa in the cytoplasm, a 17-aa transmembrane domain, and a C-terminal 405-aa extracellular domain with four potential N-glycosylation sites. Northern blot analysis indicated a close association of the 5.0-kb hBRAG mRNA transcript with RAG1 in numerous human pro-B, pre-B and mature B cell lines assessed, but not in human T cell lines. In human tissues, hBRAG is expressed at highest levels in B cell-enriched tissues, but is not expressed in fetal or adult thymus. Southern blotting analysis revealed that this gene is conserved across eukaryotes, is expressed as a single copy in the human genome, and is likely not a multigene family member. The hBRAG gene was localized to the long arm of chromosome 10 (10q26). Transfection of the full-length hBRAG cDNA increased levels of human RAG1 transcripts in the B cell line OCI LY8-C3P, but not in the non-lymphoid line K562, suggesting a B cell-specific role for the hBRAG product in regulating RAG expression.

Isolation of genes negatively or positively co-expressed with human recombination activating gene 1 (RAG1) by differential display PCR (DD RT-PCR).

Differential display PCR (DD RT-PCR) has been extensively used for analysis of differential gene expression, but continues to be hampered by technical limitations that impair its effectiveness. In order to isolate novel genes co-expressing with human RAG1, we have developed an effective, multi-tiered screening/purification approach which effectively complements the standard DD RT-PCR methodology. In 'primary' screens, standard DD RT-PCR was used, detecting 22 reproducible differentially expressed amplicons between clonally related cell variants with differential constitutive expression of RAG mRNAs. 'Secondary' screens used differential display (DD) amplicons as probes in low and high stringency northern blotting. Eight of 22 independent DD amplicons detected nine independent differentially expressed transcripts. 'Tertiary' screens used reconfirmed amplicons as probes in northern analysis of multiple RAG-and RAG+sources. Reconfirmed DD amplicons detected six independent RAG co-expressing transcripts. All DD amplicons reconfirmed by northern blot were a heterogeneous mixture of cDNAs, necessitating further purification to isolate single cDNAs prior to subcloning and sequencing. To effectively select the appropriate cDNAs from DD amplicons, we excised and eluted the cDNA(s) directly from regions of prior northern blots in which differentially expressed transcripts were detected. Sequences of six purified cDNA clones specifically detecting RAG co-expressing transcripts included matches to portions of the human RAG2 and BSAP regions and to four novel partial cDNAs (three with homologies to human ESTs). Overall, our results also suggest that even when using clonally related variants from the same cell line in addition to all appropriate internal controls previously reported, further screening and purification steps are still required in order to efficiently and specifically isolate differentially expressed genes by DD RT-PCR.

Up-regulation of recombination activating gene expression by signal transduction through the surface Ig receptor.

Recent evidence has demonstrated that a signal transduced through the lymphocyte Ag receptor may regulate the expression of the recombination activating genes (RAG). Although several groups have shown that such a signal may be required to down-regulate RAG-1 and RAG-2 after a functional Ag receptor has been generated in early mature T or B cells, recently it has been suggested that under some circumstances, cross-linking the B cell Ag receptor may result in up-regulation of RAG expression. To study this possibility, we used a unique set of cell variants isolated from a human mature B cell line, which differ in their expression of both the surface Ig receptor (sIg) and RAG-1 and RAG-2 genes. Two forms of stimulation were employed to generate a signal; either a soluble F(ab')2 anti-mu fragment or the combination of PMA and ionomycin. Northern blot analysis demonstrated that RAG-1 mRNA levels were increased in sIg+ variants after cross-linking with anti-mu. Increases were also observed in all variants after stimulation with PMA and ionomycin. Further analysis of cross-linked sIg+ variants suggests that the observed up-regulation in RAG expression was a reversible event. Furthermore, we have determined that both increased transcription and transcript stabilization contributed to this inducible up-regulation. We thus describe a mature B cell line in which RAG expression is up-regulated after sIg cross-linking. This finding is discussed in the context of its potential role in situations where sIg+ B cells may undergo secondary rearrangements for the purpose of "editing" their sIg receptors.

Rearrangement and expression of the human psi C lambda 6 gene segment results in a surface Ig receptor with a truncated light chain constant region.

The constant region of the human Ig lambda locus consists of seven tandemly organized J-C gene segments. Although it has been established that the J-C lambda 1, J-C lambda 2, J-C lambda 3, and J-C lambda 7 gene segments are functional, and code for the four distinct Ig lambda isotypes found in human serum, the J-C lambda 4, J-C lambda 5, and J-C lambda 6 gene segments are generally considered to be pseudogenes. Although one example of a functional J-C lambda 6 gene segment has been documented, in the majority of cases, J-C lambda 6 is rendered nonfunctional by virtue of a single duplication of four nucleotides, creating a premature translational arrest. We show here that rearrangements to the J-C lambda 6 gene segment do occur, and that such a rearrangement encodes an Ig lambda protein that lacks the terminal end of the constant region. We also show that this truncated protein is expressed on the surface with the IgH chain, creating an unusual surface Ig (sIg) receptor (sIg delta CL). Cells that express this receptor on the surface do so at significantly reduced levels compared with clonally related variants, which express sIg receptors with conventional Ig lambda L chains. However, the effects of sIg cross-linking on tyrosine phosphorylation and surface expression of the CD25 and CD71 Ags are similar in cells that express conventional sIg receptors and in those that express sIg delta CL receptors, suggesting that the latter could possibly function as an Ag receptor.