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 cc-expresse d with human recombination activating gene-1 (RAGI). A 5.0-kb transcri pt detected by the differential display amplicon 3G1 was found to corr elate strongly with RAG1 mRNA expression in various human cell lines. Subsequent screenings of a pre-B cDNA library with 3G1 led to the iden tification of a complete cDNA we have termed hBRAG (human B-cell RAG-A ssociated Gene). The hBRAG cDNA encodes a 503-amino acid (aa) protein with no known homology to any nucleotide or protein sequence. The pred icted molecular mass of 55 kDa was confirmed by in vitro translation. Based on sequence analysis, the predicted open reading frame encodes f or a type II transmembrane spanning glycoprotein with the N-terminal 8 1-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 hB RAG mRNA transcript with RAG1 in numerous human pro-B, pre-B and matur e B cell lines assessed, but not in human T cell lines. In human tissu es, hBRAG is expressed at highest levels in B cell-enriched tissues, b ut is not expressed in fetal or adult thymus. Southern blotting analys is revealed that this gene is conserved across eukaryotes, is expresse d 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 chromos ome 10 (10q26). Transfection of the full-length hBRAG cDNA increased l evels of human RAG1 transcripts in the B cell line OCI LY8-C3P, but no t in the non-lymphoid line K562, suggesting a B cell-specific role for the hBRAG product in regulating RAG expression.