Subtractive cDNA cloning of a novel member of the Ig gene superfamily expressed at high levels in activated B lymphocytes

Abstract

Using subtractive cDNA cloning we have isolated a series of cDNA clones that are exclusively or selectively expressed in B lymphocytes. mRNA transcripts from one such cDNA clone, referred to as BL11, were found to be expressed at low levels in RNA from normal B lymphocytes, but at very high levels in RNA from in vitro activated B lymphocytes. One major 2.5-kb BL11 mRNA transcript was detected, while low levels of 4.8-, 1.8-, and 1.6-kb transcripts were also found. BL11 mRNA transcripts were absent or present at low levels in RNA prepared from resting or mitogen activated T cells, a variety of lymphoid cell lines including several B-cell lines, and several different tissues. Low levels of BL11 transcripts were found in poly(A) RNA purified from brain and lung. A study of the kinetics of BL11 mRNA accumulation in B lymphocytes stimulated in vitro with Staphylococcus aureus Cowan strain I showed a rapid induction of BL11 mRNA within 2 hours of stimulation with peak expression by 16 hours and a mild decrease with time following the peak levels. Consistent with the in vitro data, in situ hybridization using antisense BL11 RNA probes and human tonsillar tissue localized BL11 transcripts in B-cell-enriched areas. Multiple BL11 cDNA and genomic clones were isolated and sequenced to complete and verify the BL11 cDNA sequence (2,404 bp). A 615-nucleotide open reading frame predicted to encode for a 205-amino acid protein with a molecular weight of 23 Kd was identified. Search of protein data bases with the predicted BL11 protein showed homologies to several members of the Ig superfamily. Analysis of the predicted protein showed a likely signal peptide, a single membrane spanning region, and one V-like Ig domain with three predicted n-glycosylation sites. Southern blot analysis of human genomic DNA suggested that BL11 is a single copy gene without evidence of rearrangement. Primer extension and S1 nuclease mapping identified four tightly clustered transcriptional start sites approximately 40 bp upstream of the predicted translation start site. The first 270 bp of the promoter region were sequenced and found to contain a CATAA box rather than a TATAA box and several DNA motifs found in activation genes. BL11 should prove to be an interesting gene that likely encodes for a protein involved in B-cell activation. This is a US government work. There are no restrictions on its use.