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Subtractive cDNA cloning of a novel member of the Ig gene superfamily
expressed at high levels in activated B lymphocytes
EJ Kozlow, GL Wilson, CH Fox and JH Kehrl
Laboratory of Immunoregulation, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
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.
Volume 81,
Issue 2,
pp. 454-461,
01/15/1993
Copyright © 1993 by The American Society of Hematology
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