Isolation ofcoordinately regulated genesthatareexpressed in discrete stages ofB-cell development

Wehaveutilized subtractive hybridization to isolate 16distinct cDNAsequences representing genesex- pressed inpre-B-cell lines butnotmyeloma cell orfibroblast lines. Thesesequences represent RNAtranscripts that varyin abundance inpre-B-cell lines from0.001% to0.05%. Fiveof these sequences werenotrelated toanyknowngenes. Onewas related tobutdistinct fromknownmyosin regulatory light chain genes andanother encoded aprotein with lectin domains. Threerepresented previously identified genesencoding car- bonic anhydrase typeII,thymosin, andCD2;these genes were notpreviously knowntobespecifically expressed inearly stages ofB-cell development. Otherisolated genescorresponded to pre-B-cell-specific orpre-B-cell/B cell-specific genes recently described byothers. Theisolated cDNAsequences maybe divided into twogeneral categories-those representing genes expressed only inthepre-B-cell stage ofB-cell development and those expressed inboththepre-B-cell andB-cell stages. Thein vivo expression patterns oftheidentified genes suggest that somefunction specifically inlymphocytes while others may haveroles inadditional lineages. Differentiation ofB-lineage cells occurs inthree general stages-the precursor B-cell (pre-B-cell) stage, theB- lymphocyte stage, andtheplasma-cell stage (1). Inmammals, pre-B-cell development occurs intheliver ofthefetus and shifts tobonemarrowinadults. Pre-B-cells first assemble heavy(H)chain variable (V)region genes fromcomponent variable (VH)diversity (D), andjoining (JH) genesegments andsubsequently assemble light (L)chain V region genes fromVLandJLsegments (2). Expression ofH andLchains bypre-B-cells leads tothegeneration ofB lymphocytes, which express complete immunoglobulin molecules ontheir surface (3). B lymphocytes migrate toperipheral lymphoid organs, suchasthespleen andlymph nodes (1). Binding of specific antigen tothemembrane immunoglobulin caninduce aBlymphocyte toproliferate andmature, ultimately gener- ating terminally differentiated, immunoglobulin-secreting plasma cells. T lymphocytes differentiate bya program somewhat analogous tothat ofBcells; inparticular, genes encoding T-cell receptor (TCR) Vregions also areassembled ina programmed fashion inpre-T-cells (4). Assembly of immunoglobulin andTCRV region genesismediated bya shared recombination activity (V-D-J recombinase (5)); re- cently, aV-D-Jrecombination-activating genecalled RAG-1 wasisolated andfound tobeexpressed specifically inpre-B- andpre-T-cells (6). Staging ofB-lineage cells often isbased onhistological analyses, expression oflineage orstage-specific antigens (1, 7,8), thenature ofexpressed immunoglobulin, andthestatus ofimmunoglobulin H andLchain generearrangements (3). Expression patterns ofcertain genes suchasterminal deoxy- nucleotidyltransferase, germ-line VHgenesegments, and certain protooncogenes also havebeencorrelated withdis- crete stages ofB-cell development (9-12). Mostrecently, subtractive hybridization/di fferential screening methods led toisolation oftwogenescalled A5andVPrC-B; specifically expressed inpre-B-cells (13, 14); asingle genecalled mb-1, expressed inbothpre-B-cells andBcells butnotinplasma cells (15); andaB-lineage-specific genecalled B29,expressed inallB-cell stages (16). Although nofunctions havebeen defined fortheA5,VpreB, orB29products, alldisplay homology toimmunoglobulin Lchain domains. Present ev- idence suggests mb-iprotein complexes withmembrane immunoglobulin (15, 17), while ASandVpre Bproteins may associate with kLH chain proteins (18). Identification ofadditional genes expressed specifically in discrete stages ofB-cell development should allow further definition ofthis pathway andelucidation ofnovel stage- specific processes. Toisolate suchgenes, wehaveutilized subtractive hybridization techniques (19-21) toisolate se- quences specifically expressed incelllines representing pre-B-cell butnotplasma-cell stages. We further optimized this technology topermit isolation ofcDNAsrepresenting mRNAsoflowabundance, reasoning that rare mRNAsmight represent regulatory genesnotreadily accessible byother methods.