Proc. Natl. Acad. Sci. USAVol. 86, pp. 5552-5556, July 1989Immunology

Immunoglobulin A light-chain-related genes 14.1 and 16.1 areexpressed in pre-B cells and may encode the humanimmunoglobulin t light-chain proteinGREGORY F. HOLLIS*t, ROBERT J. EVANS*, JEANNINE M. STAFFORD-HOLLISt, STANLEY J. KORSMEYER§,AND JOHN P. MCKEARNt*Department of Biological Sciences, Monsanto Co., Saint Louis, MO 63198; tMolecular and Cellular Biology Department, G. D. Searle Co., Saint Louis, MO63198; and §Howard Hughes Medical Institute, Department of Medicine and Department of Microbiology and Immunology, Washington University School ofMedicine, Saint Louis, MO 63110

Communicated by Howard A. Schneiderman, April 10, 1989 (received for review December 6, 1988)

ABSTRACT Human pre-B cells, which produce immuno-globulin heavy chain but do not produce immunoglobulin lightchain, are shown to contain a 1-kilobase transcript homologousto immunoglobulin A light-chain genes. Detailed analysis ofRNA and cDNA clones derived from these transcripts revealsthat they originate from the distinct immunoglobulin A-likegenes 14.1/16.1. Sequence analysis of these clones reveals along open reading frame, beginning with an ATG, capable ofencoding a protein of 214 amino acids with an unprocessedmolecular weight of 22,944. The C-terminal half of this pre-dicted protein is highly homologous to immunoglobulin Alight-chain joining and constant region protein sequence, whilethe amino-terminal end does not share homology with variableregions. Unlike immunoglobulin genes, these genes do notundergo rearrangement prior to expression. Analysis ofa panelof 26 hematopoietic cell lines reveals that expression of 14.1/16.1 is limited to pre-B cells and one B-cell line, which, like thepre-B cells, is surface immunoglobulin negative. Antiseraraised against a peptide whose sequence was predicted from the14.1 cDNA sequence identifies a 22-kDa protein in humanpre-B cells. Immunoprecipitation of immungobulin Iz-chainfrom these pre-B cells with anti-immunoglobulin la antibodycoprecipitates a 22-kDa protein, which is a candidate for thehuman immunoglobulin t light-chain protein and may be theprotein product of the 14.1/16.1 genes.

The developmental stages by which B cells mature frompluripotent stem cells can, in part, be distinguished by therearrangement and expression ofimmunoglobulin heavy- andlight-chain genes (1-6). In this ordered progression, therearrangement and expression ofheavy-chain genes precedesthat of light-chain genes. Pre-B cells have rearranged heavy-chain genes and express a cytoplasmic form of immunoglob-ulin ,z-chain in the absence of light chain. The rearrangementand expression of light chain, which enables the formation ofH2L2 immunoglobulin tetramer (H, heavy chain; L, lightchain), is a mark of the transition from the pre-B- to B-cellstage of development.

In our characterization of the human immunoglobulin Alight-chain locus, we discovered and cloned two genes, 14.1and 16.1, which are related to the A light-chain genes (7).These genes contain open reading frames homologous tothose of the immunoglobulin A joining and constant regions(JA and CA).

In this report, we describe an analysis of the expression ofthe 14.1/16.1 genes and their protein product. Our resultsindicate that these genes are expressed in human pre-B cellsas a 1.0-kilobase (kb) transcript, which contains homology to

immunoglobulin JA and CA. Unlike immunoglobulin genes,which undergo rearrangement prior to expression, the maturetranscript is derived from unrearranged 14.1/16.1 genes.Immunoblotting and immunoprecipitation analysis suggeststhat the protein product of these genes may be the immuno-globulin c-chain, which is associated with the immunoglob-ulin pL-chain in pre-B cells (8).

MATERIALS AND METHODSCell Lines. The human pre-B-cell lines Reh, RS4;11,

Nalml, Nalm6, and HPB Null have been characterized indetail including the state of immunoglobulin gene rearrange-ment as well as studies of their immunological markers(9-11). Of importance, Reh and RS4;11 have rearrangedheavy-chain loci but do not express heavy-chain protein. Theremaining cell lines used in this study are listed in Table 1(12-14).DNA and RNA Extraction Analysis. DNA was prepared and

analyzed directly from the cell lines as described (15, 16).RNA extractions and blots were performed as described (17,18). DNA and RNA blots were hybridized to the following32P-labeled human DNA probes: (i) 300-base-pair (bp) SstI/BamHI fragment from clone 16.1, which contains homol-ogy to human immunoglobulin CA (probe A); (ii) 200-bp SmaI fragment from clone 16. 1, which is 72% homologous to the39-bp coding region of the human immunoglobulin JA (probeB).cDNA Library Construction. cDNA was prepared essen-

tially by the method of Gubler and Hoffman as modified byShaper et al. (19, 20). The cDNA was ligated into the EcoRIrestriction site of Agtl0 and packaged (Promega Biotec).Recombinant bacteriophage (250,000) were screened usingthe 200-bp Sma I fragment from clone 16.1 (probe B), and 20positive clones were isolated.

Nucleotide Sequence Analysis. The DNA sequence analysiswas determined by the dideoxynucleotide chain-terminationmethod using the United States Biochemical SequenaseDNA sequencing kit. Nucleic acid alignments and transla-tions were done using the University of Wisconsin Sequenceanalysis software package (21).

Immunoblotting. Rabbit antisera were generated to a pep-tide (TGPRCWPRGFQSKHNS) whose sequence was pre-dicted from the 14.1 cDNA sequence. B and pre-B cells werelysed, electrophoresed, and electroblotted onto nitrocellu-lose membranes and immunoblotted essentially as described(22). The nitrocellulose blot was probed with a 1:10,000dilution of immune rabbit serum, 5 ,ug of immunizing peptide

Abbreviations: J, C, and V, joining, constant, and variable regions.tTo whom reprint requests should be addressed at: AA4C, 700Chesterfield Village Parkway, Monsanto Co., Saint Louis, MO63198.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Table 1. Cell lines analyzed for the expression of 14.1/16.1Differentiation type/ 14.1/16.1

name of clone Origin expressionPre-B cellReh Acute lymphoblastic leukemia +RS4;11 Acute leukemia +Nalml Chronic myelogenous leukemia +Nalm6 Acute lymphoblastic leukemia +HPB-Null Acute lymphoblastic leukemia +

Mature B cellSUDHL4 Diffuse histiocytic lymphomaSUDHL9 Diffuse histiocytic lymphoma +DHL6 Diffuse histiocytic lymphoma -

Robinson Chronic lymphocytic leukemia -

Ferrel Chronic lymphocytic leukemia -

IARCLY67 Burkitt lymphomaRaji Burkitt lymphomaBL33 Burkitt lymphomaBHM23 Burkitt lymphomaRPMI 8432 EBV transformedSB EBV transformedRPMI 8392 EBV transformedCESS EBV transformedBALM1 Acute lymphoblastic leukemia -

T cellRPMI 8402 Acute lymphoblastic lymphoma -

HSB-2 Acute lymphoblastic lymphoma -

MOLT-4 Acute lymphoblastic lymphoma -

HuT 102 Mycosis fungoidesOther lineage cellsU-937 Monoblastic leukemia -

HL-60 Promyelocytic leukemia -

K-562 Chronic myelogenous leukemia -

EBV, Epstein-Barr virus.

plus a 1:10,000 dilution of immune rabbit serum or a 1:10,000dilution of preimmune rabbit serum, labeled with '25I-proteinA and autoradiographed.

Immunoprecipitations. Cells (10 x 106) were labeled with[35S]methionine and lysed in 1 ml of 1% Nonidet P-40 inphosphate-buffered saline containing 1 mM phenylmethyl-sulfonyl fluoride. The cell lysate was clarified and preclearedwith Sepharose 4B normal goat serum beads, and 200-1.lpacked volume of Sepharose 4B beads coupled to goatanti-human IgM (Sigma) was added and shaken overnight at40C. The beads were spun down and washed, and 100 ,ul ofreducing sample buffer [15 mM Tris HCl, pH 6.8/2% SDS/10% (vol/vol) glycerol/10% (vol/vol) dye solution/5% (vol/vol) 2-mercaptoethanol] was added and the mixture wasboiled for 2 min. Samples were electrophoresed on a 15%reducing SDS/polyacrylamide gel, the gel was dried, and thebands were visualized by autoradiography.

RESULTSWe have previously reported the cloning and initial charac-terization oftwo gene sequences (14.1 and 16.1), which shareextensive homology with the human immunoglobulin A genes(7). We were interested in determining whether these geneswould exhibit a B-cell lineage-specific expression pattern. Todetermine this, we prepared total RNA from five humanpre-B-cell lines (Reh, RS4;11, Nalml, Nalm6, and HPBNull), as well as a more mature A-producing B cell repre-sented by the Burkitt lymphoma line BL33, and performed aNorthern blot analysis. Probe A, from a region of the 16.1clone, which shares strong homology with 14.1(96%) and thehuman immunoglobulin CA (CAl, 88%), revealed that all of thepre-B-cell lines, as well as the Burkitt lymphoma line BL33,expressed 1-kb transcripts that strongly hybridized to this

probe (unpublished results). Because probe A is 88% homol-ogous to CA genes (7, 23, 24), it is expected to cross-hybridizeto transcripts from this gene locus. The hybridization seen tothe BL33 line RNA most likely represents this cross genehybridization. Thus, an immunoglobulin A homologous tran-script was evident among pre-B cells including those withgerm-line configuration of the classic immunoglobulin Alocus.Northern Analysis with a Probe from an Upstream Exon.

Characterization of the 14.1 and 16.1 genes located a se-quence that shares homology with an immunoglobulin JAregion (7). This area of the genomic clone was a likelycandidate for an upstream exon. A 300-bp Sma I fragment(probe B) that includes this region was prepared from clone16.1. This fragment shares >95% homology with the sameregion from clone 14.1 (R.J.E., G.F.H., and H. Chang,unpublished results). This probe should not cross-react withimmunoglobulin A transcripts under our experimental condi-tions because homology is imperfect and is limited to the39-bp J coding region. When this fragment was used to probeRNA from the pre-B cells and the human immunoglobulinA-producing B cell, BL33, a 1-kb band was identified in all ofthe pre-B-cell lines (Fig. 1), but no hybridization with BL33RNA was observed. Northern analysis onRNA isolated from14 mature B-cell lines, 4 T-cell lines, and 3 other leukemic celllines indicated that these cell lines, with the exception ofSUDHL9, do not express this transcript (Table 1) (12-14).SUDHL9, previously characterized as a mature B cell, hasrearranged immunoglobulin 'y and K genes, but it has germ-line immunoglobulin A genes (14). Interestingly, this line, like

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FIG. 1. Northern blot analysis ofRNA from five pre-B cells with16.1 J region-containing exon. Total RNA from five pre-B-cell lines(Reh, RS4;11, Nalml, Nalm6, and HPB Null) and one immunoglob-ulin A-producing Burkitt lymphoma (BL33) were analyzed by North-ern blot. The blot was hybridized with probe B, which is a 200-bpSma I fragment from 16.1 that contains homology to human immu-noglobulin JA. A 1-kb transcript is seen in all five pre-B-cell lines butnot in BL33.

Immunology: Hollis et A

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the pre-B cells, does not express surface immunoglobulin.These results demonstrate that the transcripts seen in thepre-B-cell lines are distinct from human immunoglobulin Agene transcripts and are in fact the product of 14.1/16.1 genetranscription.cDNA Cloning and Sequence Analysis. We prepared a

cDNA library in AgtlO from poly(A)+ RNA isolated from thepre-B-cell line Nalm6. Recombinant bacteriophage (250,000)were screened with probe B from 16.1, which contains the JAregion homology. Twenty positive clones were purified andsix were characterized in detail. A restriction map of one ofthese clones, Hom-1, is shown in Fig. 2 (Lower). CloneHom-1 was sequenced in its entirety and revealed an insertof 847 nucleotides (Upper). Translation of the nucleotidesequence revealed the presence of a long open reading framebeginning from an ATG triplet at nucleotide 119 and extend-ing to nucleotide 760. Nine nucleotides upstream from thisATG is an in-frame stop codon. This open reading frame iscapable of encoding a protein 214 amino acids long with apredicted molecular weight of 22,944. Nucleotide alignmentprograms revealed that this cDNA contains identical se-quence to the immunoglobulin C and J region homology fromclone 14.1 and therefore identifies Hom-1 as a transcriptderived from the 14.1 gene. The immunoglobulin C regionhomology of the Hom-1 cDNA appears to encode an immu-noglobulin protein domain that includes conserved cysteines

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Proc. Natl. Acad. Sci. USA 86 (1989)

normally involved in intrastrand disulfide bond formation.This sequence also contains a cysteine residue at the penul-timate amino acid. A cysteine in this position of immuno-globulin A proteins is involved in the disulfide bond linkingimmunoglobulin heavy and light chains and suggests a similarrole for this amino acid in the 14.1 protein. In contrast to aclassic immunoglobulin A transcript, the region 5' of theHom-1 J region is not homologous to an immunoglobulin VA(V, variable). Sequence analysis of the five additional inde-pendently isolated cDNA clones demonstrated that theywere all derived from the 14.1 gene.

Southern Analysis. Immunoglobulin genes undergo geneticrearrangement events as a prerequisite for expression (1-6).To determine whether the 14.1 and 16.1 genes are rearrangedin these four pre-B-cell lines, total genomic DNA was pre-pared from these lines as well as BL33 and a T-cell line,MOLT-4. When probe B was used in a Southern analysis ofthese DNAs, a germ-line pattern was observed for all of thepre-B-cell DNAs as well as the B- and T-cell DNA controls(unpublished results).Immunoblot Analysis. Antiserum was raised in rabbits to a

peptide 5' of the J region homology, which is unique to the14.1 gene product. When this antiserum was used to probetotal protein extracts from five human pre-B cells (Reh,RS4;11, Nalml, Nalm6, and HPB Null) and two B-cell lines(BL33 and DHL6), a 22-kDa protein unique to the pre-B cells

90

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GCCCCACATGGACTGGGGTGCAATGGGACAGCTGCTCCAGCGAGKAGCAGGCACCA¢GiCTCTCTA¢GGA¢CCCACACTGCAC¢TCAG¢CCACAAGGAC

116 130 160 170 190CTCTGACCCTGAGGGCCGATGAGGCCAGGGACAGGCCAGGGGGGCCTTGAGGCCCCTGGTGAGCCAGGCCCCAACCTCAGGCAGCGCTGGCCCCTGCTOC

i R P C T C Q C C L E A P C E P C P N L R Q R W P L L L

210 230 260 270 290TGCTGGGTCTGCCGTGGTAACCCATGGCCTGCTCCCcCACACATOCATCGCAGAGCAGGCCCTG CCTGGAGCCCCTGGAGGAAOCAGCCGGTC

L G L A V V T H G L L R P T A AS Q S R A L G P G A P G G S S R S

310 330 360 370 390301 CAGCCTGAOGAGCCGT¢G~GCAGGTTCCTGCTCCAOC~CGOCTCCTGOiCTGCcCCAGG¢TCTG¢CCcC¢CGOGTCATCCAAGCCTAACTCAGTG

S L R S R W G R F L L Q R G S W T G P R C W P R G F Q S K H N S V

410 430 460 470 490

401 ACGCATGTGMGGCAGCGCGACCCAOCTCACCGTTTTAMGTCAGCCCAMGCCACCCC¢TCGGTCCTCGTTCCCOCCGTCCTCTGAGAC.CTCCMGT H V F G S G T Q L T V L S Q P K A T P S V T L F P P S S E E L Q A

I--------------J Region------------I -------------------------------------------.

516 630 560 670 690601 CCAACAAGGCTACGCTGGTGTGTCTCATCAATGACTTTATCCGGOMTCTTOACGGTCACCTGGMAGCAGATGGTACCCCCATCACCCAOGGCGTGGA

N K A T L V C L M N D F Y P C I L T V T W K A D C T P I T Q G V E-------_----------------------------C R*cion---------------------------------------------------------

816 630 656 676 606

601 GATGACCAC¢CCCTCCAAGCCAA^CCAACAATAC¢GCGCCA¢CA¢CTACCTGAGCCTGAC¢CCCCGAGCA¢TGGACCTCCCGCAGAAGCTACAGCTGCi T T P S K Q S N N K Y A A S S Y L S L T P E Q W R 'S R R S Y S C

716 730 760 770 790702 CAGGTCATCCiACGAAC¢~CACCGT¢>CoAGACOCTC¢CCCCCTOCAC.AoTTTCATA¢CTTCCCAICiCCGCACCCCCC~iCAAGC¢CCTGACT¢CA¢

Q V i H E C S T V E K T V A P A E C S

810 830

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CI 0./I.

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FIG. 2. Sequence and re-striction map of Hom-1 cDNA.(Upper) Sequence analysis ofthe Hom-1 cDNA clone is shownwith predicted amino acid se-quence. One long open readingframe capable of encoding a pro-tein of 214 amino acids (single-letter code) is presented.(Lower) A restriction map andsequencing strategy, showingtranslation start and termina-tion, for the Hom-i cDNA cloneis depicted. UT, untranslatedregion.

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was identified (Fig. 3). Addition of the immunizing peptide tothe immunoblot incubation mixture completely inhibitedbinding of the antiserum to the 22-kDa protein. Althoughseveral other protein bands are identified in this immunoblot,they are also identified by the preimmune serum and are notaffected by the addition of the immunizing peptide (Fig. 3).This experiment demonstrates that the product of the 14.1gene is a 22-kDa protein expressed in pre-B cells.

Immunoprecipitations with Anti-Immunoglobulin it-Chain.Pre-B cells synthesize a cytoplasmic form ofimmunoglobulinpL-chain that has traditionally been believed to exist with noassociated immunoglobulin light chain. However, Pillai andBaltimore (8) have recently shown that in murine pre-B cells,heavy-chain 1 protein exists in association with a light chainthey have called w. The presence ofan immunoglobulin A-liketranscript and protein product in human pre-B cells suggeststhat this protein might exist in complex with the humanintracellular immunoglobulin 1L-chain. We tested this idea byusing anti-immunoglobulin .L-chain to immunoprecipitate theintracellular heavy-chain protein a from four pre-B cells andtwo B cells. From the control immunoglobulin A- and K-producing B cells, BL33 and DHL6, the heavy-chain ,uprotein was precipitated in complex with the expected light-chain proteins (Fig. 4). Of the four pre-B cells, only two(Nalm6 and HPB Null) have precipitable ,u protein. Interest-ingly, two additional proteins, whose sizes are 22 and 16 kDaand which migrate faster than the immunoglobulin K and Aproteins, were coprecipitated with ,u from these pre-B cells(Fig. 4). Consistent with published reports, the remaining twopre-B-cell lines, Reh and RS4;11, contained no immunopre-cipitable ,u heavy chain. In these cells, the 22- and 16-kDabands were not seen. The coprecipitated 22-kDa protein isidentical in size to the 14.1 gene product, which suggests thatthe 14.1 gene may encode this p-associated protein.

DISCUSSIONWe have previously characterized two genomic clones, 14.1and 16.1, which contain open reading frames homologous toimmunoglobulin JA and CA regions. In this report, we dem-onstrate that these genes are expressed in human pre-B cellsas a 1-kb transcript. Analysis of cDNA clones made fromthese transcripts reveals a long open reading frame beginningfrom an ATG triplet that is capable of encoding a protein of214 amino acids. This protein resembles an immunoglobulin

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FIG. 4. Anti-ju immunoprecipitations of pre-B-cell proteins. Fourpre-B cells (Reh, RS4;11, Nalm6, and HPB Null) and immunoglob-ulin A- and K-producing B cells (BL33 and DHL6, respectively) werelabeled with [35S]methionine and heavy-chain .t was precipitatedwith anti-s antibody. An autoradiograph of a 15% reducing SDS/polyacrylamide gel loaded with the precipitates is shown. A prom-inent it heavy-chain band is seen in BL33, DHL6, Nalm6, and HPBNull. The lanes loaded with the BL33 and DHL6 immunoprecipita-tion have coprecipitated bands whose migration is consistent withbeing the associated immunoglobulin A and K light chains, respec-tively. Nalm6 and HPB Null contain 22- and 16-kDa protein bands.

A light chain in its C-terminal half sharing homology with bothJ and C regions. The area upstream of this region does notexhibit extensive homology to VA regions. We have analyzedsix cDNA clones and all are derived from the 14.1 gene.Analysis of the 16.1 gene has not revealed any structuralreason that would prevent its appearance as a cDNA clone.One explanation for its absence may be that the 16.1 gene isexpressed at lower levels than the 14.1 gene in the cell lineNalm6. Its absence from our cDNA clones may be due to thesmall number of samples. In our Northern analysis, wecannot distinguish between 14.1 and 16.1 transcripts becausethe genes are so similar (>96%), and therefore transcriptsfrom both may be present.We have examined 26 hematopoietic cell lines for the

expression of the 14.1/16.1 genes. In this survey, all 5pre-B-cell lines express the 1-kb 14.1/16.1 transcript. Of theremaining 21 cell lines, only SUDHL9, characterized as amature B cell, contains this transcript. SUDHL9 has rear-

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FIG. 3. Immunoblot of pre-B cellswith anti-14.1 antiserum. Total proteinextracts from five pre-B cells (Reh,RS4;11, Nalml, Nalm6, and HPB Null)and two B cells (BL33 and DHL6) wereanalyzed by immunoblotting with a rab-bit anti-14.1 antiserum. A 22-kDa proteinwas identified by the antiserum in the fivepre-B cells but not in the two B-cell lines(Immune Serum lanes). The specificity ofthis binding is demonstrated by blockingwith the immunizing peptide (ImmuneSerum + Peptide lanes). Backgroundbands are identified by immunoblottingwith preimmune serum (Pre-immune Se-rum lanes).

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ranged immunoglobulin y and K but germ-line configurationof the immunoglobulin A genes and, like pre-B cells, has nosurface immunoglobulin. Previously, this cell was believed toexpress an immunoglobulin A RNA (14); however, our resultssuggest that this transcript is derived from the 14.1/16.1genes. In fact, analysis of immunoglobulin A expression ofNorthern blotting or anti-immunoglobulin A antibodies mayincorrectly score cells expressing the 14.1/16.1 genes asimmunoglobulin A positive, because the 14.1/16.1 genesshare such extensive homology with immunoglobulin A.Immunoglobulin genes undergo a series of rearrangements

that are essential for the formation and production of heavy-and light-chain protein (1-6). Southern analysis of thesegenes in the pre-B cells in which they are expressed did notreveal gene rearrangement. Based on this Southern analysisand comparison of the Hom-1 cDNA clone to the germ-lineclones, it appears that these genes are expressed from agerm-line gene.

Recently, Melchers and his colleagues (25-27) have de-scribed a gene termed A5 from the mouse. This gene isexpressed exclusively in pre-B cells and the C-terminal endof the encoded protein shares homology with the mouseimmunoglobulin A light-chain J and C regions. The N-terminal half of the As protein does not encode an immuno-globulin VA region, but it does share 38% homology at theprotein level with the 14.1 gene. The similarity of the expres-sion pattern and structure of the 14.1 and A5 genes suggeststhat they may be human/mouse homologs.

Pillai and Baltimore (8) have shown that intracellularimmunoglobulin A in mouse pre-B cells is associated with alight-chain protein of 18 kDa, which they have termedimmunoglobulin w. We have shown that in human pre-Bcells, ,u heavy chain is associated with two proteins 22 and 16kDa in size. Our immunoblot analysis demonstrates that the14.1 gene encodes a protein of 22 kDa. This protein containsa cysteine as the pre-terminal amino acid, which is theposition involved in the immunoglobulin light-chain-heavy-chain disulfide bridge. The size and structure of theprotein encoded by the 14.1 gene suggests that it may encodethe 22-kDa protein associated with intracellular ,u heavychain in pre-B cells and therefore is a candidate for the humanimmunoglobulin w gene.Melchers and his colleagues (28-30) have identified a

pre-B-cell-specific gene, termed VpreB. The predicted pro-tein of this gene contains a single immunoglobulin domain,which is homologous to immunoglobulin V regions. Whenexamined together, the VpreB gene and 14.1 gene productsencode V-J-C region sequences, the components found in animmunoglobulin light-chain protein. We are using our immu-nological probes to determine whether these two gene prod-ucts are functioning as "light-chain surrogates" in pre-B cellsand whether they encode the 16- and 22-kDa immunoglobulin,u coprecipitated bands.There is mounting evidence that immunoglobulin heavy-

chain allelic exclusion is mediated by a functionally rear-ranged immunoglobulin heavy-chain gene in pre-B cells,which shuts off further V to DJ recombination (D, diversityregion) and allows light-chain gene rearrangement to begin(31-35). The formation of a A heavy-chain surrogate lightchain(s) complex in pre-B cells may be an essential step inthis process. The work presented here suggests that theproducts of the 14.1/16.1 genes may be an integral part of thiscomplex.

We thank Dr. L. Mengle-Gaw for critically reading this manu-script, Dr. P. Manning for helpful discussions, and Mr. J. Bullock andDr. S. Adams for antigen preparation.

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