Expression of J chain RNA in cell lines representing different stages of B lymphocyte...

Cell, Vol. 23, 369-378. February 1981, Copyright 0 1981 by MIT

Expression of J Chain RNA in Cell Lines Representing Different Stages of B Lymphocyte Differentiation

Elizabeth L. Mather,*+ Frederick W. AIt,+ Alfred L. M. BothwelLS David Baltimore* and Marian Elliott Koshland* *Department of Microbiology and Immunology University of California Berkeley, California 94720 *Center for Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary

During B cell differentiation to pentamer IgM secretion, synthesis of the pentamer joining component, the J chain, is initiated. We investigated the mechanism for initiating J chain synthesis by analyzing murine cell lines representing different stages in B cell differentiation. The expression of functional J chain mRNA was evaluated by cell-free translation and specific immunoprecipitation of a J chain product. The expression of precursor mRNA was examined by hybridization with a J chain probe obtained by molecular cloning of cDNA. No J chain-specific RNA could be demonstrated in a lymphoma line representative of an undifferentiated B lymphocyte, but three species of J chain RNA were identified in hybrid cell lines representative of IgM-secreting plasma cells: a mature message of approximately 1.5 kb and two minor components of 2.5 and 0.92 kb. The encounter of a B cell with antigen or mitogen must therefore trigger events that effect either transcription of J chain sequences or their intranu- clear stabilization.

The immunoglobulin J chain has been shown to play a critical role in B cell differentiation to pentamer IgM secretion. The first evidence for such a role was obtained from studies of the mechanism of pentamer assembly (Koshland, 1975). Analyses of the reaction in vitro showed that the pentamer molecule is assembled by crosslinking five IgM monomers and a single J chain through disulfide bonds. The chain initiates the polymerization by forming a disulfide bridge between two IgM monomers, and the resulting J chain- containing dimer then promotes the disulfide bonding of additional monomers to complete the pentamer structure.

A second line of evidence was obtained from the relationship between B cell differentiation and J chain synthesis. The unstimulated B lymphocyte synthesizes monomer IgM and inserts these molecules into the plasma membrane, where they serve as antigen receptors (Vitetta et al., 1971). Upon contact with the

t Present address: The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 191 11

appropriate antigen or mitogen, the cell switches from the synthesis of receptor IgM to the production of pentamer IgM antibody (Melchers and Andersson, 1974). This process is accompanied by significant changes in J chain content. Radioimmunoassays showed that unstimulated populations of splenic lymphocytes contain little or no J chain (Mather and Koshland, 1977). After mitogen exposure, however, there is an immediate increase in intracellular J chain, which precedes the appearance of detectable pentamer IgM in the external medium (Roth et al., 1979). These data indicate that one of the steps involved in 6 cell differentiation to IgM secretion is the activation of J chain synthesis.

The results obtained with normal lymphoid cells were supported by analyses of cell lines representing different stages of B cell differentiation. B cell lympho- mas that display the characteristic membrane IgM phenotype of unstimulated 6 cells were found to have no detectable J chain (Raschke et al., 1979). As expected, plasmacytomas secreting pentamer IgM contained large amounts of J chain, and even plasmacytomas representing a later IgG-synthesizing stage in B cell differentation were found to synthesize significant quantities of J chain (Kaji and Parkhouse, 1974). The latter finding was used in studies of J chain function by somatic cell hybridization (Raschke et al., 1979). By fusing a B cell lymphoma with an IgG- secreting plasmacytoma, it was possible to obtain hybrid cells that secreted pentamer IgM. Analysis of the hybrids showed that the IgM was expressed as a result of complementation between the synthetic capacities of the parental lines. The hybrid ceils synthesized both monomeric IgM and J chain and assembled these components into a pentameric molecule with the correct stoichiometry. These findings provide further evidence that J chain is a differentiation-induced cell product required for the assembly and secretion of pentamer IgM.

There are several possible mechanisms by which J chain synthesis is activated: induction could occur at the cytoplasmic level by unblocking translation of J chain mRNA; alternatively, induction could occur at the nuclear level by stabilizing or processing J chain hnRNA, or by transcribing a previously silent J chain gene. As a first step toward distinguishing among these possibilities, a B cell lymphoma and its daughter hybrid lines were compared for the presence of J chain-specific RNA sequences. The analyses were carried out by isolating poly(A)+ RNA from the lines and assaying the preparations for their capacity to synthesize J chain in a cell-free system and to hybridize with cloned J chain cONA.

Results

Sources of RNA The murine cell lines characterized in previous studies of J chain function (Raschke et al., 1979) were chosen

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as the sources of RNA. Table 1 summarizes the lg- synthesizing capacities of the lines. The WEHI 231 lymphoma has the properties of a mature 6 cell that synthesizes monomer IgM as a membrane receptor, but it does not express J chain. On the other hand, the hybrid lines derived from the fusion of the WEHI 231 lymphoma and the MPC 11 myeloma resemble IgM-secreting plasma cells; both the MXW 231 .l a and the MxW 231 .l b clones synthesize large amounts of IgM and J chain and export correctly assembled pentamer IgM. These characteristics of the lymphoma and hybrid lines make them particularly suitable for assessing changes in the expression of J chain RNA associated with B cell differentiation to pentamer IgM secretion.

Translation of J Chain-Specific RNA The possibility that J chain synthesis is under trans- lational control was investigated by comparing the capacity of the RNA preparations to synthesize J chain in a reticulocyte cell-free system. For these experiments, poly(A)+ RNA from the lymphoma and hybrid lines was translated in a rabbit reticulocyte lysate pretreated with Sl nuclease. The 35S-methionine-labeled products were immunoprecipitated, and the precipitates were analyzed by electrophoresis on alkaline urea/polyacrylamide gels and by fluorogra- phy. The choice of the alkaline gel system was dic- tated by the abnormal behavior of J chain in SDS gel electrophoresis. Although the J chain has a molecular weight of 15,000 daltons, it migrates on SDS gels with an apparent molecular weight of 25,000, similar to that of light chains (Wilde and Koshland, 1973). On alkaline urea gels, however, the J chain exhibits a rapid rate of migration that is highly characteristic.

As expected, poly(A)+ RNA from the hybrid cell lines was found to direct the synthesis of J chain. The

Table 1. lmmunoglobulins Produced by a B Cell Lymphoma. WEHI 231, and Daughter Hybrid Lines, M x W 231 .la and M X W 231.lb

lmmunoglobulin MxW MxW

WEHI 231 231.la 231.lb

Cellular Ig’ Monomer IgM @. IO 3.3 X lo* 1.0 x 106 7.5 x 105

J chain Ob 1.2 x 106 1.3 x 106

IgG (yztn ~‘1 0” 4.2~10~ 0’

K’ chain fragment 0 + +

Secreted Ig” Pentamer IgM 0 3.2 x lo5 1.3 x IO5

IgGzb 0 + 0

Free I(’ chain 0 + +

a Determined by radioimmunoassay; units are molecules/cell. b Less than 100 molecules/cell. ’ Less than 1500 molecules/cell. ’ Determined by radioimmunoassay; units are molecules/hr/cell.

autoradiogram in Figure 1 shows that a fast-moving, component was specifically precipitated from the translation mixture by anti-J chain antibody (lane a). This component was not precipitated by normal rabbit IgG (lane c), and it was readily distinguished by its more rapid mobility from the K chain fragment (Kuehl and Scharff, 1974) that was precipitated by anti-K

chain antibody (lane b). The J protein produced in vitro gave an electrophoretic pattern similar, but not identical, to that of J chain synthesized in vivo (lane h). The differences observed could be attributed to the glycosylation of the intracellular J chain (Neider- meier et al., 1972). The presence of variable amounts of negatively charged sugar residues would produce the faster migration rate and the more diffuse pattern exhibited by the J chain from cell lysates.

No component with the mobility of J chain was observed in comparable analyses of the WEHI 231 translation products (Figure 1 lane d). Moreover, no J chain product could be detected when the sensitivity of the assay was increased by using 5 fold volumes of the translation lysate for immunoprecipitation and 5 fold exposure times (data not shown). From the J chain positive controls, it was estimated that these conditions should have permitted the detection of J chain synthesis at levels less than 1% of those observed with the hybrid cell RNA.

Preparation of a J Chain cDNA Probe As a second approach to analyzing the induction of J chain synthesis, a J chain cDNA probe was synthesized and used to isolate cDNA clones. Several lines of evidence indicated that the probe could not be prepared by the usual procedure of purifying an mRNA template and synthesizing a complementary cDNA. Cell-free translation studies showed that the J chain mRNA constituted a relatively small proportion, 0.2-0.3%, of the hybrid cell poly(A)+ RNA. Moreover, sucrose gradient fractionation studies showed that the J chain mRNA did not have sedimentation properties distinct enough to permit significant additional purification. These data were obtained by translating the RNA sequences in each sucrose gradient fraction and analyzing the products by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. To ensure that the J and K chain electrophoretic patterns could be distinguished, the analyses were carried out sep- arately; the translation mixtures were first reacted with anti-p and anti-K chain reagents, and the supernatants were then treated with anti-J chain antibody. As the yields of translation products in Figure 2A show, the p chain mRNA was concentrated in fractions 6 and 7, the K chain mRNA in fractions 9 and 10 and the K

chain fragment mRNA in fraction 11. The J chain mRNA was found to be located primarily in fraction 9 (Figure 2B) and, because of the overlap with the more abundant K chain mRNA, could not be purified further by size fractionation.

Expression of J Chain RNA in Lymphoid Lines 371

‘/2 K -

-J

Q b c d e f g h i

Figure 1. Translation of J Chain-Specific mRNA

Micrococcal nuclease-treated rabbit reticulocyte lysates were pro- grammed with poly(A)+ RNA at a ratio of 10 ng/gl lysate and a final concentration of approximately 8pg/ml. The reaction volumes ranged from 12.5 to 100 ~1. After incubation for 75 min at 37°C the reaction was stopped by making the solution 1% in Nonidet-P40 and 0.1% in SDS. Aliquots were then immunoprecipitated and the precipitates were analyzed by 4.15% alkaline urea/polyacrylamide gel electrophoresis. Shown are translation products of 50 ng of MxW 231 .l b poly(A)’ RNA precipitated with (a) anti-J chain antibody, (b) anti-k chain antibody and (c) normal rabbit IgG: translation products of 50 ng of WEHI 231 poly(A)+ RNA precipitated with (d) anti-J chain antibody, (e) anti-k chain antibody and (f) normal rabbit IgG; and biosynthetically labeled products from MxW 231 .la cell lysates precipitated with (g) anti-k chain antibody, (h) anti-J chain antibody and (i) normal rabbit IgG. % K = K chain fragment: J = J chain.

In view of these data, an alternative method of probe preparation was used (Alt et al., 1978, 1979). 32P- labeled cDNA was transcribed from the total poly(A)’ RNA of the hybrid line, MXW 231.1 b, and the cDNA sequences complementary to J chain mRNA were then enriched by the negative and positive selection procedures diagrammed in Figure 3. As the first step, the 3’P-labeled cDNA was depleted of sequences not specific to lymphoid cells by hybridization to a large excess of poly(A)’ RNA isolated from L cells, a mouse fibroblast line. The hybridization was allowed to pro- ceed to completion and the remaining single-stranded cDNA was isolated by hydroxyapatite chromatography. Eighteen percent of the radioactivity was recovered in the single-stranded fraction. As the next step, the J chain cDNA sequences were separated from those specifying 1-1 and K chains by hybridizing the 32P-labeled cDNA to poly(A)+ RNA isolated from

vi

A ye and IC Chain Content B J Chain Content

J- Of-

&

6 7 9 IO II 12 7 6 9 IO II 12

Fraction Figure 2. Translation Products of 231 .l b PolyfA)’ RNA after Frac- tionation by Sucrose Gradient Centrifugation

Cell-free translation was carried out as described in the legend to Figure 1, using 2.5~1 of sucrose gradient fraction/l 0~1 of reticulocyte lysate. Equal aliquots of each reaction mixture were incubated overnight at 4°C with anti-IgM b. K) antibody, the immune complexes were separated by Staphylococcus aureus precipitation and the supernatants were then incubated overnight at 4°C with anti-J chain antibody. The immunoprecipitates were analyzed on SDS 12.5% polyacrylamide gels. (A) Results with anti-IgM antibody. a = A chain: K’ = MPC 11 L chain; J = J chain; K = WEHI 231 L chain: ‘/2 K’ = MPC 11 K chain fragment. (6) Results with anti-J chain antibody. Some J chain was co-precipitated in (A) perhaps because of IgM polymerization in vitro, and some K’ chain was co-precipitated with J chain in (B).

the IgA-secreting plasmacytoma, MOPC 315. This cell line was chosen because it synthesizes J chain, but not p or K chains or their respective RNAs (Alt et al., 1980b), and thus J chain 32P-labeled cDNA should form cDNA/RNA hybrids, whereas the p and K cDNA should remain single-stranded. The hybridization was carried out to a Rot of IO mole-set/l, which preliminary experiments indicated was the optimum value for selecting J chain cDNA and minimizing the hybridization of low levels of non-J chain cDNA to less abundant RNA sequences. The hybrid molecules were isolated by hydroxyapatite chromatography, eluted from the resin with high-salt buffer and depleted of RNA by base hydrolysis. The yield of selected cDNA was 12% of that applied to the resin and approximately 1.5% of the original cDNA preparation.

Specificity of the Probe Preparation The specificity of the selected 32P-labeled cDNA was assessed by hybridization to the poly(A)+ RNA from cell lines of the following phenotype: pL+ K+ J+ (M x W 231 .l b); pL+ K+ J- (WEHI 231); and pcL- K- J- (L cells). At a Rot of 10 mole-set/l, 78% of the selected cDNA

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231.lb poly A+ RNA ( K+,J, “housekeeping”)

Negative Selection !

*cDNA

1 Hybridized to Lcell mRNA

(fibroblost)

HAP

J\ *EDNA *cDNA-RNA

l- (K+,J) (“housekeeping”)

Positive

Selection

Hybridized to MOPC 315 mRNA (Q,J)

HAP /\

1 *cDd LA RNA (4 (J:

*cDNA probe Figure 3. Protocol for Enriching for J Chain cDNA Sequences l cDNA = labeled cDNA.

was protected from Sl nuclease digestion by MxW 231.1 b poly(A)+ RNA, whereas at the same Rot, 28% and 17% of the selected cDNA was protected by WEHI 231 and L cell poly(A)+ RNA, respectively. These results showed that some nonlymphoid sequences, as well as some lymphocyte-specific sequences, remained in the selected cDNA. The majori of the selected cDNA sequences, however, hybridized to poly(A)+ RNA species present in moderate abundance in M XW 231 .l b cells, a result that would be expected of cDNA specific for J chain.

More convincing evidence for the specificity of the cDNA probe was obtained by measuring the extent of its hybridization to M X W 231 .l b poly(A)+ RNA fractionated by sucrose gradient centrifugation. For these experiments, aliquots of the selected cDNA were reacted with equal portions of the sucrose gradient fractions described in Figure 2. The conditions of the reaction were chosen such that the extent of hybridization would be roughly proportional to the concentration of complementary RNA sequences (Fan and Baltimore, 19731, and the percentage of hybrid molecules formed was determined by Sl nuclease digestion. The results obtained are compared with the synthetic capacities of the fractionated RNA (Figure 4). The total protein synthesized by each fraction was quantitated from the iadioactivity precipitable by trichloroacetic acid, and the J chain from the radioactivity precipitable by anti-J chain antibody. The comparison shows that the extent of cDNA/RNA hybridization closely paralleled the J chain synthetic capacity of the

fractionated RNA. Maximum hybridization was obtained with the fraction richest in J chain mRNA, and considerably less hybridization was observed with adjacent fractions, which had a high content bf p or K mRNA and a high protein output (see Figures 2 and 4).

Selection and Characterization of J Chain cDNA Clones The J chain cDNA probe was then used to screen bacterial colonies, which were transformed with pBR322 plasmids containing plasmacytoma cDNA inserts. The inserts represented the 0.5-l .O kb fraction of cDNA synthesized from the total poly(A)+ RNA of the IgGnb-secreting cell line, X63. Of approximately 1200 colonies examined, 12 hybridized with the J chain probe. Plasmids were isolated from the positive colonies and analyzed for cDNA inserts by digestion with Pst I and Hinf I restriction enzymes and electrophoresis of the digests on agarose gels. Nine of the 12 clones yielded inserts ranging in size from 0.53- 1.09 kb and three were negative within the limits of detection of the analysis, approximately 0.1 kb.

The specificity of the cDNA inserts was first examined by hybridization selection (Ricciardi et al., 1979). Plasmids from the three clones giving the most intense hybridization signal (pJc clones 1, 2, 3) were immobilized on nitrocellulose filters and hybridized to poly(A)+ RNA isolated from the J chain-synthesizing MxW 231 .l b cells. The bound RNA was eluted and translated in vitro, and the 35S-methionine-labeled products were assayed by gel electrophoresis of the translation mixtures. Analyses of alkaline urea gels (Figure 5 lanes a, b, c) showed that the selected mRNA encoded a polypeptide migrating in the characteristic position of J chain. Although a number of other bands were observed, these bands were also present in the translation products of the control lysate (Figure 5 lane d), and thus they probably reflected the proteins synthesized by a small amount of endoge- nous RNA remaining in the reticulocyte lysate. The identification of the fast-moving polypeptide as J chain was confirmed by immunoprecipitation of the translation mixtures with anti-J chain antibody. As shown in Figure 5 (lanes a’, b’, c’), only a single labeled product was recovered with a mobility identical to the bands seen in the untreated lysates and in the J chain- positive control (lane e). Moreover, comparable results were obtained with the use of SDS gel electrophoresis; both untreated and immunoprecipitated samples of the translation mixtures exhibited single bands that migrated with an apparent molecular weight of 25,000. No other protein is known to have the particular electrophoretic properties of J chain- that is, a very rapid rate of migration on alkaline gels and an anomalously slow rate of migration on SDS gels; these data therefore provide strong evidence


80 1

I I I I I I I I

6 8 IO 12

Sucrose Gradient Fractions

e

,

Figure 4. Analysis of Probe Specificity by Hybridization to MXW 231 .l b Poly(A)’ RNA Fractionated by Sucrose Gradient Fraction

Three microliters of each sucrose gradient fraction were hybridized with the ‘*P-labeled selected cDNA sequences (1300 cpm/aliquot) for 30 min at 45°C and the extent of reaction was assayed by Si nuclease digestion and trichloroacetic acid precipitation of the hybrid RNA/cDNA. (A----A) Percentage of selected cDNA hybridized. (O--O) Total protein synthesized in cell-free translation of the fractionated RNA, as determined by the ‘?S-methionine radioactivity precipitable by trichloroacetic acid. (O----O) J chain synthesized in cell-free translation of the fractionated RNA, as determined by the ?S-methionine radioactivity precipitable with anti-J chain antibody.

that the three plasmids examined contained J chain cDNA sequences.

The cDNA sequences in the six other plasmids (pJc clones 4, 5, 6, 9, 10, 11) were characterized by comparison with one of the clones of established specificity, pJc3. After digestion with the restriction endonucleases, Pst I and Hinf I, the plasmid fragments were separated by agarose gel electrophoresis, transferred to nitrocellulose and reacted with 32P-labeled pJc3 insert. All six inserts and their restriction fragments were found to hybridize with the probe. More- over, the sequences shared with the pJc3 cDNA could be located by restriction mapping as shown in Figure 6. On the basis of these findings, the nine clones selected were judged to be J chain specific.

Hybridization of Cloned J Chain cDNA with Lymphoma and Hybrid Cell RNA Once J chain cDNA clones were obtained, it was possible to compare the lymphoma and hybrid cell RNA for the presence of J chain-specific mRNA and nuclear RNA. Poly(A)+ RNA from these lines and a number of control preparations was size-fractionated on methylmercury hydroxide gels, transferred to diazobenzyloxymethyl (DBM) paper, and hybridized to

a b c d e a' b' c' d' Figure 5. Identification of J Chain cDNA Clones by Hybridization- Selection

M x W 231.1 b p&y(A)+ RNA was hybridized to the immobilized plasmid from three presumptive J chain cDNA clones and to nitrocellulose alone, and the bound RNA was examined for its capacity to direct J chain synthesis in a reticulocyte cell-free system, as described in the legend to Figure 1. Half of the translation mixture was reduced and alkylated and then electrohporesed directly on 4.15% alkaline urea/ polyacrylamide gels; the other half was immunoprecipitated with anti- J chain antibody, and the precipitates were analyzed by alkaline urea/polyacrylamide gel electrophoresis. Shown are entire translation products from RNA selected by (a) pJc1 plasmid. (b) pJc2 plasmid. (c) pJc3 plasmid and (d) nitrocellulose; immunoprecipitated translation products from RNA selected by (a’) pJc1 plasmid, (b’) pJc2 plasmid. (c’) pJc3 plasmid and (d’) nitrocellulose. Included as a positive control (e) are the translation products of 50 ng of I.4 x W 231 .l b poly(A)+ RNA precipitated with anti-J chain antibody.

clone pJc3 plasmid that had been 32P-labeled by nick translation. As expected, the J chain cDNA did not hybridize with RNA from the nonlymphoid L cell line, which does not express J chain. However, the probe did hybridize with the RNA from all the J chain-synthesizing plasmacytomas tested, including the two MxW 231 hybrid cell lines (Figure 7). Three J chain RNA species could be identified: a major band of 1.5 kb and minor bands of 2.5 and 0.92 kb, respectively. The size of each RNA species was determined by comparison with the position of secreted IJ chain mRNA (2.4 kb), K chain mRNA (1.2 kb), and K chain fragment mRNA (0.8 kb) observed when nick-translated K and p chain cDNA plasmids were hybridized to the same DBM paper (Schibler et al., 1978). The relative abundance of the J chain RNAs suggested that the 2.5 kb species could represent a nuclear precursor from which introns are excised to yield a mature message of 1.5 kb. This deduction was consistent with sucrose gradient analyses, which showed that J chain mRNA sedimented slightly more rapidly than did the 1.2 kb K chain mRNA (Figures 3 and 5).

Cell 374

c Ions

pJc3

pJc9

pJc II

pJc6

pJc5

pJc IO

pJc 4

I I I I J

t : I 1

I I

I : I 1 1 I

t :

1 I

I I

I : 25 Kb

Lsnglh of insrrt tbp)

1060

1060

1090

660

640

600

530

Figure 6. Preliminary Restriction Maps of pBR322 Plasmids Containing J Chain cDNA Inserts

The orientation of the pJc3 insert in the vector plasmid was determined by partial sequencing of plasmid-cDNA junction fragments. The 3’ and 5’ designations refer to the mRNA template sequence. The sequences shared between the pJc3 insert and each of six other cDNA inserts were determined by digestion with restriction endonucleases. as indicated by the vertical bars. The identification of the pJc4. pJc9 and pJcl1 sequences did not re- quire the use of the Alu I endonuclease. and thus the absence of a vertical bar in these cases does not signify the absence of Alu I cleavage sites.

Thus the mature J chain message was found to be considerably larger than that necessary to code for a J protein of approximately 120 amino acid residues (Mole et al., 1977). In this respect the message may be similar to several eucaryotic mRNAs that have been shown to have a large 3’ untranslated region, such as mouse p-globin mRNA (Proudfoot and Brownlee, 1976) and dihydrofolate reductase mRNA (Nunberg et al., 1960).

In contrast to the results obtained with the hybrid cell poly(A)+ RNA, the J chain cDNA probe failed to hybridize with the poly(A)+ RNA from the WEHI 231 lymphoma. No bands were detected after 14 hr of exposure as shown in Figure 7, or after six days of exposure (data not shown). The non-poly(A)-containing RNA from the lymphoma cells was equally negative. From the conditions used for hybridization it was calculated that the limit of detection of the analyses was 3 x 1 O5 molecules, or less than one molecule of J chain RNA per 100 cells. Thus the hybridization data supported the finding from radioimmunoassays that the 6 cell lymphoma contains no detectable J protein, and the finding from cell-free translation experiments that the lymphoma has no detectable mRNA coding for J chain. Moreover, the hybridization data suggested that the B cell lymphoma does not tran- scribe the J chain gene.

Discussion

The studies presented in this paper are consistent with a transcriptional mechanism for activating J chain synthesis. The evidence was obtained by comparing the expression of J chain RNA in cell lines representing different stages of B cell differentiation. J chain- specific RNA was readily demonstrable in hybrid cell lines resembling plasma cells differentiated to pentamer IgM secretion. Cell-free translation analyses showed that RNA from these cells can direct the

synthesis of a protein that is precipitable by anti-J chain antibody and is similar in charge and size to the J chain produced in vivo. RNA blotting procedures showed that the hybrid cells contain three species of J chain RNA, a mature message of 1.5 kb, a larger, presumably precursor, molecule of 2.5 kb and a smaller 0.92 kb molecule of unknown function.

In comparison, no J chain RNA sequences could be demonstrated in a lymphoma line that has the properties of an undifferentiated B lymphocyte. Assays for J chain translation products or for J chain cDNA/RNA hybrids were negative, even though the assay conditions used maximized the chance of detecting J chain RNA. The failure to demonstrate J chain RNA in the lymphoma line was not due to the deletion of the J chain gene; when DNA from the lymphoma line was digested with restriction endonucleases, the fragments hybridized with cloned J chain cDNA in a pattern identical to that obtained with mouse embryo DNA (M. Yagi and M. E. Koshland, manuscript in preparation).

The absence of detectable J chain RNA in the lymphocyte-like lymphoma cells strongly suggests that new gene transcription is required to initiate J chain synthesis. If synthesis were initiated by a trans- lational mechanism, the lymphoma cells would be expected to synthesize detectable amounts of J chain mRNA and mRNA precursors. Moreover, the mRNA should be functional under the deregulated conditions of a cell-free translation system. A similar argument can be made against the induction of J chain synthesis by mechanisms involving RNA processing. Nuclear RNA intermediates would be expected to be present in the lymphoma cells, as has been observed in p+- thalassemic individuals with a block in &globin RNA processing (Maquat et al., 1960). There are, however, possibilities for control of J chain synthesis that are not ruled out by these studies of steady-state RNA populations. If J chain expression is induced by mech-


a b C e f

Figure 7. J Chain-Specific RNAs in Lymphoma. Hybrid Cell and Plasmacytoma Lines

Poly(A)+ RNA (6 pg samples) was fractionated by electrophoresis through methylmercury hydroxide agarose gels, transferred to DBM paper and hybridized with lo6 cpm of nick-translated insert from pJc3 plasmid (insert spec. act. approximately 5 x 10’ cpm/pg). RNA is shown from (a) L cells, (b) HOPC 1, (c) MOPC 315. (d) MPC 11, (e) MxW 231.la, (f) MxW 231.lb and (g) WEHI 231. p = p chain; x = WEHI 231 L chain: ‘/z K = K chain fragment.

anisms that stabilize the primary RNA transcript, the synthesis of short-lived transcripts by the lymphoma cells might not have been detected by the analytical procedures used. Pulse-labeling experiments will be required to exclude these possibilities and to provide definitive evidence for the activation of J chain synthesis at the transcriptional level.

The absence of J chain RNA from the WEHI 231 lymphoma also serves to emphasize the role played by J chain in B cell differentiation to pentamer IgM secretion. Analyses of the p chain RNA in this lymphoma have shown that two mRNA species are present, and in vitro translation studies have shown that two species of p chain are synthesized, one corresponding in size to the membrane-associated form of p chain and the other corresponding in size to the secreted form of p chain (Alt et al., 1980a). Thus the WEHI 231 lymphoma produces the p chain component, as well as the light chain component, of pentamer IgM, but is incapable of assembling and secreting pentamer in the absence of J chain expression.

De novo synthesis of cellular components other than the J chain may also be required for the differentiation of B cells to IgM secretion. Likely candidates would include regulatory proteins and intermediates necessary to effect IgM polymerization, such as the enzyme that has been shown to catalyze disulfide bond formation between IgM subunits and J chain (R. A. Roth and M. E. Koshland, manuscript in press). The techniques used to clone J chain cDNA should prove useful in cloning the sequences encoding such critical gene products. By appropriate absorption of

cDNA from cells differentiated to IgM secretion with RNA from undifferentiated cells, it should be possible to enrich for sequences that are expressed only in the more differentiated cells.

The comparison of the synthetic capacities of the lymphocyte-like lymphoma and its daughter hybrid lines has important implications for understanding the signaling of differentiation. From the data obtained to date, it appears that the interaction of antigen or mitogen with the B cell membrane must be communi- cated to the nucleus. Moreover, the communication has been shown to have two different consequences at the nuclear level: the recently described shift in p chain RNA expression to the secreted form (Alt et al., 1980a; Rogers et al., 1980; Early et al., 19801, and the activation of J chain synthesis described here. These combined findings indicate that the membrane interaction generates either multiple signals or a single signal with a cascade effect. By extending the comparison of the lymphoma and hybrid lines to their nuclear contents, it may be possible to obtain clues to the identity of the signaling component(s) and clarify the mechanisms involved.

Experimental Procedures

Origin and Maintenance of Cells The murine B lymphoma cell line, WEHI 231 01, K) was obtained from N. Warner (Warner et al., 1975) and the IgG-secreting plasmacytoma line, MPC 11 (y2,,, K) was the gift from M. Scharff. The hybrid cell lines, MXW 231.la and MXW 231.lb. were subclones of the MxW 231 hybrid that was produced by the fusion of the WEHI 231 lymphoma and the MPC 11 plasmacytoma (Raschke et al., 1979). The plasmacytoma tumors. MOPC 315 (a, A,,) and HOPC 1 (yna, A,). were obtained from M. Gefter and were established as cell lines by E. Siden. All the lymphoid and hybrid cell lines were maintained as suspension cultures in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum. For growth of the WEHI 231 cells the medium was also supplemented with 5 x 10m5 M 2-merceptoethanol. The mouse fibroblast line (L cells) was maintained as a suspension culture in Joklik’s modified Eagle’s medium containing 10% fetal calf serum.

RNA isolation Total RNA was prepared from the lymphoid and hybrid lines by the guanidine hydrochloride extraction procedure (Strohman et al., 1977). and from the fibroblast cell line by the urea/SDS extraction procedure (Holmes and Bonner. 1973). Poly(A)+ RNA was selected by oligo(dT)-cellulose chromatography as described by Alt et al. (1978). except that 1 mM EDTA was included in the buffers. For some experiments the poly(A)’ RNA was further fractionated by sucrose gradient centrifugation. Portions of 50-l 00 pg in 100 mM NaCI, 10 mM Tris-HCI. pH 7.6,1 mM EDTA. 0.5% SDS, were brought to a concentration of 3 mM in methylmercury hydroxide, allowed to stand for 15 min at 22°C. and applied to 5 ml 5-20% (w/w) sucrose gradient prepared in the same buffer. The gradients were centrifuged at 2O’C for 4 hr at 45,000 rpm in an SW50.1 rotor, and 0.3 ml fractions were collected. The RNA in each fraction was precipitated with 2.3 volumes of ethanol in the presence of 0.4 M NaCl and 5 pg of rabbit liver tRNA carrier. The pellets were washed and dissolved in 0.2 mM EDTA. pH 7.0.

Assays for in Vitro Translation of RNA Poly(A)+ RNA was translated in the micrococcal nuclease-treated rabbit reticulocyte lysate system (Pelham and Jackson. 1976) supplemented with ?S-methionine. essentially as described by Philipson et al. (1978). Aliquots of the translation mixture were incubated

Cdl 376

overnight at 2°C with rabbit anti-mouse J chain antibody or rabbit anti-mouse IgM @, K) antibodies prepared as described by Raschke et al. (I 979). or with rabbit anti-mouse K chain antiserum purchased from Gateway. The immune complexes formed were precipitated by the addition of formalin-treated Staphylococcus aureus (Kessler, 1976) prewashed with SA buffer (0.5% Nonidet-P40. 0.05% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 50 mM methionine in phosphate-buffered saline), and were resuspended in the same buffer supplemented with 0.1% bovine serum albumin. After the precipitated pellets were washed twice with SA buffer containing albumin and once with SA buffer lacking albumin, the immune complexes were eluted from the bacteria by treatment with 9.5 M urea, 30 mM dithiothreitol, 0.05 M Tris-HCI, pH 8.0, for 15 min at 68°C. The supernatant solutions were brought to a concentration 72 mM in sodium iodoacetate, incubated for 30 min at 22°C and precipitated by the addition of five volumes of acetone at -20°C (Mosmann et al.. 1978). The acetone precipitates were dissolved in IO M urea for analysis on alkaline urea 4.15% polyacrylamide gels (Reisfeld and Small. 1966) or in 1% SDS, 2% 2-mercaptoethanol. 10% glycerol, 100 mM Tris-HCI, pH 6.8, for analysis on 12.5% SDS-polyacrylamide gels (Laemmli. 1970). Gels were fluorographed by the PPO-DMSO method of Bonner and Laskey (1974). In order to compare the properties of the Ig chains synthesized in vitro and in vivo. the proteins of the M X W 231.1 a hybrid line were labeled by incubating the cells at a density of 3.3 x 10’ cells/ml with 300 PCi of 35S-methionine (spec. act. 1120 Ci/mmole) for 3 hr at 37’C. The cells were pelleted, washed and lysed with 1% Nonidet-P40. 0.1% SDS, 100 mM sodium iodacetate. 0.1 mM phenylmethylsulfonyl fluoride in phosphate- buffered saline. Appropriate portions of the lysate were analyzed by immunoprecipitation and gel electrophoresis as described above.

cDNA Preparation ‘*P-labeled cDNA was prepared essentially as described by Alt et al. (1978) and modified by Alt et al. (1980a). Fifty-two micrograms of MXW 231 .l b poly(A)+ RNA were brought to a concentration of 3 mM in methylmercury hydroxide before addition to the reaction mixture (Payvar and Schimke, 1979), which contained 250 pM dNTP and 1 mCi each of s-32P-labeled dATP and dGTP (New England Nuclear: spec. act. approximately 400 Ci/mmole).

RNA/cDNA Hybridization Analytical RNA/cDNA hybridizations were carried out using the procedures described by Alt et al. (1978; 1979). At the end of the hybridization reaction, single-stranded sequences were digested with Sl nuclease and the extent of hybrid formation was determined from the radioactivity precipitable by trichloroacetic acid. Preparative hybridizations were carried out in a similar manner. For the negative selection step, L cell polytA)+ RNA (6 fold excess over the amount of M XW 231 .I b poly(A)+ RNA used as template for the cDNA transcrip tion) was hybridized to the 32P-labeled cDNA until a Rot of 5200 mole- set/l was attained. For the positive selection step, MOPC 315 poly(A)+ RNA (2.4 fold excess over the amount of M x W 231.1 b RNA used as template) was hybridized to the single-stranded cDNA obtained from the negative selection until a Rot of 10 mole-set/l was achieved. The reaction mixtures were brought to a concentration of 0.1 M in Na2P0,. pH 6.8, and applied to 1.2-I .6 ml hydroxyapatite columns equilibrated with the same buffer and maintained at 60°C. Single-stranded cDNA was eluted with 0.1 M or 0.12 M Na2P01, pH 6.8, and RNA/cDNA hybrids were eluted with 0.2 M Na2POI, pH 6.8. The eluted material was desalted by passage through Sephadex G- 100 and was concentrated by ethanol precipitation. RNA was re- moved by base hydrolysis.

Selection and Characterization of J Chain cDNA Clones and Plasmids A library of plasmacytoma cDNA clones was used for the selection of cloned J chain sequences. The library was constructed from the cDNA complementary to poly(A)+ RNA from the IgGzb-secreting plasmacytoma. X63.2b-7 (Radbruch et al., 1980). The cDNA was made double-stranded and the 0.5-I kb fraction was selected by sucrose

gradient centrifugation. inserted into the Pst I site of pBR322 by the G-C tailing method and used to transform E. coli strain Xl 776 (Villa- Komaroff et al., 1978). The details of the library preparation will be described elsewhere (P. Schreier et al., manuscript in preparation). Bacterial colonies were screened by hybridization (Grunstein and Hogness, 1975) to the “P-labeled cDNA probe enriched in J chain sequences. Plasmid DNA was isolated by the procedures of Rambach and Hogness (1977) and Clewell and Helinski (1972). The cDNA inserts were excised by digestion with Pst I endonuclease. and their size was determined by electrophoresis on 15% agarose gels (Sharp et al., 1973). For restriction mapping the plasmid DNA was digested with Pst I, Hinf I. Taq I and Alu I endonucleases. either singly or in various combinations, and was analyzed on agarose gels of the appropriate percentage. The fragments were transferred to nitrocellulose (Southern. 1975) and hybridized to the J chain cDNA insert from pJc3 (Wahl et al., 1979). which was *‘P-labeled by nick translation (Rigby et al., 1977). Procedures used conformed to the NIH guidelines.

Hybridization-Selection of J Chain RNA The procedures of Ricciardi et al. (1979) were followed with slight modifications. Plasmid DNA was digested with Eco RI endonuclease, phenol-extracted, ethanol-precipitated and redissolved to a concentration of 0.5 mg/ml in 10 mM Tris-HCI, pH 7.6, 1 mM EDTA. The DNA was boiled for 3 min, frozen on pulverized dry ice and thawed, and 10 Al were spotted onto 1 cm’ nitrocellulose filters. After the filters were dried and baked, they were wetted with 50 pl of 65% deionized formamide, 10 mM 1.3~piperazinediethane sulfonic acid, pH 6.4, 0.4 M NaCI. and fragmented. Twenty micrograms of WEHI 231 .I b poly(A)+ RNA in 100 ~1 of the same buffer were added. The filters were incubated for 90 mip at 45’C and washed as described by Ricciardi et al. (I 979). except that 0.5 ml volumes were used for the final two washes. To elute the RNA from the hybrids, the mixture was boiled for 1 min in 0.2 mM EDTA, ethanol-precipitated with 5 Fg of yeast tRNA carrier and dissolved in water. The RNA was heated to 68’C for 3 min. frozen on dry ice, thawed and translated in vitro.

Identification of Size-Fractionated RNA Poly(A)+ RNA was size-fractionated on methylmercury hydroxide 1% agarose gels according to the procedure of Bailey and Davidson (1976). except that 0.05 M boric acid was omitted from the electrophoresis buffer. RNA was transferred from the gel to DBM paper and the coupled paper was pretreated and then hybridized with nick- translated (Rigby et al., 1977) recombinant plasmid following the procedures of Alwine et al. (I 980).

Acknowledgments

We would like to thank Peter Schreier for providing access to the plasmacytoma cDNA library and Gordon Cann for the restriction mapping of the cloned J chain cDNA. This work was supported by grants from the American Cancer Society and the National Institutes of Health. F.A. is a special fellow of the Leukemia Society of America. A.L.M.B. was a postdoctoral fellow of the National Cancer Institute and the Medical Foundation. D.B. is a research professor of the American Cancer Society.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received September 8, 1980; revised November 17. 1980

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