The constant (C) regions of the heavy chains are encoded by a cluster of C region genes. (5 -iA 69 'Y3i'Y'Y2b ..... (Laskov et al., 1981;Word and Kuehl, 1981).
The EMBO Journal Vol.2 No.2 pp.167-172, 1983
Simultaneous expression of At- and y-chain mRNA in cloned murine B-lymphoma cell lines R.Laskov*, R.Ishay-Michaeli, M.Wallach, D.Givol1 and K.J.Kim2 The Hubert H.Humphrey Centre for Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem 91010, 1Department of Chemical Immunology, Weizmann Institute of Science, Rehovot 76100, Israel, and 2Laboratory of Microbial Immunity, National Institutes of Health, Bethesda, MD 20205, USA Communicated by D.Givol Received on 3 November 1982
The expression of immunoglobulin heavy chain genes was studied in five murine B-lymphomas known from previous studies to express either y (38C-13), A + 6 (L1OA, K46, BCL,) or y chains (A20). The presence of It- and -y-nRNAs in these tumors was determined by Northern blot analyses of the total cell poly(A) + mRNA, using the appropriate 32P-labeled recombinant plasmid probes. In four out of the five lymphomas examined, both p- and y-mRNAs were dejected. The A-mRNA appeared as multiple discrete bands of 1.9-3.0 kb. In three out of the four lymphomas, the y-mRNA appeared as two bands, a major one of 1.9 and a minor one of 3.9 kb. Three myelomas examined by similar methods did not contain more than one class of heavy chain mRNA. Reexamination of the Ig chains produced by the B-lymphomas which expressed both A- and -y-mRNAs revealed that two of them preserved their original phenotype and expressed A (38C-13) or y chains only (A20). In contrast, two of the cell lines previously shown to express A but not 'y chains (i.e., L1OA and K46R) had changed during growth in culture and 'switched' to the production of -y chains only. These results indicate that, in contrast to myelomas, B-lymphomas possess two classes of mRNA. However, the production of heavy chain mRNA in B-lymphomas is not necessarily accompanied by synthesis of the corresponding polypeptide chains. More studies are necessary to find out whether the expression of 'non-productive' heavy chain mRNA molecules in B-lymphomas is related to the phenomena of 'allelic exclusion' and/or the 'heavy chain switch' which occurs during the maturation of B-cells. Key words: B-lymphoma/gene expression/heavy chain
switch/ immunoglobulin/mRNA Introduction Recent studies on the structure of the immunoglobulin (Ig) genes have confirmed the clonal nature of antibody synthesis which allocates a single antibody specificity for each antibody-forming cell (Burnet, 1959). The genes encoding the variable region of the heavy chains are composed of clusters of separate genetic elements: the variable (VH), diversity (D) and the joining (JH) DNA segments. The constant (C) regions of the heavy chains are encoded by a cluster of C region genes (5 -iA 69 'Y3i 'Y 'Y2b Y2aq, E, cx-3') located on the same chromosome at some distance downstream from the VH segments (reviewed by Adams, 1980). During immunodifferentiation multiple DNA recombination events are *To whom reprint requests should be sent. IRL Press Limited, Oxford, England.
responsible for the formation of complete, active heavy and light chain genes. In each B-cell both heavy and light chains are the product of only one of the two alleles ('allelic exclusion') (Pernis et al., 1965), thus ensuring the formation of Ig molecules of a single antigen-combining specificity. This situation is unique to Ig genes since, with the exception of the female X-linked genes, both alleles of other known genes are expressed. At the molecular level only one of the VH, D and JH segments are selected and recombined on one allele to form a VHDJH-coding sequence in a single pre-B cell, resulting in the production of a single type of a 14 heavy chain (Burrows et al., 1979). Recombinational events also occur in the heavy chain gene segments on the other allele; however, these do not usually lead to the formation of a complete it chain (Hurwitz et al., 1980; Perry et al., 1981; Nottenburg and Weissman, 1981). Another remarkable feature of the immune system is the ability of a single antibody-forming cell to switch from the production of IgM to other classes of Ig, while preserving the same antigen-combining specificity ('heavy chain class switch') (Davis et al., 1980). Recent studies (Moore et al., 1981) have shown that the expression of u and 6 heavy chains on the same lymphocyte is due to an alternative RNA splicing mechanism of a large primary RNA transcript. On the other hand, a gene deletion mechanism was proposed to explain the production of 6, -y or a chains by mature plasma cells (Moore et al., 1981). According to the 'deletion model' the mature VH gene (i.e., the VHDJH sequence) which constitutes part of the / gene can translocate to the vicinity of each of the other C region genes further down the row and thus form an active heavy chain gene of a different class. This results in the deletion of a DNA segment containing the intervening C region genes (Rabbitts et al., 1980; Kataoka et al., 1980; Coleclough et al., 1980; Hurwitz et al., 1980). However, the evidence for the deletion model came from studies done on DNA derived from plasmacytomas which are the malignant counterparts of normal plasma cells and thus the results obtained may not reflect the situation in young B-cells. Moreover, the sequential deletion of the heavy chain genes as found in plasmacytomas may be the final outcome, rather than the primary event in the isotypic switching, since B-lymphocytes may express both A and y heavy chains (Premkumar et al., 1975; Abney et al., 1978). Therefore, to delineate the primary events in Ig gene expression, it would be useful to investigate B-lymphoma cells which are less mature than plasmacytomas. We have studied several clones of B-lymphoma cell lines which were previously found to produce either A or -y chains (Laskov et al., 1981). Analysis of the content of ,- and 'ymRNA revealed that, in contrast to plasmacytomas, four out of the five B-lymphomas produced both W- and 7y-mRNAs. 'Non-productive' -y heavy chain mRNAs were found in one B-lymphoma expressing the i chain and 'non-productive' AtmRNAs were found in three y-producing lymphomas. These findings indicate that heavy chain gene expression is less restricted in B-lymphoma compared to myeloma cells and that the early events in the phenomenon of 'isotopic switching' may not involve deletion of the u chain gene. 167
R.Laskov et al. Kb
40
Wii.
I
.2:
e1
Fig. 2. Analysis of g-mRNA in
B- and T-lymphomas. Poly(A)+ mRNA of the lymphomas was analyzed as in Figure 1. Multiple bands of y-RNA sequences of 1.9- 3.0 kb in size are evident in both B- and T-lymphomas. The third lane (BCL) was identical to the first lane but for longer exposure time of the autoradiograph. The seventh lane [L1OA(D)] was identical to the eighth lane but for longer exposure time.
Fig. 1. Analysis of t-mRNA in B-lymphomas. Poly(A)+ mRNA of B-lymphomas (20 Ag/lane) and myelomas (8-18 jig/lane) was electrophoresed on agarose gel containing formaldehyde, blotted to nitrocellulose paper and hybridized with nick-translated 32P-labeled C-region It probe.
Results B-lymphoma clones express both st- and y-mRNA Total poly(A) mRNA derived from various B-lymphoma cell lines was electrophoresed on agarose gels under denaturating conditions, transferred to nitrocellulose paper and hybridized to nick-translated plasmids containing the C region sequence of the / or the 'Y2b heavy chains. The autoradiographs in Figures 1 and 2 show that all the B-lymphomas tested contained W-RNA sequences. As expected, only MOPC104E (/,X) but not MPC1 1 (,yx) myeloma cells contained t-mRNA. Figure 1 shows the presence of major hybridization bands 2.4-2.7 kb in size in MOPC104E, 38C-13, A20.3 and BCL1 cells. Some of the bands were better resolved in Figure 2. BCL1 cells contained two major bands of similar intensity and approximate sizes of 2.7 and 2.4 kb. These bands probably represent A-mRNA for the membrane and secreted forms of the it heavy chain (Alt et al., 1980a; Rogers et al., 1980). Similar doublet bands were also found in 38C-13 cells (not shown). Prolonged exposure revealed an additional /-RNA band of 1.9 kb in BCL1 cells (Figure 2, lane 3). Multiple j4-RNA bands were also found in K46R(D) and L1OA(D) cells. Their approximate sizes were 3.0 and 2.1 kb for K46R(D) (Figure 1), and 3.0, 2.4 and 1.9 kb for L1OA(D) cells (Figure 2). Similar bands were also found in two subclones of L1OA(D) cells (not shown). Since recent studies have revealed the presence of i-RNA sequences in some T-lymphomas and normal T-cells (Kemp et al., 1980), it was of interest to compare the W-RNA sequences in the B-lymphomas with those in T-lymphomas. Figure 2 shows three faint pt-RNA bands of an approximate size of 3.3, 2.4 and 1.7 kb in YAC but not in COVA 62 T-lymphoma. Hybridization with 'Y2b probe revealed a completely different pattern (Figure 3). Unexpectedly, it was found that all the B-lymphoma cells except BCL1 contained 'y-mRNA sequences. Two bands of 'y-mRNA were observed in 38C-13, L1OA(D) and A20.3 lymphomas, a major band of 1.9 kb and 168
Flg.
rymRNA in B-lymphomas. Poly(A)' mRNA of the were electrophoresed, blotted and hybridized to Figure a C-region -ya, probe. The two left lanes are from the same experiment as in Figure but th t probe was melted off and the paper rehybridized to the probe. 3.
same
Analysis of
tumors as in
Y,
a
minor
band
of
-3.9
kb.
Similar
bands
were
found
in
the
sY2b-producing MPCi myeloma, but not in MOPCOi4E and MOPC315 which produce IgM and IgA, respectively. Probably these two bands represent y-mRNA for the secreted and the membrane forms, respectively, of the 'y heavy chain (Rogers et al., 1981). Multiple bands ranging in size from 1.3 to 3.0 kb were present in K46R(D) cells (Figure 3). Hybridization to a plasmid probe containing the cDNA sequence of the heavy chain (pSI07) revealed a-mRNA only in MOPC315 myeloma (a,X) but not in LIOA(D), K46R(D), 38C-13, or BCL1 lymphoma cells (Table I). Since the y2b probe cross-reacts with 73, y1 and 'y2 sequences (Kataoka et al., 1979), it is unlikely that BCL1 cells contain any 'y-mRNA sequences. On the other hand, it is not 1
a
Co-expression of heavy chain mRNAs in B-ymphomas Table I. Analysis of y- and -y-polypeptides and heavy chain mRNAs in murine B-lymphomas and myelomas Cells
Type
Heavy chainsa A
BCL1 38C-13 LIOA 6.2 L1OA(D) K46R(D) A20.3 MPCll (68.5)
MOPC104E MOPC315
lymphoma lymphoma lymphoma lymphoma lymphoma lymphoma myeloma myeloma myeloma
+ + + -
+ -
y
Heavy chain-mRNAb a -y Ai
+ + + + -
+ + NDC + + + + -
+ ND + + + + -
I
ND 4
-E
i-N_
ND
ND +
aDetermined by immunofluorescence and/or biosynthetic labeling assays. bDdetMined by RNA blotting and hybridization to specific plasmid probes. cNot done. -
ae
-
Fig. 5. Isotype swich in L1OA lymphoma cells. L1OA 6.2 and L1OA(D) cultured cells were labeled with [35S]methionine for 12 h and the cellular Ig precipitated with class-specific rabbit antibodies to the -y or i chain and goat anti-rabbit IgG (Materials and methods). The immune precipitates were dissolved and electrophoresed on 6%1o spacer, and 15% polyacrylamide main slab gel, and fluorographed. LIOA(D) cells produced two y heavy chains of -67 000 and 53 000 daltons; LIOA 6.2 cells produced / chain of apparent mol. wt. 67 000 daltons. Both lines produced light chains (L) and a protein which is known to bind to antigen-antibody complexes (M) (Premkumar et al., 1975) and has an apparent mol. wt. of 40 000 daltons.
osee o..
Fig. 4. Relative content of t-RNA in B-lymphomas. Increasing quantities of poly(A)+ mRNA derived from various lymphoma and myeloma tumors, spleen cells (SPN) and A20.3 cultured cells (C) were dried on nitrocellulose paper, hybridized to plasmid containing the cDNA insert of the C-region of the A sequence, and autoradiographed (left). The blackened spots were scanned and the absorbance plotted versus the amount of mRNA/spot (rght). Channel
known what subclasses of -y-mRNA are present in the B-lymphomas. The presence of both j- and -y-mRNA species in B-lymphoma cells was not due to an inherent genetic heterogeneity of the tumor cells since 38C-13 and A20.3 are cloned cell lines. In addition, two clones of LlOA(D) cells contained the A and -y heavy mRNA sequences (not shown). Quantitation of the A-mRNA in B-lymphoma and myeloma cells The relative amount of it-mRNA present in the various B-lymphoma cells was determined by the 'RNA-dof assay. The amounts of it-mRNA in LIOA(D), 38C-13 and A20.3 lymphoma cells were 5-10 times lower than in MOPC104E myeloma cells (Figure 4). On the other hand, BCL1 tumor cells contained as much ,-mRNA as did MOPC104E myeloma cells. Spleen cells contained, on average, more mRNA than the B-lymphoma cells, but less than MOPC104E myeloma cells. However, since only 600o of spleen lymphocytes are of the B-lineage, splenic B-cells may have approximately the same average amount of yt-mRNA as do myeloma cells. MPC1 1 (,y,x) and MOPC315 (y,,y) myeloma cells served as negative controls and contained only very little t4mRNA sequences, which may have been derived from in-
number
Fig. 6. Microfluorimetric analysis of membrane Ig chains in LIOA cell lines. Viable L1OA 6.2 and L1OA(D) cells were stained directly by fluorescein-labeled goat antibodies to mouse 72 and y chains. The stained cells were analyzed in the cell sorter. L1OA 6.2 clone represents the original L1OA culture and expresses y chains. L1OA(D) cells represent cells which have 'switched' and express -y- and not ti-chains. Fluoresceinated goat antirabbit IgG and unstained cells served as unstained controls and gave profiles identical to those obtained for L1OA 6.2 stained with anti-y2 and for L1OA(D) stained with anti-/t.
filtration of the tumors by host B-cells. The question of whether the presence of i-mRNA in -yexpressing B-lymphoma cells was due to host B-lymphocytes rather than to tumor-derived cells was examined by analysis of mRNA derived from cultured A20.3 cells. Figure 4 shows that the amount of pi-mRNA in the cultured A20.3 cells was as high as that of mRNA derived directly from the in vivo growing tumor cells, indicating that the 14-mRNA detected was not contributed by host B-lymphocytes. In conclusion, quantitative analysis of several lymphoma and spleen cells showed that some lymphomas and splenic B-lymphocytes had similar concentrations of pt-mRNA as did fully mature myeloma cells. However, on a per cell basis, 169
R.Laskov et al.
L 10 A( D,ir
A 20.3
38 C -- 13
..P.. 1
'0004*
10 00 0 4
'Y
C.-
n
a
0% A L-
S
_
L 4-,
A.
0
-N f
g
C -Y
c
:vA
g
OVA -C Vi
Fig. 7. Biosynthesis of Ig chains by B-lymphoma cell lines. L1OA(D), A20.3 and 38C-13 cells were labeled with [5S]methionine for 12 h. The cytoplasmic lysates were preprecipitated with rabbit anti-ovalbumin (OVA) and goat anti-rabbit IgG (contro) and then divided and precipitated with rabbit anti-mouse-Ig or class-specific anti-y and rabbit anti-it and goat anti-rabbit IgG. The precipitates were dissolved and electrophoresed on polyacrylamide slab gels and fluorographed. Two types of 'y-chains (mol. wts. 67 K and 53 K) were produced by L1OA(D) and A20.3 cells and two types of A-chains (74 K and 69 K) were formed by 38C-13 cells.
myeloma cells contained more W-mRNA, since they are larger than lymphoma cells. Spontaneous switch of i to ey chain expression in Blymphoma cell lines In previous studies, L1OA and K46 cells were found to express both jA and 6 but not y chains, using immunofluorescence (Kim et al., 1979), biosynthetic (McKeever et al., 1979), and lactoperoxidase iodination methods (Laskov et al., 1981). In other studies, BCL1 cells were shown to express membrane A and small amounts of 6 chains (Knapp et al., 1978), 38C-13 cells exhibited membrane /t but not 6 chains (Bergman and Haimovich, 1978), and A20 cells produced 'y chains only (Laskov et al., 1981; Word and Kuehl, 1981). Since seemingly normal size y-mRNAs were found in several of these B-lymphomas, it was decided to reexamine the production of -y heavy chains by these cells. This was of particular importance since 3 years had elapsed between the examinations made on the polypeptide chains and the analysis of the Ig-mRNA, as described in the present work. Moreover, the L1OA(D) and K46R(D) cells analyzed in the present work were maintained for several months in Dulbecco's medium and not in the original Click's medium prior to their growth and transfer as solid tumors in BALB/c mice. Biosynthesis labelling assays made on several of the B-lymphomas revealed that L1OA(D) cells had changed their phenotype and produced 'y rather than A heavy chains. In contrast, a clone derived from the original L1OA culture (6.2) and maintained in Click's medium continued to synthesize 'O chains and did not produce detectable amounts of the 'y heavy chains (Figure 5). These findings were confirmed by analysis of Ig expression on the surface of L1OA cells. Figure 6 shows that L1OA 6.2 and L1OA(D) cell lines were each composed of a single population of cells expressing either / or 'y but not both heavy chains; also very few if any null cells were detected. A similar change to 'y production was also evident in K46R(D) cells (not shown). In contrast, 38C-13 and A20.3 preserved their original phenotype and continued to syn-
170
thesize either it or oy chains, respectively (Figure 7). In L1OA(D), K46R(D) and A20.3 cells, two y-bands were detected: a main band of -53 000 and a minor band of 67 000 dalton. These bands probably represent the cytoplasmic and membrane forms, respectively, of the -y heavy chain (Word and Kuehl, 1981). In 38C-13 cells, two A chain bands of similar intensity were revealed, their approximate sizes being 69 000 and 74 000 dalton, and they represent the cytoplasmic and membranous forms of the ,u heavy chain (Bergman and Haimovich, 1978)
(Figure 7).
Discussion The results presented show that most B-lymphoma cells produce both ,- and -y-mRNA, in contrast to plasmacytoma cells which produce mRNA for only one heavy chain isotype. However, in these lymphomas only one of the heavy chain mRNAs is translated into a detectable full size heavy chain. Thus, only A chains were expressed in 38C-13 cells and only 'y chains in L1OA(D), K46R(D) and A20.3 cells, yet all these cell lines contained both A- and Py-mRNAs (Table I). Multiple forms of productive and non-productive 'y- and AmRNAs appeared in the various B-lymphomas. Two main types of y-mRNA (3.9 and 1.9 kb) were found in L1OA(D) and A20.3 lymphomas which probably code for the membrane and the secreted forms of 'y-chains produced by these cells. Bands of roughly the same size also appeared in 38C-13 cells, which did not express immunologically identifiable ychains. More studies are needed to delineate the differences between the productive and non-productive -y-mRNA species. The situation seems to be even more complex with regard to the t-mRNA. The it-chain expressing lymphoma cells (BCL1 and 38C-13) exhibited two main species of ,-mRNA, namely, the 2.4 and 2.7 kb which code for secreted and membrane forms of the it-chain, respectively (Perry and Kelly, 1979; Alt et al., 1980a; Rogers et al., 1980). In addition, several other ,-RNA species, ranging in size from 1.9 to 3.0 kb, were detected mostly in lymphoma cells which do not ex-
Co-expression of heavy chain mRNAs in
press A chains. These findings agree with previous studies of Pre-B and B-lymphomas in which multiple forms of untranslated it-RNA molecules were identified (Perry and Kelly, 1979; Kemp et al., 1980). Recently, a detailed study of Abelson Pre-B cell lines showed that the non-productive yRNA molecules can be grouped into several categories. While the 'aberrant' i-RNA contains the VH segment and sometimes codes for short A-chains, the 'sterile' type is devoid of the VH gene segment (Alt et al., 1982b). Untranslated forms of A-RNA which lack the VH segment have also been demonstrated in some T- and myeloid cells (Kemp et al., 1980; Walker and Harris, 1980; Alt et al., 1982b). These species seem to be analogous to the 'sterile' A4-RNA found in Abelson lymphomas. However, in contrast to the latter, yRNAs in T-cells are usually transcribed from unrearranged 14 genes (Alt et al., 1982b). The significance of the occurrence of the various types of aberrant W-RNA is discussed below. It is thought that various B-lymphomas represent different stages of the B-cell maturation pathway. Thus, 1t-expressing 38C-13 cells which represent an early stage of B-cells (Bergman and Haimovich, 1978), contain a non-productive -y-RNA, and A20.3 cells expressing y-chains and representing mature B-cells (Kim et al., 1979), contain non-productive yRNA. Therefore, non-productive Ig-mRNA molecules may appear at both early and late maturation stages of the B-cell lineage. A somewhat analogous phenomenon, in which an 'early' mRNA is present at a later stage, has been described for light chain mRNAs (Alt et al., 1980b; Laskov et al., 1982). Thus, in addition to the X-mRNA, X-producing lymphomas and myelomas were found to contain x-mRNA. It was suggested that B-cells make sequential attempts to form a productive xchain gene. If these attempts are unsuccessful, efforts are made to form a X-chain gene. Formation of an intact light chain will inhibit, by a feedback mechanism, further attempts to rearrange the light chain genes (Alt et al., 1980b). Thus, the presence of a non-productive x-mRNA in X producing cells seems to be reminiscent of the previous maturation stage of these cells. An example of early appearance of 'late' mRNA is the production of the it-mRNA for the secreted form of As heavy chain in B-lymphoma cells which does not secrete A chains. In this case, however, the secreted ut-mRNA is translated (Sidman, 1981) but the chain is rapidly degraded and is not secreted (Dulis et al., 1982). The early production of the secreted ,-mRNA is probably aimed at rapidly meeting demands for antibody formation. An additional observation made was that two of the B-lymphoma cell lines (L1OA and K46) revealed a spontaneous switch from /t to y chain expression. These switches may have been enhanced by replacing the original Click's medium by Dulbecco's medium which lacked a mixture of the four ribonucleosides and 2-mercaptoethanol (2-ME). Another example of a spontaneous heavy chain switch in culture was recently reported by Burrows et al. (1981) in an Abelson lymphoma. This it chain-producing cell line segregated and produced clones expressing -y2b heavy chains. A different type of heavy chain switch has been described in myeloma cultures (Francus et al., 1978; Radbruch et al., 1980). However, these switches have the peculiar property that they occurred between neighboring -y heavy chain genes (i.e., 72b - -ya), were reversible, and evolved at the low rate of 10- 6-10- 7/cell/generation (Radbruch et at., 1980). The significance of this latter type of heavy chain switch is not
B-ymphomas
known, since it has not been demonstrated in normal B-cells. The role of the non-productive t- and -y-mRNA in B-cells is not yet understood. It is possible that the production of these molecules is related to the phenomenon of allelic exclusion. Accordingly, the non-productive mRNAs are transcribed
from the allelic silent chromosome. This possibility is supported by the findings that rearrangements of the heavy chain genes occur on both the expressed and the non-expressed allelic chromosomes in normal and malignant B-cells (Hurwitz et al., 1980; Perry et al., 1981; Nottenburg and Weissman, 1981). In some cases, an abortive rearrangement was found to involve joining of a D to a JH segment with no participation of a germline VH segment (Sakano et al., 1981). In others, however, the non-functional gene was assembled from VH, D, and JH exons, supporting the contention that allelic exclusion operates through several mechanisms some of which result in the formation of non-functional polypeptide chains (Alt et al., 1982b). Another possibility is that the formation of non-productive mRNA is related to the phenomenon of the heavy chain switch. Accordingly, productive and non-productive mRNAs are transcribed from a single allelic chromosome. Transcription of both pt- and y-mRNA from a single chromosome in the y-producing lymphomas (L1OA(D), K46R(D) and A20.3) seems to violate the gene deletion model for the heavy chain switch (Davis et al., 1980). Thus, the formation of the - and 7y-mRNAs could result from an alternative splicing event taking place in a long pre-mRNA molecule, in a similar way to that found for the u- and 6-mRNAs (Moore et al., 1981). However, it may pose a problem since the distance between the w- and 6-genes is far shorter than that between the it and e genes, which measure up to 80 kb (Shimizu et al., 1981). Giant primary transcripts of -50 kb have been described in the Balbiani rings of the polytene chromosomes of Drosophila melanogaster (Danehalt, 1975), and probably exists in heterogenous nuclear RNA (Darnell, 1979). It is also possible that an early gene deletion event brings one of the y genes within close proximity to the It gene. The latter mechanism is compatible with the observed precommitment of an Abelson lymphoma for a class-specific switch (Burrows et al., 1981). A switch operating through an RNA splicing mechanism is supported by recent studies on Abelson cell line exhibiting a change from i to 'Y2b synthesis. The 'switched' cells retained their A genes and yet expressed a y2b gene having the same VH segment as that expressed in the Iu gene (Alt et al., 1982a).
Materials and methods Tumors and cell lines The B-lymphoma cell lines L1OA, K46R and A20 were derived from spontaneous tumors of aged BALB/c mice and maintained in Click's medium supplemented with 1Oo fetal calf serum (FCS), 2 mM glutamine, 1.5 x 10- 5 M 2-ME, 50 U/ml penicillin and 50 y/ml streptomycin, as described (Kim et al., 1979). L1OA 6.2 and A20.3 are clones derived from the respective original cell lines by the limiting dilution method. LIOA(D) and K46R(D) were derived from the original cultures by growth in Dulbecco's medium supplemented with 1007o FCS, glutamine and antibiotics as above, but without 2-ME. The various cell lines were also maintained as transplantable solid tumors in BALB/c mice. The BCL, tumor was maintained by serial transfer in BALB/c mice (Knapp et al., 1978) and tumorous spleens (weight > 1.0 g/spleen) were kindly provided by S.Slavin (Medicine A, Hadassah Hospital, Jerusalem). A clone derived from the 38C-13 B-lymphoma cell line (Bergman and Haimovich, 1978) was kindly provided by J. Haimovich (Tel Aviv University). These cells were maintained as a transplantable solid tumor in C3H mice. Myelomas MOPC104E (iX), MOPC315 (AX) and MPC 1I clone 68.5 (Xx)is were maintained as transplantable solid tumors in BALB/c mice. COVA 62
R.Laskov et al. a clone of radiation leukemia virus-induced T-lymphoma grown in culture (Azar et al., 1981). Recombinant pksmid probes The plasmid Py2b(1 1)' is a -y2b-cDNA clone containing only the C region (Schibler et al., 1978). The plasmid pAl83.5 is a it-cDNA clone containing the C and part of the V region sequences of the A chain of TEPC 183 myeloma (Auffray and Rougeon, 1980). A pBR322 subclone containing the PstI fragment corresponding to most of the Ct-4 domain (codon 483 to the 3' end) was used as a probe for the C+ region. The plasmid pS107 is an a-cDNA clone containing the C and part of the V sequences of the (x chain (Adams et al., 1980). Isolation of poly(A)+ mRNA Total RNA and poly(A)+ mRNA was extracted from solid tumors, spleens and cultured cells according to a modification of the method of Nienhuis et al. (1974), as described (Wallach et al., 1982). RNA blotting and hybridization Poly(A) + mRNA samples were electrophoresed on 1% agarose gels containing formaldehyde and transferred to nitrocellulose paper, as described (Wallach et al., 1982). The dried papers were hybridized to nick-translated 32P-labeled plasmid probes (Rigby et al., 1977) (sp. act. 100-300 c.p.m./pg) according to Alwine et al. (1977), and autoradiographed. In some experiments the hybridized probe was melted by immersing the nitrocellulose paper in 0.05 M NaOH, 0.1 M NaCl for 15 min at 20°C. The paper was washed with water and hybridized to a second probe. RNA dot assay The assay was performed according to Thomas (1980). The paper was hybridized to 32P-labeled probes, autoradiographed and the blackened spots on the film (Agfa PR-2) were scanned at 525 nm. The area under each peak was determined and plotted versus the amount of poly(A)+ mRNA per spot. Biosynthesis of Ig chains B-lymphoma cells (2-5 x 109 were labeled for 12 h in 1.0 ml of RPMI-1640 medium lacking methionine and containing 1 7% FCS and 50 fiC of [35S]methionine (Amersham, UK). The cells were lysed in the presence of 0.5% Nonidet P40(NP-40) and the 100 000 g supernatant was precipitated at 0°C using rabbit anti-ovalbumin (5X for 2 h) and goat anti-rabbit IgG sera (50X for 20 h). The cleared supernatants were divided into three equal portions and immuno-precipitated for 2 h at 0°C with (1) rabbit anti-mouse-Ig (2.5X); (2) affinity-purified rabbit anti-mouse-M (4 pg); and (3) rabbit antimouse-y (7 tg). Goat anti-rabbit IgG serum (50X) was then added for 20 h at 0°C. The immune precipitates were washed with phosphate buffered saline containing 0.1I% NP-40, dissolved in 4% SDS, 10% glycerol, 0.1 M Tris HCI pH 6.8, 0.3 M 2-ME, 0.02% bromphenol blue, boiled for 1 min and electrophoresed on 6% spacer, 15% main polyacrylamide slab gels (Laskov et al., 1980). The gels were stained and fluorographed (Bonner and Laskey, 1974). Fluorescence activated cell sorter (FACS) analysis Affinity purified, class-specific anti-mouse Ig antibodies were prepared and conjugated to fluorescein dye as described (Morse et al., 1977). Cells (2-5 x 109 were stained with these reagents and analyzed (FACS II, Beckton & Dickinson).
Acknowledgements We are most grateful to Dr.F.Rougeon for providing the recombinant plasmid pA183.5; Dr.R.Perry for the plasmid p-y(I 1)7; and Dr.J.Adams for the plasmid pSIO7. We are also grateful to Dr.R.Asofsky for the fluoresceinated class specific antisera, Dr.E.Vitetta for the rabbit antibodies to A- and y-chains, and to Dr.Y.Benbassat for help in writing the manuscript. This work was supported by Mrs.Stella Lehrer Endowment Fund and by the basic research fund of the Israel Academy of Sciences and Humanities.
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