encoding a domain - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 77, No. 1, pp. 554-558, January 1980

Immunology

Intervening sequences divide the gene for the constant region of mouse immunoglobulin ,u chains into segments, each encoding a domain (gene cloning/rearranged DNA/heavy chain joining region)

NICHOLAS M. GOUGH, DAVID J. KEMP, BRETT M. TYLER, JERRY M. ADAMS, AND SUZANNE CORY Molecular Biology Laboratory, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia

Communicated by Gustav J. V. Nossal, September 4, 1979

probably plays a central role because the precursors of all antibody-forming cells express the ,u chain (20). Immunoglobulin genes, like most eukaryotic genes studied to date, are segmented by sequences not retained in the corresponding mRNA. Although the function of such "introns" or "intervening sequences" remains speculative (21-23), their location in immunoglobulin genes shows an intriguing correlation with the polypeptide structure. Both light and heavy chains consist of regions of partially homologous amino acid sequence about 110-115 residues long which fold into globular domains, one for a V region, one for CK and CA, three for Ca and Cy chains, and four for the CQu chain (1, 24, 25). Intervening sequences not only separate the genes encoding a V and C region but, more remarkably, also divide the genes for the C regions of a and 'y1 heavy chains into segments encoding each of the three domains (17, 18), as well as a short segment encoding the Cyl "hinge region" (18). This raises the question of whether the domains in other heavy chain genes are separated by intervening sequences. We show here that CQt genes undergo somatic rearrangement and report the cloning of a rearranged C~ugene from an IgMsecreting plasmacytoma. We show that the CIA gene is divided by intervening sequences into four coding segments and that each coding segment specifies one of the four domains (homology units) of the ,u polypeptide, as identified by Kehry et al. (25) from the amino acid sequence. In addition, we have found that the mRNA sequence coding for amino acid residues near the V-C junction is absent from the CAL clone and from clones bearing homologous embryonic VH sequences. We suggest that this mRNA segment is derived from an independent genetic element, a joining region (JH) gene for heavy chains. MATERIALS AND METHODS Cloning of EcoRI Fragments of HPC 76 Tumor DNA. An EcoRI digest of DNA from the plasmacytoma HPC 76 (26) was fractionated by sucrose gradient centrifugation. Fragments 8-20 kilobases (kb) long were cloned in the phage vector Charon 4A (27) as described (16). Hybridization to Restriction Fragments. Restriction fragments were fractionated by electrophoresis on horizontal slab gels, then blotted onto nitrocellulose filters (28). Hybridization was carried out as described (29), except that the mixture contained denatured Escherichia coli DNA (6.6 ,gg/mt), 5 mM EDTA, and poly(C) at 20 Ag/ml instead of poly(A). Filters were washed for 5 hr at 650C in 0.30 M NaCI/0.030 M sodium citrate/0.1% sodium dodecyl sulfate/5 mM EDTA and then, for cellular DNA, for 30 min at 650C in the same buffer diluted

ABSTRACT To elucidate the structure of the gene for the constant region of immunoglobulin g chains, we have cloned a 9.9-kilobase-pair fragment of mouse DNA bearing a gene for the constant region of the ;& chain (Cp gene) from an IgM-secreting mouse plasmacytoma. The sequence around this gene has apparently undergone somatic rearrangement; the gene occurs in an EcoRI restriction endonuclease fragment of a different size from that in embryo or liver DNA and no Citbearing fragment of embryo size remains in the plasmacytoma. The cloned sequence lacks a variable region gene; hence, if this Cp gene is active, its position within the clone indicates that the gene for the variable region of a heavy chain (VH gene) must be more than 3.7 kilobase pairs away. The CQ gene is divided by three intervening sequences into four coding segments, each of which encodes one of the domains (homology units) of the polypeptide. The nucleotide sequence coding for amino acids near the V-C junction is not present within the Cp clone or clones bearing homologous embryonic VH genes. This suggests that an immunoglobulin heavy chain, in common with light chains, is encoded not only by a V and C gene, but also by an independent joining region (JH) gene. Immunoglobulins consist of light and heavy chains, each having a variable (V) region, which determines antigen-binding specificity, and a constant (C) region, which mediates biological effector functions (see refs. 1 and 2 for reviews). For both X and K light chains, molecular cloning has confirmed the longstanding hypothesis (3, 4) that V and C genes are distant in the germline (albeit genetically linked) and that rearrangement of DNA during lymphocyte differentiation brings a V and C gene closer together (5-11). Unexpectedly, light chain V regions themselves have been found to be encoded by independent DNA segments, one specifying the NH2-terminal 97 or 98 amino acids of a classical VA or V,< region, and another, now termed "joining" (J) region, specifying the remaining 15 amino acids (10, 12-14). In the germline, the J segment is actually closer to the C than the V gene. Somatic rearrangement, which is apparently required for gene expression, joins the V and J genes without altering the spacing between J and C (6, 7,

11). The heavy chain system, -which has been studied to a lesser extent, is of particular biological interest, because a single VH region apparently can be associated with different types of CH region in the same cell or cell lineage (4, 15). We have shown by cloning that there are multiple embryonic VH genes which are closely linked to each other but distant from CH genes (16). There is evidence that heavy chain genes undergo rearrangement (17, 18), and indirect evidence from several VH amino acid sequences suggests that there is a joining region for heavy chains (19). The gene for the constant region of ,u heavy chains

1:10.

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

Abbreviations: V, variable; C, constant; J, joining region; kb, kilobase(s).

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Proc. Natl. Acad. Sci. USA 77 (1980)

Detailed Restriction Mapping of C. Gene. The CM gene occurs within clone Ch-H76A1 (see Results). DNA from this clone was digested with BamHI, the 3' termini were labeled by use of E. coli DNA polymerase I, dGTP, and 2 ,uM [a32P]dATP (Amersham, 2-3 Ci/Amol; 1 Ci = 3.7 X 1010 becquerels), and two large labeled fragments (16 and 19 kb) containing the CAu gene were separated from smaller vector fragments by sucrose gradient centrifugation. Further digestion with Xba 1 yielded two fragments spanning the Cju gene (0.9 and 3.4 kb; A and B in Fig. 4) which were resolved on a 5% polyacrylamide gel. Partial restriction endonuclease digests (30) of A and B were analyzed by autoradiography after electrcphoresis on 5% polyacrylamide gels. Biological and Physical Containment Work was conducted in facilities classified CII and CIII by the Australian Academy of Science Committee on Recombinant DNA (ASCORD) (both classified P3 on NIH guidelines) with EK2 host-vector systems, in compliance with both ASCORD and NIH guidelines. RESULTS AND DISCUSSION Cs Genes Undergo Somatic Rearrangement. To serve as a pure hybridization probe for ,u genes, we have constructed a recombinant plasmid (pH76A17) that bears a nearly fulllength cDNA copy of a ,u mRNA isolated from the mouse plasmacytoma HPC 76 (unpublished data). Its identity has been confirmed by several lines of evidence; for example, nucleotide sequences of over 150 residues in this clone (unpublished results) fit exactly with part of the known amino acid sequence for the mouse Au constant region (25). We prepared restriction fragmerits of the cloned cDNA as probes for specific portions of the V and C regions, as indicated in Fig. 1. To determine whether the CM gene undergoes somatic rearrangement, we digested BALB/c mouse embryo, adult liver, and HPC 76 DNA with EcoRI, fractionated the resulting fragments by gel electrophoresis, blotted them onto a nitrocellulose filter (28), and then detected the CM gene by hybridization with a 32P-labeled CIA probe (Fig. 2). Embryo DNA yielded a single --12.5-kb CM fragment, whereas HPC 76 DNA contained fragments of 410 and ;7.9 kb. The HPC 76 fragments were labeled by both 5'- and 3'-C/A probes (c and d in Fig. 1), indicating that each contains a complete CM gene. The 7.9-kb CM band was 3 times as intense as the 10-kb band, as determined by densitometry, suggesting that HPC 76 DNA contains several copies of the 7.9-kb fragment. Thus, HPC 76 DNA contains at least two types of fragments bearing rearranged CM genes. DNA from adult liver gave only a fragment of embryo size, as expected if the rearrangement were restricted to immunoglobulin-synthesizing cells. Because no CM fragment of embryo size remains in HPC 76 DNA, rearrangement must

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FIG. 2. Detection of CM gene in EcoitI fragments of mouse DNA. The autoradiograph shows the fragments that hybridized to a CM probe (fragment c in Fig. 1) in digests (1 ,ug) of DNA from BALB/c embryos (E), adult liver (L), and HPC 76 plasmacytoma (H76). The sizes of CQ-bearing fragments (given in kb) were determined by comparison with appropriate DNA markers run on the same gel.

have occurred on each of the homologous chromosomes bearing

CH genes. Presumably not all the rearranged CM genes are ac-

tive, because heavy chain expression is confined to one allele (32). Hence, not all rearrangements of immunoglobulin genes may lead to expression. A Clone Bearing a Rearranged CM Gene. To isolate a rearranged CM gene, we cloned 8- to 20-kb EcoRI fragments of HPC 76 DNA in the X phage vector Charon 4A. On packC

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FIG. 1. Probes for specific regions of ki genes made from cloned ji cDNA. The At cDNA segment of plasmid clone pH76ju17 is shown with the V region (stippled), a proposed J region described here (solid), and the Cg region (hatched). Restriction endonuclease sites defining specific fragments used as probes are indicated. The entire cDNA insert served as a VH76 + CQu probe; Mbo II fragments a, b, and c served as V, J, and 5'-Cut specific probes, respectively. A related plasmid, pH76$7, bearing only that portion of the CQ sequence indicated as fragment d, provided a 3'-Cg specific probe. Fragments were labeled by nick translation (31).

3 4 5 6 7 8 9 1 2 FIG. 3. Identification and orientation of the cloned Cy fragment. (A) Detection of a 9.9-kb fragment bearing the CMA gene in Ch-H76,M1. An EcoRI digest of Ch-H76MA1 DNA (0.8 Mig) was electrophoresed on a 1% agarose gel and hybridized with the VH76+CMI probe. Track 1, ethidium bromide-stained gel; track 2, autoradiograph. (B) Relationship of the cloned fragment to CM fragments of mouse DNA. EcoRI digests of Ch-H76M1 (350 pg; track 3), Ch-H76MA1 plus embryo DNA (350 pg + 15Mg; track 4), and HPC 76 DNA (15 Mg; track 5) were fractionated on a 0.7% agarose gel. The fragments were detected with a CMA-specific probe (fragment c in Fig. 1). (C) Polarity of the CMu gene within the 9.9-kb fragment. HindIll-digested samples of Ch-H76M1 DNA (0.8 Mg) were electrophoresed on 1% agarose and then hybridized with a VH76+CM (track 7), 5'-CMA (track 8), or 3'-CM (track 9) probe. Track 6, ethidium bromide-stained fragments; tracks 7-9, autoradiographs.

Proc. Natl. Acad. Sci. USA 77 (1980)

Immunology: Gough et al.

556

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3' 5' C/ FIG. 4. Restriction map of the 9.9-kb mouse DNA insert in CH-H76A1. The direction of transcription of the CA gene (hatched) is indicated. The second HindIII site from the left occurs at one of the positions marked with broken lines. Fragments A and B (0.9 and 3.4 kb) were those used to drive the more detailed restriction map in Fig. 6.

aging the recombinant DNA into phage coats in vitro (33), we obtained 250,000 clones. Screening by plaque hybridization (34) with our , V+C probe revealed one clone (Ch-H76A1) that

hybridized unusually strongly, making it a likely candidate for a Cp clone. An EcoRI digest of Ch-H76A1 DNA contained two fragments (9.9 and 6.8 kb), in addition to the vector arms (Fig. 3, track 1). The 6.8-kb fragment was shown by heteroduplex analysis to be an internal fragment of Charon 4A (27). The 9.9-kb fragment hybridized to the V+C probe (track 2) and to a CA probe (track 3), but failed to hybridize to a V region probe (fragment a in Fig. 1), indicating that this clone bears only a Cp gene. Fractionating the cloned fragment together with a digest of embryo DNA (Fig. 3B, track 4) clearly established that the cloned fragment was smaller than the embryo CA fragment and indistinguishable in size from the larger HPC 76 CA fragment (track 5). Thus, Ch-H76A1 contains a CA gene within a 9.9-kb segment of mouse DNA that has undergone rearrangement from the embryo context. A.

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FIG. 5. Segmentation of the CQ gene revealed by R loops. Electron micrographs of R loops between HPC 76 Ai mRNA and Xho Idigested Ch-H76,u1 DNA. In the diagrams the mRNA is depicted by dashed lines. The RNA tails distal to the Xho I site in A is the unhybridized V region. C1-C4 are coding segments described in the text. The arrow indicates a probable intervening sequence. In Xho-cut Ch-H76,gl, the CQg gene is located 3.3 kb from the Xho end and 21.8 kb from the natural (right) end of the recombinant. Hence, in each molecule it was easy to distinguish the Xho end from the other end unambiguously at magnifications lower than that shown here. R loops were formed by incubating mRNA (10 pLg/ml) and DNA (5 pg/ml) in 80 mM 1,4-piperazinediethanesulfonic acid (Pipes)/5 mM Tris.HCl, pH 7.9/0.42 M NaCl/3 mM EDTA/60% formamide for 3 min at 90'C, for 5-6 hr at 580C, then for 45 min at 30'C and spread as described (36).

We positioned the Cp gene within Ch-H76A1 by constructing a restriction endonuclease cleavage map of the 9.9-kb fragment (Fig. 4). The gene could be positioned accurately, because the only BamHI cleavage site in the cloned fragment occurs within the gene (see Fig. 1). The orientation of the Cp gene was determined by hybridization to HindIII fragments of Ch-H76A1 DNA (Fig. 3C). A probe representing the entire Cp region hybridized to two fragments (track 7), indicating that HindIII cuts within the gene region, whereas a 5'-CAuprobe only hybridized to only one of these (track 8) and a 3'-CAtheprobe order of to the other (track 9). Because we had determined the HindIII fragments, this unambiguously established the orientation shown in Fig. 4. The clone contains 3.7 kb of mouse DNA to the left of the Cp gene (Fig. 4). Thus, if this rearranged gene is expressed in HPC 76 cells, the V and C genes must be separated by an intervening

sequence of at least 3.7 kb which contains an EcoRI site. Such a separation would not be unexpected, because a Ca gene is 6.8 kb from the VH gene in an IgA-secreting tumor (17). We do not know, however, whether this rearranged CAu gene is ex-

pressed. The CM Gene Contains Three Intervening Sequences. The first indication that the CA gene contained intervening sequences came when we examined R loops (35) formed between HPC 76 A mRNA and Ch-H76A1 DNA that had been digested at the unique Xho I site (see Fig. 4) to provide an orientation point. The 48 R loops examined revealed molecules containing one to three hybrid loops spanning all or part of the Cp gene. Their location in different molecules suggested that the gene was divided into four roughly equal segments by three small intervening sequences. For example, a number of molecules, like that in Fig. 5A, had two short hybrid loops distal to the Xho I site, suggesting two coding segments in the 5' half of the gene; a few like that in Fig. 5B were observed with two short loops proximal to the Xho I site, indicative of two more coding segments in the 3' half. We did not see four clear hybrid loops in any single molecule, presumably because two of the intervening

sequences are very short (see below). To confirm the presence of intervening sequences and to position them precisely, we constructed a detailed restriction map of the Cp gene by partial digestion (30) of two fragments spanning the gene (A and B in Fig. 4). We had previously that all the C region mapped the cloned cDNA and determinedrelated to the amino restriction sites could be unambiguously acid sequence of the MOPC 104E p chain (25), and the alignment has been verified in several regions by nucleotide sequencing (unpublished results). We could identify coding segments of the gene by matching the pattern of restriction sites within the gene to that of the cDNA. Fig. 6 shows that the gene has four coding segments (C1-C4), in which the restriction sites intervening secorrespond to those in the cDNA, and threethat contain sites defined and 13), regions I2, by quences (I1, not present in the cDNA. For regions C2, C3, and C4, the spacing between restriction sites in the gene and cDNA agree very well, but C1 appears to be t60 bp larger than expected (370 in contrast to 310 bp). This discrepancy may result from error in

measurement or the presence of a further small inter-

in vening sequence within C1. I1, I2, and I3 are comparable size (135, 365, and 60 bp) to intervening sequences within the Cyl and Ca genes (17, 18). The patterns of restriction sites within I1, I2, and I3 differ from those within the Cyl gene (18),

but this does not exclude sequence homology. Each Coding Region Specifies a Polypeptide Domain. Having previously aligned our restriction maps with there-g amino acid sequence (25), we could show that each coding gion specifies one of the four domains (homology units) of the

Immunology: Gough et al.

Proc. Natl. Acad. Sci. USA 77 (1980)

557

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FIG. 6. Intervening sequences in the CQg gene revealed by comparison with the corresponding cDNA and mRNA. (Top) Mouse Al chain mRNA, showing the V region (stippled) and four C region domains, CH1-CH4 (hatched). A candidate joining region (J) described in the text is shown at the V-C junction (solid). Presumptive noncoding regions (NC) are shown at the 3' and 5' ends of the mRNA. (Middle) Restriction map of the cDNA segment in clone pH76,u17. (Bottom) Restriction map of the CM gene region within clone Ch-H761. The restriction sites that define the boundaries of the coding sequences, C1-C4 (hatched), are related to equivalent sites in the cDNA restriction map by solid lines. Sizes of coding regions and intervening sequences (11-13) are given in base pairs. Region C4 can be related to the cDNA restriction map up to the rightmost Hae III site and is thus at least 340 bp long. Estimates from R-loop analysis suggest that this region is 550 + 80 bp long.

polypeptide (Fig. 7). Kehry et al. (25) propose that homology unit CH1 begins at residue 126 and that CH1, CH2, and CH3 end after residues 232, 335, and 446, respectively. Our mapping data on the gene segments indicate that C1 begins between residues 130 and 135 and that Cl, C2, and C3 terminate between amino acid residues 224-229, 331-335, and 450-453, respectively. This is a very good correlation, given that the ends of polypeptide homology regions cannot be defined precisely. Thus, in common with the constant regions of 'yl (18) and probably a (17) chains, each domain of the ki constant region is encoded by a separate gene segment. Gene segment C4 probably also specifies the 3' noncoding region of the mRNA. The restriction mapping data indicate that region C4 must be at least 340 bp long (as defined by the terminal Hae III site), and we estimate from R-loop analysis that the total length of C4 is 550 ± 80 bp. Thus, C4 appears to be 100-200 nucleotides longer than needed to encode the final portion of the polypeptide chain. A Candidate Joining (J) Region for Heavy Chain Genes. We have identified a ji mRNA segment immediately 5' to the N

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5 4 5 1 2 4 3 3 B A Fic.. 8. Absence of a presumptive J region sequence from the Cy clone and three VH clones. Restriction fragments from the Cy clone (track 1), from the y cDNA plasmid (track 2), and from three clones hearing VH76 genes (tracks :3-5) were fractionated on the same 1% agarose gel. (A) Ethidium bromide-stained gel; (B) autoradiograph after hybridization with the J region probe (fragment b in Fig. 1). I)NA samples contained: track 1, 0.8 Mg of a HindIII digest of ChH76M1IDNA; track 2,30 ng of the entire cDNA insert from pH76,u17 which contains both V and C sequences; tracks 3-5, 0.8 Mg of EcoRI digests of ChMe-M48, ChMe-M20, and ChMe-Ml1, respectively (16). Hybridization and washing conditions were 1.5 M NaCl/0.15 M sodium citrate at 550C. 1

2

558

Immunology: Gough et al.

C region U in Fig. 6) which appears to represent a joining region for heavy chain genes, because it is not derived from the CM gene or homologous VH genes. We had positioned the 5' end of the CAL gene just to the left of the Mbo II site indicated by the asterisk in Fig. 6, because the restriction maps of the gene and cDNA diverge thereafter. We therefore tested whether the sequence extending 80 bp leftward from that site in the cDNA clone (fragment b in Fig. 1) was present anywhere in the CA genomic clone. Fig. 8 shows that fragment b did not hybridize to Ch-H76A1 DNA (track 1), although it hybridized very strongly to a molar equivalent of cDNA plasmid (track 2). Because the sequence was absent from the entire CM clone, we tested whether it represents part of a VH region by using embryonic clones bearing VH sequences homologous to that in the HPC 76 mRNA (16). Tracks 3-5 show that fragment b failed to hybridize to DNA from three clones bearing different VH76 genes, which hybridized well with a VH76 probe (fragment a in Fig. 1) or smaller probe segments (43-119 bp long) derived from it (see figure 6 in ref. 16). Each VH clone contains 16-19 kb of embryo DNA, and one bears two VH76 genes 15 kb apart and may represent an entire repeating unit of VH76 gene structure (16). Because theM mRNA sequence bridging V and C regions is not derived from a DNA sequence near the CM or embryonic (untranslocated) VH genes, we propose that it is derived from a JH gene, an independent genetic element similar to the J segments implicated in translocation of light chain genes (6, 7, 11, 13, 14). By analogy with light chain J regions, it is likely that the JH gene expressed in HPC 76 is located together with a VH76 gene but separated from the expressed CM gene by a large intervening sequence. The JA gene is separated from the CA gene by about 1.2 kb (7) and five different J. genes are separated from the CK gene by 2.5-4 kb (13, 14), but the separation between a JH and CH gene may well be somewhat larger, judging by the 6.8-kb separation between an expressed VH and Ca gene (17). The 80-base-pair restriction fragment we used as a J region probe would correspond to amino acid residues 106-132 of the MOPC 104E chain; hence, the J region lies within that region. Although our results clearly point to the presence of JH genetic elements, their number and organization remain to be established. Differences between VH amino acid sequences near the V-C junction suggest that there are at least four different JH regions (19), and these appear to assort with different types of CH region (19, 37). Hence, CH genes may share a single pool Of JH as well as VH genes. Alternatively, different CH genes may possess separate pools of similar JH genes. We thank Elizabeth Webb for useful discussions and Jill Jackson, Jan Holton, and Sue Kyne for excellent technical assistance. The research was supported by the National Health and Medical Research Council (Canberra), the U.S. National Cancer Institute (RO1 CA 12421), and the American Heart Association. 1. Gaily, J. A. (1973) in The Antigens, ed. Sela, M. (Academic, New York), Vol. 1, pp. 162-298. 2. Spiegelberg, H. L. (1974) Adv. Immunol. 19,259-294. 3. Dreyer, W. J. & Bennet, J. L. (1965) Proc. Nati. Acad. Sci. USA

54,864-869.

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