Mrna for Surface Immunoglobulin Y Chains Encodes a Highly Conserved
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nati Acad. Sci. USA Vol. 79, pp. 2008-2012, March 1982 Immunology mRNA for surface immunoglobulin y chains encodes a highly conserved transmembrane sequence and a 28-residue intracellular domain (alternative RNA processing/B-lymphoma cDNA clones/antigen receptor/memory B cells/membrane gene segments) BRETT M. TYLER, ALAN F. COWMAN, STEVEN D. GERONDAKIS, JERRY M. ADAMS, AND ORA BERNARD Molecular Biology Laboratory, The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria 3050, Australia Communicated by G. J. V. Nossal, December 18, 1981 ABSTRACT To probe the structure of the y heavy chain of (16) that 2PK-3 synthesizes a 1.9-kb y2aS mRNA and a 3.9-kb membrane IgG and the mRNA and gene segments that encode it, y2am mRNA with distinct 3' ends. 2PK-3 also contains a 3.5-kb we have analyzed cDNA clones derived from a yV membrane RNA membrane Vy RNA (16) which is probably analogous to certain of B lymphoma 2PK-3. The nucleotide sequence of the clones in- transcripts of the C, gene that lack V regions but are correctly dicated that membrane yV chains bear a COOH-terminal 71-res- spliced in the C and membrane regions (17). We have now idue segment that is absent from secretory yV chains. This ter- isolated several membrane yV cDNA clones from this RNA. minus includes a 26-residue hydrophobic transmembrane region Their nucleotide sequences indicate that membrane y chains homologous to that ofmembrane ,I chains and, significantly, a 28- have a hydrophobic transmembrane sequence surprisingly ho- residue intracellular domain found only on y chains. The extra mologous to that of, plus a 28-amino acid intracellular domain domain suggests that receptor IgG, on memory B cells, may gen- A, erate adifferent signal on bindingantigen than does receptor IgM, specific to ychains. This extra domain suggests that membrane on virgin B cells. The Vi membrane terminus is encoded by two IgG generates a signal different from that of IgM on binding gene segments 1.5 and 2.4 kilobase pairs downstream from the C,,1 antigen. We also show that membrane gene segments for dif- gene, and -homologous segments occur 3' to the C-,2a and C-A ferent subclasses of y chain are organized similarly and that ym genes. Small amounts of membrane y mRNAs persist in plasma- mRNA occurs even in IgG-secreting plasmacytomas. cytomas secreting IgGI, IgG2a, or IgG2b, suggesting that com- petition between alternative RNA processing pathways governs MATERIALS AND METHODS the synthesis of membrane and secretory y chain mRNAs. Construction of cDNA Clones. Poly(A)+RNA (140 ,ug) iso- lated (17) from 2PK-3 tissue culture cells was size fractionated Immunoglobulin can either be secreted as antibody or serve on on a 10-40% sucrose gradient. Double-stranded cDNA was the membrane as the antigen receptor of a B lymphocyte (1). constructed (18) from RNA fractions (-10 ,ug) containing mem- The COOH-terminal constant (C) regions of the heavy chain brane y sequences and cloned by dGdC tailing (18) into the Pst determines whether the molecule will be secreted or bound to site of pBR322. Transformants identified by colony hybridiza- the membrane (2). In the best studied case, IgM (2-6), the se- tion (19) with membrane y sequences (see text) were purified cretory heavy chain (ij) has a 20-residue hydrophilic terminus and plasmid DNA was isolated (18). and the membrane ,u chain (Im) has a 41-residue hydrophobic Nucleotide Sequence Determination. Insert sequences from terminus that spans the membrane. The /im and 1,i mRNAs the clone VyML.3 were isolated on 5% acrylamide gels after have correspondingly distinct 3' termini generated by alter- digestion with Rsa I or Hpa II. The fragments were cut further native RNA processing pathways (4-6). The ,us terminus is en- as outlined in Fig. 1B, labeled at their 5' ends by using polynu- coded contiguously to CH4 whereas the two gene segments for cleotide kinase (20) or at their 3' ends by using the large frag- the gum terminus lie 1.85 kilobases (kb) downstream (6). The ment of DNA polymerase I (20), recut with another enzyme, other major membrane immunoglobulin, IgD, also has distinct and purified on 8% acrylamide gels. Sequence determination mRNAs and gene segments for the membrane and secretory by partial chemical degradation was essentially as described chains (7-8). (20). For sequence determination by chain termination (21), the Whereas IgM and IgD occur primarily on virgin B cells (9), large Rsa I fragment was digested with Hae III or Sau3A and the other surface immunoglobulin classes, such as IgG, appear cloned into the HincII or BamHI sites, respectively, of to be associated with memory B cells (10, 11). Little is known M13mp7.1 (a gift from G. Brownlee). Recombinant plaques ofthe structure of membrane IgG or whether the signal that it were analyzed as described (21). The sequencing primer, generates on binding antigen differs from that transmitted by dNTPs, and ddNTPs were from Collaborative Research. membrane IgM (12). The size of the ym polypeptide suggests a specific membrane terminus of at least 7,000 daltons (13, 14) RESULTS and the recently published nucleotide sequences of putative membrane Vy and V2b gene segments suggest that part of this Isolation ofMembrane yV cDNAClones. To clone sequences terminus is similar to the gUim terminus (15). from the membrane Vy or V2& RNAs of 2PK-3 (16), approxi- To define the structure ofmembrane ychains, a prerequisite mately 40,000 cDNA clones were constructed from poly(A)+RNA to understanding howreceptor IgG functions, we have analyzed enriched for membrane species on sucrose gradients. By using mRNA from the B lymphoma 2PK-3, which produces both genomic membrane V2a or y3 probes (b and c in Fig. 2) defined membrane and-secretory IgG2a (13). We have previously shown previously by hybridization to 2PK-3 mRNA (ref. 16; S. Nakai and T. Honjo, personal communication), five membrane y The publication costs ofthis article were defrayed in part by page charge cDNA clones were identified, with inserts of 0.4 to 2.5 kb. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: C region, constant region; kb, kilobase(s). 2008 Downloaded by guest on September 24, 2021 Immunology: Tyler et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2009 A 1 200bp 1 ao "s 0 cf 0y X a. i: >- 0 6)~~~~V I~ I I II I £I I,r,I T xM1.1 I CH II CH2 I CH3 I M I 3'UT H I XM1.2 I I XM1.3 B 100 bp CH3 M 3'UT Avall AvaI Ava \ m Haem Sau3A m Sau3A Hinf H aem Sau3A Rsa Sau3A Hif K I l l 1 1- 1 r a I I --_, - - - -- -- -I I---. FIG. 1. Membrane yj cDNA clones from 2PK-3. (A) Restriction maps of three clones. Only one of the three Hinf sites in yM1.1 is shown. The unmarked horizontal line indicates a probe fragment. (B) Sequence determination strategy for yM1.3. Solid and broken lines indicate fragments analyzed by chemical degradation from the 5' and 3' ends, respectively; wavy lines indicate those analyzed in M13. Although the yj and Y2a RNAs were ofcomparable abundance, membrane Yi sequences, the 3' half of yM1.1 (Fig. 1A) was it happened that all five clones derived from the ==3.5-kb mem- hybridized to mRNA from 2PK-3 and from plasmacytoma brane yj RNA. Restriction maps of three ofthem are shown in MOPC 21, which produces both membrane and secretory IgGC Fig. 1A. All restriction sites in CH1 to CH3 fit the C.Y sequence (23). The cDNA clone hybridized strongly to the 3.5-kb mem- (22). The two longest clones, yM1. 1 and 'yMl.2, contained 1.5 brane )yi RNA of2PK-3 (arrow, Fig. 3, lane a). Moreover, lane kb 3' to CH3 not present in secretory Yi mRNA (22) and hence b shows that the probe hybridized strongly to the 3.9-kb pre- included the 5' 70% of the "2-kb 3' untranslated region (16). sumptive membrane Yi mRNA in MOPC 21. (The hybridization yM1.3 contained only the 5' 0.8-kb of this region. to the 1,000-fold more abundant yls mRNA presumably reflects To confirm that the 3' region of the clones corresponded to trace CY,1 contamination of the probe.) 5- Aval Sma 3' CXWI| td[amRa Sma Hha Kpn Hha Hha EcoRi Aval Hha Aval Kpn B912 CH1 ^ H| CH2 CH3_ 3S ~~~~~~~1.50kb 0.81kb Cv lM2 o2a Barnan PtPst Sn~Sma Hhah 1 638Bgl12 Hha BgI2 Hind Xba Barn I ~~~~~~1.38kb m0-47kb ~ii.- - C b ' 1 "63 Barn Kpn Pst Hha Rsa Xba Hind3 I ~~~~~~1-42kb m0-50kb a c 0X2b Bam I >- Kpn Kpn Hha Bam EcoRl - * _- l Ia I _I .I - - - 1-36kb - 0 57ko -- COu I lkb -aZ..-E .fX- 1f .164 :.::.::.:.:.:.:3 1-85kb - - CH1 CH2 CH3 CH4 S Ml M2 FIG. 2. Gene segments for membrane immunoglobulin heavy chains. CH1, H, CH2, and CH3 encode the C region domains, S encodes the se- cretory mRNA 3' terminus, and Ml and M2 encode the membrane mRNA 3' terminus (see text). The CH3-M1 and M1-M2 distances shown derive from measurements of >20 heteroduplex molecules each, the standard errorbeing always less than ± 0.05 kb. In the M2 segments, solidbars indicate coding sequences, stippled bars are 3' untranslated regions, and open bars are stretches of low homology (<50%) to the y1 cDNA clones.