Proc. NatL Acad. Sci. USA Vol. 79, pp. 2623-2627, April 1982 Genetics

Nucleetide sequences of gene-segments encoding membrane domains of immunoglobulin y chains (conserved hydrophobic segment/intervening sequence-mediated domain transfer) YURIKO YAMAWAKI KATAOKA*, SUMIKO NAKAI*, TAKASHI MIYATAt, AND TASUKU HONJO* *~Department of Genetics, Osaka University Medical School, Nakanoshima, Kita-ku, Osaka 530, Japan; and tDepartment of Biology, Faculty of Science, Kyushu University, Fukuoka8l2, Japan Communicated by Motoo Kimura, December 28, 1981 ABSTRACT The nucleotide sequences of the exons encoding MATERIALS AND METHODS membrane-bound IgGI, IgG2a, and IgG2b of mouse were deter- Materials. Recombinant DNA clones that contain membrane mined and compared with the sequence ofmembrane-bound IgM. were ob- The sequences indicate that membrane-bound y chains bear an exons of mouse immunoglobulin genes previously additional 71 residues at the COOH termini, including 17-residue tained and characterized: IgH2 bearing the C.1 gene (14), acidic, 26-residue hydrophobic, and 28-residue hydrophilic por- Igy2a-9 bearing the CY2. gene (15), and IgH22 bearing the C,2b tions. The hydrophobic portion, which seems to be anchored in gene (16). All the clones were obtained from BALB/c mouse the bilayer of the membrane, is highly conserved among DNA. An EcoRI fragment [4.9 kilobases (kb)] of Igy2a-9 was membrane-bound ji and y chains. We propose the presence of a inserted into pBR322 and subeloned. The insert (6.6 kb) of membrane that recognizes the conserved hydrophobic IgH22 was subcloned in pBR322 previously (17). Phage and segment and anchors the membrane-bound immunoglobulin. plasmid DNAs were prepared as described (14, 17). Sources of Comparison of the nucleotide sequences revealed another exam- restriction enzymes were as described previously except that ple of intervening sequence-mediated domain exchange around HindIII, HincII, Pst I, and Acc I were obtained from Takara one of the membrane exons. Shuzo Co. (Kyoto, Japan). [y-32P]ATP was purchased from the Radiochemical Centre (Amersham, England). Immunoglobulin molecules such as IgM, IgG, and IgA exist in Methods. Nucleotide sequence was determined according two different forms, as a surface receptor and as a se- to Maxam and Gilbert (18). Thin gels (0.5 mm) were used as creted (1-10). The membrane-bound forms of the described (14). Nucleotide sequences ofthree y subclass genes heavy (H) chains- 4., y.' and am-are larger than the se- were aligned by using a computer program (12). The extent of creted forms of the heavy chains-,us, ys, and as. Recent anal- the nucleotide sequence divergence was calculated as described yses of cloned immunoglobulin genes demonstrated that (13). mRNAs encoding membrane-bound and secreted forms of the ,u chain are transcribed from the same C, gene,. and the two ;RESULTS AND DISCUSSION forms ofthe y2a chain are transcribed from the same Cy2a gene (3-6, 9, 10). (C indicates the gene for the constant region ofthe Nucleotide Sequences of C,, Membrane Exons. Locations of heavy chain.) There are two membrane exons in the 3' flanking membrane exons in the C, genes had been determined by R- regions of the C, and C ,2a genes. The alternative pathways of loop and heteroduplex studies (9). Restriction enzyme cleav- RNA splicing give rise to two mRNAs encoding separately the age maps around membrane exons of the Cyl, Cy2a, and Cyb membrane-bound and secreted forms of the heavy chains. genes were constructed by conventional procedures. The The amino acid-as well as nucleotide sequence of the u.~ ranges and restriction sites used to determine the nucleotide chain revealed a 26-residue hydrophobic segment at the COOH sequences of the membrane exons are shown in Fig. 1. terminus, which seems to be anchored-in the lipid bilayer ofthe The nucleotide sequences ofthe membrane exons ofthe C-,,, membrane (3, 4). Previously, we showed by heteroduplex anal- C12a, and C,2b genes were determined, compared, and aligned yses that the nucleotide sequences of the membrane exons are to maximize homology as shown in Fig. 2. We looked for an conserved among four CY subclass genes (9, 11). open coding frame that can be translated into the amino acid We have now determined the nucleotide sequences of the sequence homologous to that in the C. membrane exon. Such membrane exons ofthe Cy2a, Cy2b, and C,, genes; the sequences a region is shown by amino acid residues predicted from DNA clearly demonstrate that the highly conserved 26-residue hy- sequences ofthe Cy2a gene. The precise splice sites as indicated drophobic segments are also present in these C, genes. Com- by vertical lines in Fig. 2 were determined by comparison to parison ofthe nucleotide sequences ofthe membrane exons and the nucleotide sequence of the cDNA coding for the ylm chain neighboring introns revealed that not only the Ml exon but also (19). The amino acid as well as nucleotide sequences of three about 160 base pairs (bp) ofthe intron immediately 5' to the M1 C, genes are so homologous to each other that the M1 exons exon are highly conserved between the C,, and Cy2b (or C,2,) of the Cy2a, Cy2b, and CYj genes were unequivocally identified. genes. Such sequence homology at limited portions of a gene The M2 exons of the Cy2a and C,,2b genes were also easily as- indicates that another intervening sequence (IVS)-mediated signed by comparison with the yl cDNA sequence (19). Al- domain exchange took place around the Ml exon, in addition though the M2 exon ofthe C, gene encodes only two amino acid to the one proposed around the CH1 exon (12, 13), between the residues, the M2 exons of the C 2aand Cy2b genes encode 27 CYj and Cy2b genes. amino acid residues. Because the M2 exon is about 1,000 bp

The publication costs ofthis article were defrayed in part by page charge Abbreviations: subscripts m and s, membrane-bound and secreted payment. This article must therefore be hereby marked "advertise- forms of immunoglobulin heavy chains, respectively; IVS, intervening ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. sequence; bp, base pair(s); kb, kilobase(s). 2623 Downloaded by guest on October 3, 2021 2624 Genetics: Yamawaki-Kataoka et al. Proc. Nad Acad. Sci. USA 79 (1982) kb 0 I 2 3 4

CHI H CH2 CH3 Ml M2 flv9.

l, Hha I y2a PvuII AvfoU Alul B0 AMu~~~~~~~SmaHo Hinf I AMlulIWo1 Hin I Sm I Hinf I MboI Mboll_____ rS-|'14M1 S 11 ' Snfl s~~' I I - 1X7h u Pz II11U1AillAkA~I BolilAII All |HhIJ'& Hijf I AMul MbI IBIll AMuMON HinfI SmoI Hinf I Mbo 11 Mo 11 Hinf I i

- I .D V1 Hiff I Ava I] Alul1 Ad ||Hol Accl If MboI MboIl _. i 4 100 bp FIG. 1. Restriction maps and sequencing strategy of membrane exons of C,2a, C,2b, and C,1 genes. Open, filled, and dotted rectangles indicate exons encoding secreted domains, membrane-bound domains, and untranslated sequences, respectively. The direction of transcription is from left to right. Arrows indicate ranges and directions of the sequence read. long, the M2 exon must contain a 3' untranslated sequence of The Membrane Domain Is a Common Structure of the Im- more than 900 bp; the sequence of this region has not been munoglobulin CH Gene. The membrane exons ofthe Cyl C 2b, determined completely. and Cy2a genes, which were indicated by R-loop and hetero- 20 40 60 80 100 y2a CCICCACCliClCClGIACiGAAGGAAlGAAAGA1GAGACAAGCA1AGAGGGCAC11GAAlAA1CCAGG1CAC1C1GAGG1CCA------***vC y2b CClCCACCllClCC161AC1GAAGGAAlGAAAGAlGAGACAAGCAlAGA6GGCAC11GAAIAAlCCAAGlCAClCIGAGGICCA.------C yl llGClClCCAG1ClCAAAAC1GAAGGClGGAGCACACAGAAlAAGClCClACACAGCCCAGGCCAGlAlCGGGlCCAGIG1G1ClGAAlGAGC y2a CCAAGGCAllAllGGAClCAGG-lGGGAAGCTGAGAClGG1GlCCCAGAGGGAA-GGAAGGAAAGCAGG-CCCCGGGGAGGGlCIGC161.------C 200 y2b CCAAGGCA11A11G6AClCAGG-1GGGAAGC1GAGAC1GA1GlCCCAGACGGAAAGGAAGGAAAGCAGG-CCCCGGGGAGGGIClGC1G61.*-----C yl ClAGGGAAAAAAIGGCAGCAC111GGGGAAC1GAGG11IC1G*----*GICCAAGAAGGAGAGklGGA6GCCCAGGGAGGITC1GC1GACCGCCCAGC y2a CCAG1CAGGC1GGAG'A1ClCICClCIGAA1CCAIGCAGACA1GlClGCC1CACAG66AAIC1CCCCAGCACCAACCAIG11GGGACAAAC1GAC'IG 300 y2b CCAGICAGGC1GGAGAIC1CICClClGAClICAlGCAGACA1GlCIGCClCACAGGGAA1ClClCCC-AGCAICAACCA1G11GGGACAAACACIGAC1G y1CCAGCCCAGClGCAGCll1C1CCIGGGCCICCAIGCAG--C1ICCIGCCACACAGG6AA1*-GGCCC1AGCCCCA-CC11AlIGGGACAAAwACI6ACCG L D L D D V C A E A ' D G E L. D G l 1W 11 1 1 I F I S l y2a ICC1ClCI1G1CAI G6ClAGACClGGAlGAIGlC16GC16GAGGCCCAGGACGGGGAGC1GGACGGCCIC1G6ACGACCA1CACCA1lAiCA1CAGCC 400 .y2b ICCIClC1G11CAt GCI1AGACC16GAIGAlAICIGlGCIGAGGCCAAGGAIGGGGAGC1GGACGGCCIClGGACGACCAICACCA1C1 1CAITCAGCC1 C Y1 CCCI lCICTGICCA GGClGCAACIGGACGAGACCl Gl GCIGA6GCCCA6GACGGGGAGCl GGACG'GGCI ClGGACGACCAICACCAI CI11CA1lCAGCILI- C F L l S V, C Y S A S V I l F K y2a 11TCCI GCTlCAGCGl GlGC1 ACAGCGCC1 ClG1 CACACIC1TTCA'A G1*lGGCAClT61C1CCCACCClC16CITGlGAI GGC1IACACI GACCACAAAAIG61C 500 Tb11CC16C1CAGCGlG1TGC1IACAGCGCC1 CIG1 CACAC1 ClI CAA G1*1G6CACIGlClCCCACCC1 C1GC1 G1GA1 AGC1 ACAC1 GACCACAAAAI Gl yi ITCC1GC1CAGC6161GC1ACAGCGCtGCIGICACAClCl1CAAt GlCAGCCAlAC1GlCCCCACA-GGlICGAC y2a CICICACICCICCCCAGAIGlAGIAGGACGIACITIGCCCCIACICIGICCCACACACCAII CClCCAl1CCC1GA6CCA1CCCACAIGIGC1A 600 y2b CTICAC1CC1CCCC1GA161AG1AG6A1Gl1ACI11C1GCCCCCACIC1GlACCACACACCGlI1CC1CCA1TCCClGA6CCA1CCCACAIGCIClA y2a IGlGACICCACA1161GICCCA1ACAG1C1GCCC11C1GlCIClC1GGCG1CC1GCG1GATCClGAIACIG1ClIA1GAGACCAAACCICC11GCA1 1CC 700 y2b IGlGACICCACA1161CCCAlACA61C16CCCllC161Cl'ClACGC1GlCClGCAlGAlCCTCAlAC1GIC1 AlGCAACCAAACCICCIIGCAIICC y2a ACAClAGCCl1CA1GAGG1lCAAlGC1G1C11AGACACAA1CCCClCA -----GCClCACCA1GGClCAAGGlAClClG1GAGCIA1CCICAIACCAIC 800 f2b ACAC1A6CC1ICAlGA61ICA'I6C1GlClAACACACAAlCC'CCIIGA1G11CGCC1CACCA1AAC1CAAGG1AClClGlGAGCIAAIClACCAIC y2a 1CCACClCAAClCCCACAAIC1CCAC1C1GACCCC-1CCCA1ACCCAG1ClCCIACC1G1A1GAAGGGA-A11GAA6GAGAGACAGGCCIClGlCl 1 900 y2b 1CCACClCAAClCCCACAAlA1C1CCAC111GACCACllCCCA1ACCCAGJC1CCIACC1G1A1GAAAGGAAA11GAAGCAGAGACAG6CCICl61Cl1C V K W I F S S VV E L K O CCCACA-6A1 16AGGGlClGAGCAIGG6CGlGGlCClGAC11CCICACIICCCCACAIAAAG1GGA1C1 ClCClC1GlG61GGA6CIGAAGCA 1000 y2ay2b CCCACAG6AliGGAGG61C1TGAGCA1GG1G1GGlClC1GAC1'-C1 CAC11CCCCACAI6IAAAG1GGA1C11C1CClCAGlG61GGAGCIGAAGCA 1 I S P D Y R N M I G O G A y2a GA'CGA1CCCCCl6AC1ACAGAAACAJGA11GGGCAGGGAGCC1AGGCCAC1lCCIClGGGAlCAGAAGAGC1CC1 1100 y2b 6AA6AlClCCCCl6ACTACAGAAACATGAIG66CA666A6CC1A66CCAC11CC1C1GGGAlCAGAAGAGClTCClAAGlCClGCAGAA6CCCA1CCA1 y2b CClAC1GlG FIG. 2. Nucleotide sequences of the membrane exons of C,,2., C,2b, and CY1 genes. Deletions (*) are introduced to maximize homology of the three genes. Vertical lines indicate splice sites. The amino acid residues of the membrane exons of the CY2a gene are shown by the one-letter code: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine. Downloaded by guest on October 3, 2021 Genetics: Yamawaki-Kataoka et aL Proc. Natl. Acad. Sci. USA 79 (1982) 2625

duplex analyses (9), were unequivocally identified by nucleo- Table 1. Divergence of amino acid sequences between the ,u and tide sequence analysis. It is very likely, therefore, that the con- y chains of mouse* served regions in the 3' flanking region of the C,, gene also Heavy chains contain the membrane exons. We have also identified the mem- compared CH1 CH2t CH3 M brane domains of the C6 gene by R-loop analysis and Southern ,t and yl 0.76 0.75 0.63 0.47 blot hybridization (unpublished data). The membrane exons of ,u and y2a 0.78 0.75 0.68 0.47 the C, gene were identified recently by nucleotide sequence 0.63 0.47 determination (3, 4). The membrane exons ofthe Cs gene seem 1A and y2b 0.76 0.73 to be present as indicated by R-loop analyses (20). The mRNA * Amino acid residues predicted from nucleotide sequences (21) were encoding the am chain was recently identified (8). We have also compared. Numbers are difference per residue. shown by Southern blot analyses that the 3' flanking region of t The CH2 domain of the y chain was compared with the CH3 domain the C,, gene has a restriction fragment homologous to the mem- of the 1L chain. brane exons ofthe C,, gene (unpublished data). Taken together, these results indicate that all the CH genes have membrane side of the helix is, therefore, extremely conserved among the exons that are transcribed and spliced to form mRNAs encoding C. and C, genes. The striking conservation of the hydrophobic membrane-bound immunoglobulin heavy chains. segment in the three-dimensional structure leads us to propose Amino Acid Sequences of Transmembrane Segments Are the presence ofa receptor protein in the membrane that anchors Conserved. The amino acid sequences of the membrane do- the um and ym chains. When bind to surface receptor mains of the Cy2a), Crdb, C,, and C, genes were compared as immunoglobulin molecules, either IgM or IgG, a similar signal shown in Fig. 3. The membrane domains were separated into may be transmitted through the membrane anchor protein to three parts as suggested by Rogers et al. (3)-i.e., the acidic, proliferate B . The difference in the lengths of the hydrophobic, and hydrophilic parts, which seem to be located intracellular portions ofthe Am and Ym chains may indicate that in the outside ofthe cell, the intramembrane layer, and the in- other qualitatively distinct types of signals may also be trans- side ofthe cell, respectively. It is clear that the hydrophobic part mitted by different surface receptors, IgM and IgG. is the most conserved; 1 divergence out of 26 residues among Comparison of Nucleotide Sequences of Membrane Exons three C,, genes and 9 differences out of 26 residues between of CY Genes. We have compared the nucleotide sequences of the C,, and any one of the C, genes. membrane exons and their surrounding regions ofthe Cyl, Cy2b) This conservation of the amino acid sequence in the trans- and C,,, genes. The sequences were aligned with use ofa com- membrane segment is more striking than that in any other part puter program (12) as shown in Fig. 2. The divergence of the of the CH gene. When we compared the amino acid sequence coding region was calculated separately for synonymous (K8) and ofeach domain ofthe Cl, Cy2b, and C,,, genes with that ofthe CM gene, we found that the homology ranged between 22% and 37%, whereas the homology of the membrane domains of the A GluA) CA and C, genes is about 50% (Table 1). The results indicate that the transmembrane segment is under a strong constraint Gly Lou - Trp -Thr ofselection pressure. The stronger constraint on the membrane T lae- Thr-Ile exon was also confirmed by comparison of human and mouse - lie Ser. A chains. The amino acid sequences of the Ml exons are iden- tical in these two species (22), whereas those ofother secreted ,-Lou- Lu- r domains are calculated to be 48-78% conserved on the basis of Val - Cys Tyr- the published sequences (21, 22). The 26-residue hydrophobic region can form a-helixes, Ala - Ser - Val Thr which require 3.6 residues per turn. A proper alignment ofthe /Lo- Phe hydrophobic residues to form a-helix can bring the diverged residues to two clustered patches as shown in Fig. 4. The other Lay® 10 20 B y2a G l D l D D V C A Q D G E L D G l WFi1 I I I y2b G l D l D D I C A E A K 0 G E l D G l W 1 1 I I I membrane yl G l Q l D E I C A E A Q n G E l D G L W 1l I I protein7 - -- - -E G -E V N A E E E G F E N l W I A S I \ I / /outside

y2a F S F L L S V SCY A S V I lF K V W I F S y2b FI S L F l L S V C Y S A S V I L F K V W I F S yl F I S l F Sl l V C Y S AA VI L F K V KW I F S lipid A F I V l F l l S, l F.Y S I 1 V I l F K VK bilayer

y2a S V V E L K Q I I S P D Y R N M I G Q G A y2b S V V E l K Q K I S P D Y R N M I G Q G A ,yl S V V E L K Q I l V P E Y K N M I G Q A P I I IyurnglulubI 11 FIG. 3. Comparison of amino acid sequences of IgM and IgG mem- FIG. 4. Putative structure of transmembrane segment. (A) A pu- brane domains. The one-letter code is given in the legend of Fig. 2. tative a-helical structure of the ym chain is shown. Residues different Amino acid sequences of membrane domains of the gm (3), y'lm (this from those in the M,,,, chain are boxed. Oblique lines indicate a unit of paper; ref. 19), y2am (this paper), and y2bm (this paper) chains are pre- one turn (3.6 residues). (B) Hypothetical membrane protein that an- sented. Homologous residues are boxed. A, B, and C indicate acidic, chors membrane-bound immunoglobulin. Hatched regions indicate hydrophobic, and hydrophilic portions, respectively. diverged residues. Downloaded by guest on October 3, 2021 2626 Genetics: Yamawaki-Kataoka et aL Proc. Natl. Acad. Sci. USA 79 (1982)

amino acid-substituting (KJ) sites as shown in Table 2. It should -CH I HU .CH2 CH3 Ml M2 be noted that nucleotide divergence at the synonymous sites yl ' I l of the coding regions is free from the selective constraint. op- i erating at the protein level. The divergence of the noncoding -II I I 1 region (K,) was calculated by comparing every nucleotide y2b' =I 3 position. Because K. and K, values are free from functional constraint, FIG. 5. Schematic representation of IVS-mediated domain ex- they are directly comparable (13). It is clear that the K, and K, change between ancestors of C.1 and C,2b genes. Wider rectangles rep- values of the Ml exon and of the 3' portion of IVS 4 indicate resent exons. Filled rectangles are homologous regions that were ex- that these sequences are much more conserved between the changed between the two genes. CY, and Cy2b (or C2aj genes than any other parts are. The di- vergence values (0.36-0.51) in the less. conserved regions (the determined. The values are equivalent to those ofthe most con- 5' part of IVS 4, the IVS 5, and the M2 exons) are roughly served regions (IVS 2, CH 2, and IVS 3) in the secreted exons. equivalent to those (0.37-0.53) in the exons and introns encod- The results are consistent with models of the mouse C.Y gene ing secreted domains (13). Because it is difficult to explain such evolution that were proposed previously (13). As expected from varied divergence among different segments of the C, gene by amino acid sequence comparison, the K, value of the Ml exon selective constraint, the homology region might have been gen- is more conserved than that of any other exon (12, 13). erated by the exchange of the DNA segment due to relatively Two IVS-Mediated Domain Exchanges Between C,,1 and recent recombinational events between Cy subclass genes as C.,2b Genes. We have already shown that a contiguous DNA proposed before (12). We do not know whether the recombi- segment ofthe exon coding for the CH1 domain and the 5' por- nation took place before or after the segregation ofthe Cy2a and tion ofIVS 1 must have undergone a recombinational exchange C2b genes, which seems to be the latest event during the evo- of genetic information between the ancestors of CYj and Cy2b lution of the CY subclass genes. If it occurred afterwards, the. genes-(12). We have now shown that another DNA segment ancestral C.2b gene is a more likely partner.of the C,1 gene be- containing the M1 exon and the 3' portion of IVS 4 was also cause the CY, and C,2b genes are located next to each other (15). exchanged during evolution of the C, genes. As schematically The recombination required for such domain exchange is shown in Fig. 5, the ancestral C.Yj and C,2b genes have ex- either double unequal crossing-over or gene conversion (24). changed at least two segments of the gene, probably within a Because the sequence divergence changes drastically at the short interval because the K. values of the two conserved seg- middle of IVS 4, one site of the recombination seems to be lo- ments were almost identical (0.26 and 0.23). cated within IVS 4. The precise crossing-over point was esti- Since we reported the IVS-mediated domain exchange in the mated by comparison ofevery 50-bp segment ofIVS 4 along the C,,1 and Cyb genes of mouse (12), similar observations have gene sequence. The 3' recombination site is apparently some- been made in several other gene families of the eukaryote sys- where around the junction ofthe Ml exon and IVS 5. Because tem (24-27). It is likely that IVS-mediated domain exchange is the Ml exon is significantly more conserved than the 3' portion a universal phenomenon, probably characteristic of clustered of IVS 4, there might have been another recombination at the families of genes. junction of the Ml exon and IVS 4. At the time of submission of this manuscript the nucleotide The K. and K. values of the C.,b and Cy2a genes are almost sequences of membrane exons of the Cy,2b and CYj genes were constant throughout the whole region whose sequence has been published independently (23). We thank Dr. Y. Kyogoku of Osaka University for valuable sug- Table 2. Nucleotide sequence differences in membrane exons and gestions about the structure of the transmembrane segment, Drs. introns of Cy1, C,2b, and C.2, genes B. Tyler, 0. Bernard, and J. Adams for sending unpublished data, Ms. F. Oguni for preparation of the manuscript, and Ms. Y. Sakagami and Comparison Ms. S. Nishida for their excellent technical assistance. This investiga- Region in which Cy28 Cy2b Cya tion was supported by grants from the Ministry of Education, Science CY genes are Nucleotide and and and and Culture of Japan, from the Toray Science Foundation, from the compared* positiont Cy2b Cy1 Cz1 Mitsubishi Foundation, and from the Naito Foundation. K, or K8 1. Williams, P. B., Kubo, R. T. & Grey, H. M. (1978)J. Immunol 121, 2435-2439. IVS 4 2. Kehry, M., Ewald, S., Douglas, R., Sibley, C., Raschke, W., 5' portion 8-150 0.024 0.515 0.507 Fambrough, D. & Hood, L. (1980) Cell 21, 393-406. 3' portion 151-313 0.033 0.271 0.265 3. Rogers, J., Early, P., Carter, C., Calame, K., Bond, M., Hood, Ml exon 314-445 0.032 0.128 0.096 L. & Wall, R. (1980) Cell 20, 303-312. IVS 5 446-962 0.072 0.476 0.448 4. Early, P., Rogers, J., Davis, M., Calame, K., Bond, M., Wall, M2 exon R. & Hood, L. (1980) Cell 20, 313-319. Coding region 963-1,043 0.057 0.370 0.363 5. Singer, P. A., Singer, H. H. & Williamson, A. R. (1980) Nature 3' UT 1,047-1,077 0 0.410 0.410 (London) 285, 294-300. 6. Alt, F. W., Bothwell, A. L. M., Knapp, M., Siden, E., Mather, Ka E., Koshland, M. & Baltimore, D. (1980) Cell 20, 293-301. 7. Oi, V. T., Bryan, V. M., Herzenberg, L. A. & Herzenberg, L. Ml exon 314-445 0.02 0.06 0.06 A. (1980) J. Exp. Med. 151, 1260-1274. M2 exon 963-1,046 0.016 0.134 0.118 8. Kikutani, H., Sitia, R., Good, R. A. & Stavnezer, J. (1981) Proc. Ml + M2 exons 314-1,046 0.018 0.088 0.082 Nati Acad. Sci. USA 78, 6436-6440. 9. Nakai, S., Oi, V. T., Herzenberg, L. A., Yamagishi, H. & Honjo, * IVS sequences are numbered from 5' to 3' in the whole C. gene. UT, T. (1982) Biomed. Res. 3, 37-45. untranslated sequence. 10. Tyler, B. M., Cowman, A. F., Adams, J. M. & Harris, A. W. t Nucleotide position shown in Fig. 2. The discontinuous point in IVS (1981) Nature (London) 293, 406-408. 4 was determined by calculation of divergence of every 50-bp seg- 11. Takahashi, N., Shimizu, A., Obata, M., Nishida, Y., Nakai, S., ment starting within the sequence. Divergence values were calcu- Nikaido, T., Kataoka, T., Yamawaki-Kataoka, Y., Yaoita, Y., lated from published sequences (19, 23). Ishida, N. & Honjo, T. (1981) in Immunoglobulin , Downloaded by guest on October 3, 2021 Genetics: Yamawaki-Kataoka et al. Proc. NatL. Acad. Sci. USA 79 (1982) 2627

ICN-UCLA Symposia on Molecular and Cellular Biology, eds. 19. Tyler, B. M., Cowman, A. F., Gerondakis, S. D., Adams, J. M. Janeway, C., Sercarz, E. E. & Wigzell, H. (Academic, New & Bernard, 0. (1982) Proc. Natl. Acad. Sci. USA 79, 2008-2012. York), Vol. 20, pp. 123-134. 20. Maki, R., Roeder, W., Traunecker, A., Sidman, C., Wabl, M., 12. Miyata, T., Yasunaga, T., Yamawaki-Kataoka, Y., Obata, M. & Raschke, W. & Tonegawa, S. (1981) Cell 24, 353-365. Honjo, T. (1980) Proc. Natl. Acad. Sci. USA 77, 2143-2147. 21. Kawakami, T., Takahashi, N. & Honjo, T. (1980) Nucleic Acids 13. Yamawaki-Kataoka, Y., Miyata, T. & Honjo, T. (1981) Nucleic Res. 8, 3933-3945. Acids Res. 9, 1365-1381. 22. Rabbitts, T. H., Forster, A. & Milstein, C. P. (1981) Nucleic 14. Honjo, T., Obata, M., Yamawaki-Kataoka, Y., Kataoka, T., Ka- Acids Res. 9, 4509-4524. wakami, T., Takahashi, N. & Mano, Y. (1979) Cell 18, 559-568. 23. Rogers, J., Choi, E., Souza, L., Carter, C., Word, C., Kuehl, 15. Shimizu, A., Takahashi, N., Yamawaki-Kataoka, Y., Nishida, Y., M., Eisenberg, D. & Wall, R. (1981) Cell 26, 19-27. Kataoka, T. & Honjo, T. (1981) Nature (London) 289, 149-153. 24. Baltimore, D. (1981) Cell 24, 592-594. 16. Kataoka, T., Yamawaki-Kataoka, Y., Yamagishi, H. & Honjo, T. 25. Slightom, J. L., Blechl, A. E. & Smithies, 0. (1980) Cell 21, (1979) Proc. Nati Acad. Sci. USA 76, 4240-4244. 627-638. 17. Yamawaki-Kataoka, Y., Kataoka, T., Takahashi, N., Obata, M. 26. Liebhaber, S. A., Goossens, M. & Kan, Y. W. (1981) Nature & Honjo, T. (1980) Nature (London) 283, 786-789. (London) 290, 26-29. 18. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 27. Miyata, T. & Yasunaga, T. (1981) Proc. Natl. Acad. Sci. USA 78, 499-560. 450-453. Downloaded by guest on October 3, 2021