Sequence and Evolution of HLA-DR7- and -Drw53-Associated ,8-Chain Genes (Class II Histocompatibility Antigens/Gene Conversion) JOHN A

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4929-4933, July 1987 Genetics Sequence and evolution of HLA-DR7- and -DRw53-associated ,8-chain genes (class II histocompatibility antigens/gene conversion) JOHN A. T. YOUNG, DAVID WILKINSON, WALTER F. BODMER, AND JOHN TROWSDALE Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, England Contributed by Walter F. Bodmer, March 16, 1987

ABSTRACT cDNA clones representing products of the here the sequences of cDNA clones derived from the two DR7 and DRw53 18-chain genes were isolated from the human expressed DR /3-chain genes from cells with a DR7 haplotype B-lymphoblastoid cell line MANN (DR7,DRw53,DQw2, (corresponding to DR7 and DRw53 /3 chains) and compare DPw2). The DRw5313 sequence was identical to a DRw5313 these sequences to those of other DR types including DR4. sequence derived from cells with a DR4 haplotype. In contrast, These data suggest that differential evolutionary mechanisms the DR7,B sequence was as unrelated to DR4.8 sequence as it have acted upon the sequences of the /31 domains of the two was to other DRj3-related genes, except at the 3'-untranslated active DR /-chain loci since the divergence of the DR4 and region. These results suggest that the DR7 and DR4 haplotypes DR7 haplotypes. From serological analysis of mouse L-cell may have been derived relatively recently from a common transfectants expressing cell surface HLA-DR7 molecules, ancestral haplotype and that the DR4 and DR7 13-chain genes we also identify amino acid residues that may correspond to have undergone more rapid diversification in their 11 domains, antigenic specificities defined by monoclonal antibodies. most probably as a result of natural selection, than have the DRw53 1-chain genes. Short tracts ofsequence within the DR7 MATERIALS AND METHODS and DRw53 131 domains were shared with other DR18 sequences, indicating that exchanges of genetic information Construction and Screening of cDNA Libraries. Double- between 181 domains of DR13-related genes have played a part stranded cDNA was prepared from 5 Ag of poly(A)+ mRNA in their evolution. Serological analysis of mouse L-cell (7), extracted from the B-lymphoblastoid cell line MANN, transfectants expressing surface HLA-DR7 molecules, con- which is homozygous for the HLA region due to consan- firmed by antibody binding and allelic sequence comparisons, guineous parentage (DR7,DRw53,DQw2,DPw2). Homopol- identified amino acid residues that may be critical to the ymer poly(dC) tails were added to the cDNA (8), annealed binding of a monomorphic DR- and DP-specific monoclonal with poly(dG)-tailed Pst I-digested plasmid pBR322 (GIBCO/ antibody. Bethesda Research Laboratories), and a library of 20,000 recombinants was propagated in Escherichia coli MC1061 The HLA-D region of the human major histocompatibility cells. cDNA was also ligated, using EcoRI linkers, to EcoJ- complex encodes the class II a and ,8 glycoprotein chains that digested bacteriophage XgtlO arms (Vector Cloning Systemhs, assemble to form functional a//3 heterodimeric products. The San Diego, CA), and a library of 100,000 recombinants was region is divided into at least three major subregions, each propagated on E. coli NM514 cells. cDNA libraries were containing multiple a- and 83-chain loci that encode the screened by filter hybridization (9) using a radioactively HLA-DP, -DQ, and -DR molecules. The genetic organization labeled DR/3 gene-specific probe (donated by A. So, Depart- ofthese subregions has been confirmed using pulsed-field gel ment of Rheumatology, Hammersmith Hospital, London), electrophoresis (1). prepared by hexamer priming (10). DR,3-related cDNA The HLA-DR subregion contains a single expressed a- clones were sequenced in both directions by the chaid- chain gene and multiple /-chain genes (2, 3). Molecular termination method (11). analysis ofthe DR subregion derived from DRw52-containing Flow Microfluorometric Analysis ofMouse L-Cell Transfect- haplotypes, which almost invariably include alleles for the ants. The DR7/3 cDNA clone MAB20 and a DRa cDNA clone serological determinants DR3, -5, or -w6, has revealed two (donated by H. Ikeda, Imperial Cancer Research Fund, expressed and one nonfunctional DR,3 genes. The more London) were subcloned into cDNA expression vectors pJ4 polymorphic locus DR1B* encodes the DR/3 chains associ- (unpublished vector, provided by J. Morgenstern, Imperial ated with the DR3, -5, or -w6 serological specificities, while Cancer Research Fund, London) and pcEXV3 (12), respec- the less-polymorphic locus codes for ,8 chains associated with tively. Full details of these constructions will be published the DRw52 serological specificity. Haplotypes containing the elsewhere (D.W., unpublished results). The DR7/3/pJ4 and DRw53 supertypic serological specificity (associated with DRa/pcEXV3 recombinant plasmids were cotransfected DR4, -7, and -w9) also contain two expressed DR /3-chain with pSV2neo (13) into thymidine kinase-deficient (tk-) genes (DR1B and a gene coding for DRw53) in addition to a mouse L cells, using the calcium phosphate technique (14). nonfunctional DR,/ pseudogene (2). Stable transfectants were selected using Geneticiti (G418 DR /3-chain genes derived from individuals that share Sulfate; GIBCO/Bethesda Research Laboratories) at a con- DRw52 appear to have evolved from a common ancestral centration of 1 mg/ml and by several rounds of flow haplotype (4). Those associated with DRw53 haplotypes may microfluorometric sorting using a mixture of anti-HLA-class be similarly related since they generate similar restriction fragment length polymorphism patterns on Southern blots *HLA-class II a and p chain coding loci are respectively designated using DR,8 and DQa gene-specific probes (5, 6). We present A and B, while the locus number, where there are two or more a- or p-chain loci, is placed before the corresponding letter, hence DR1B. It is not yet established whether the DRw52 and DRw53 The publication costs of this article were defrayed in part by page charge specificities represent alleles at one locus or different loci. When payment. This article must therefore be hereby marked "advertisement" this has been established they can be assigned the designations in accordance with 18 U.S.C. §1734 solely to indicate this fact. DR2B, DR3B, etc., as appropriate. 4929 Downloaded by guest on September 26, 2021 4930 Genetics: Young et al. Proc. Natl. Acad. Sci. USA 84 (1987) -25 -15 MET Val Cys Lou Lys Lou Pro Sly Sly Ser Cys net Ala Ala Lou Thr Val Thr Leu let Val NlAP20 (DR7) CTBBTCCT6TCCT6TTCTCCA6C AT6 6T6 T6T CT6 AA6 CTC CCT 66A 66C TCC T6C AT6 6CA 6CT CT6 ACA 6T6 ACA CTG AT6 6t6 86 flAP16 (DRN53) GACCA------T-----C------T ------6 ------T -C- - .Thr... -5 -1 +1 5 15 HOIt Lou Sir Ser Pro Lou Ala Lou Ala 61y Asp Thr 61n Pro Arg Phe Leu Trp 61n Gly Lys Tyr Lys Cys His Phi Phe Asn Bly fAP20 ODR CT6 A6C TCC CCA CTU 6CT TT6 6CT 666 6AC ACC CAA CCA CST TTC CT6 T6B CA6 66T AA6 TAT AA6 T6T CAT TTC TTC AAC 666 170 fAP16 (DRN53) --T-- BA- --- -C- B- B-- -- C-- --T --- ...... u...Ala ... Cys 6u ...... Lou . tas 25 35 ------45 Thr 61u Arg Val 61n Phe Lou 61u Arg Lou Phe Tyr Asn Gin 61u 61u Phe Val Arg Phe Asp Ser Asp Val Bly 61u Tyr Arg flAP20 (DR7) AC6 6A6 C66 6T6 CA6 TTC CT6 6AA A6A CTC TTC TAT AAC CAB 6AG 6AB TTC 6T6 CBC TTC BAC ABC BAC 6T6 666 BAG TAC C66 254 APA16 (DRN53) A --- T6- AA----ATC --- TA- A------A ------A------A- A--C---T --- C------a- .a...... a Trp Asn Ile... Tyr Ile .Tyr Al ... Tyr Asn ... Lou .61n 55 65 75 Ala Val Thr 61u Lou Bly Arg Pro Val Ala 61u Ser Trp Asn Ser 61n Lys Asp Ile Lou 61u Asp Arg Arg Bly 61n Val Asp NlA,20 (DR7) 6C6 6T6 AC6 6A6 CTA 666 C66 CCT 6TC 6CC 6A6 TCC T66 AAC ABC CAB AA6 BAC ATC CT6 6A6 BAC A66 C66 66C CAB 6TB SAC 338 fAP16 (DRN53) ---- 6 ------A- T --A ------C------C66 ------C- 6------.ost .moo ....of. ....Asp Tyr. Leu . Arg. Aa 61u . 85 95 Thr Val Cys Arg His Asn Tyr Bly Val Bly 61u Ser Phe Thr Val 61n Arg Arg Val His Pro 6iu Val Thr Val Tyr Pro Ala IA,20 (DRD) ACC 6T6 T6C A6A CAC AAC TAC 666 6TT 66T 6A6 ABC TTC ACA 6t6 CAB C66 C6A 6TC CAT CCT BAB UtB ACT 6T6 TAT CCT 6CC 422 MA)16 (DRN53J --- TAC ------T - -T6 ------A --- A ------T-A Tyr . Tyr .Vl .G.i....n...... Lys. Ser 105 115 125 Lys Thr 61n Pro Lou 61n His His Asn Lou Liu Vai Cys Ser Val Ser Bly Phe Tyr Pro Bly Ser Ile 61u Val Arg Trp Phe flAP20 ODR7) AAB ACT CAB CCC cUB CAB CAC CAC AAC CTC CT6 6TC T6C TCT 6t6 A6T 66T TTC TAT CCA 66C ABC ATT 6AA 6TC A66 T66 TTC 506 hAl16 (DR53) -C------A------

s n o f s e o f m o to s a s a a a * o f s t m o g t m o A n ...... An 135 145 155 ...Arg Asn 6ly 61n 61u 6Bu Lys Ala Biy Val Val Ser Thr Bly Lou Ile 61n Asn Bly Asp Trp Thr Phe 61n thr Lou Val mlet 1AP20 (DR7) C66 AAC 66C CAB 6AA 6A6 AA6 6CT 666 BIB 6t6 TCC ACA 66C CT6 ATC CAB AAt 66A 6AC T66 ACC TTC CAB ACC Ct6 BIB AT6 590 HA,16 (DRw53) ------A------

165 175 195 Lou 61u Thr Vai Pro Arg Ser Bly 61u Val Tyr Thr Cys 61n Vai 61u His Pro Sir Val Met Ser Pro Lou Thr Val 61u Trp fA,20 (DR7) CT6 6AA ACA 6TT CCT C66 ABT 66A BAA 6TT TAC ACC T6C CAA 6t6 6A6 CAC CCA ABT 6t6 AT6 ABC CCT CTC ACA UtS 6AA TBB 674 hAP16 (DRw53) ------.6------CT-----CA------6 --- C--

195 205 215 Arg Ala Arg Ser 61u Sir Ala 61n Ser Lys Net Leu Ser Bly Val Bly Bly Phe Val Lou Bly Lou Lou Phe Lou Bly Ala Biy flAP20 (DR7) ABA 6CA C66 TCT 6AA TCT 6CA CA6 ABC AAB AT6 CT6 ABT 66A BTC 666 66C TtT 6T6CTB 66C CT CtC TTC CTT 666 6CC 666 759 HA,16 (DRN53) --T ------A-A --- Ser .thr.. 225 235 Lou Phe lle Tyr Phe Arg Asn 61n Lys Bly His Ser Gly Lou 61n Pro Thr Bly Phe Lou Ser ,lA20 (DR7) TtB TTC ATC TAC TTC A66 AAT CAB AAA 66A CAC TCT 66A CTT CAB CCA ACA 66A TTC CTB ABC TBA ABTBAABAT6ACCACATTCAAB6 847 lAP16 (DRN53) C------C-- t------C------

fAP20 (DR7) AAAAACCTTCT9CCCCA6CTTT6CA66AT6AAACACTTCCCX6CTT66CTCTCATTCTTCCACAA6A6A6ACCTTTCTCC66ACCT66TTBCTACT66TTCA6CAACTXX6955 IAP16 (DRN53) --B------AA------A ---t--CA------T------X XXX----6--A-----A-----T------CT- flA,20 (DR7) CA6AAAAT6TCCTCCCCTT66CT6CCTCA6CTCAT6CCTT66CCT6AA6TCCXXCA6CATT6At66CA6CCCCTCATCTTCCAA6TTTT6T6CTCCCC1TT0CCTAAC6164 flA,16 (DRN53) ------t------tT---T-- C--TtC------C-TCA----t------T------X---C------T------AC flA20(D7) CTCCBC- RAP20 MDR7 CTTCCTBCCTCCCATSCATCTSTACTCCTCCXTST6CCACAAACACATTACATTATTAAATrTTTCTCAAACAT66A6TTAAA(A)nCCA-CACTTATC-C-TTBCAAAAACTTCATATAATTTCTCAAAT-ATTAA-An-141143 flA,16 ODRW3) T6---- 1--C--T------C-XfXX------AAAA)n FIG. 1. (Legend appears at the bottom of the opposite page.) Downloaded by guest on September 26, 2021 Genetics: Young et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4931

1 10 20 30 40 50 60 70 80 90 DR cons. GDTRPRFL-----ECHFFNGTERV--L-R---NQEE--RFDSDVGE-RAV-ELGRP-AE-WNSQKD-LE--R--VD-YCRHNYGV-ESFTVQRR

DR7 Q WQGKYK QF E LFY FV Y T r V _S_ -I DR GQ TV G

DR1 WQLKF RL E CIY PV Y E D Y L QK GQ N G DR 2 WQPKR RF D YFY SV F T D Y I QA AA T V DR3 EYSTS RY D YFH NV F T D Y L QK GR N V DR4 EQVKH RF D YFYH YV Y T DY L QR AA T V DR5 EYSTS RF D YFY YV F T DE Y F DR AA T G DR6a EYSTS RF D YFH NV F T D Y I DE AA T V DR6b EYSTS RF D YFH YV Y T A DDI L RR AE T V

DRw52a ...... ELRKS RY D YFH FL Y T L V S L QK GR N G DRw52b ELLKS RF E HFH YA Y R D Y L QK GR N V

DRw53 ( 4) Q EQAKC L WN I YIY YA YN L YQ T D Y L RR AE T] Y V DRw53 (7) Q EQAKC L WN I YIY YA YN L YQ T D Y L RR AS T| Y V

DRP Chain

270 309 DR7/w52a GGG CGG CCT GTC GCC GAG TCC TGG AAC AGC CAG AAG GAC DR1/3/4/6a/w52b ------AT ------A------DRS ------AT -AG --- -A------DR6b ------CT --G --C GA------DRw53 --- -T -----

FIG. 2. 81-domain sequences of DR3-related genes; two possible interchromosomal gene conversion events. (A) p1-domain sequences of DR3-related genes were aligned with that of a consensus sequence in which dashes indicate the most highly variable amino acids (abbreviated with the single-letter code). DR7 and DRw53(7) P-chain sequences were derived from cDNA clones MAB20 and MA,816, respectively (Fig. 1). Other DRP sequences were published elsewhere as follows: DR1 (26), DR2 (27), DR3 (28), DR4 (29), DR5 (30), DR6a,6b and DRw52a (4), DRw52b (31), and DRw53(4) (2). Gaps in each sequence indicate identity to that of the consensus, and dots represent residues for which no sequence information was available. The amino acids were numbered from the first residue of the mature DR/3 protein. Stretches of shared nucleotide/amino acid homologies between the DR7 and DRw52a p-chain genes and between the DR6b and DRw53 p-chain genes are represented by dashed and solid boxes, respectively. (B) A 39-base-pair homology between the DR7 and DRw52a p-chain genes. The nucleotides are numbered as in Fig. 1, and the corresponding regions of the other DR,3 sequences, in which dashes represent identical residues, are shown below for comparison.

II monoclonal antibodies. Mouse L-cell transfectants were encoded by cDNA clone MAP16 contained a cysteine residue analyzed using the fluorescence-activated cell sorter as at position 13 in the first domain, in addition to the two described (15). Monoclonal antibodies used were as follows: residues at positions 15 and 79 that are thought to be involved SG465 (16), SFR16-DR7M (17), 17.3.3B (18, 19), 11.4.1 (20), in the formation of a disulfide bridge. Only the DR7,8 DA6.164 (21), DA-2 (22), B7.21 (23), and Tu22 (24). sequence contained the Arg-Phe-Asp-Ser sequence at positions 39-42 (indicated by overlining, Fig. 1), which is found RESULTS in other class II antigen 81 domains and major histocompatibility complex class I al domains, and may be important for Plasmid and bacteriophage cDNA libraries were constructed T-cell recognition (25). The corresponding sequence in the from the B-lymphoblastoid cell line MANN, homozygous for DRw53 chain was Arg-Tyr-Asn-Ser (Fig. 1). HLA region genes (DR7,DRw53,DQw2,DPw2). The libraries A comparison of the 81-domain amino acid sequences were transferred to nitrocellulose filters and screened with a predicted from cDNA clones MA,816 and MAP20 with those DRP8 gene-specific probe using high-stringency hybridization ofother DRB sequences (2, 4, 26-31) is given in Fig. 2A. This conditions. DR/3 cDNA clones, which fell into two classes, shows that the 81 domain corresponding to cDNA clone were isolated and partially characterized by DNA sequenc- MA,816 [labeled DRw53(7)] is identical to a DRw53 p1- ing. The DNA sequences of two cDNA clones, MAB20 and domain sequence derived from a DR4 haplotype [ref. 2; MAp16, that represented full-length transcripts of the DR7 labeled DRw53(4)]. This identity was also found in their and the DRw53 p-chain genes, respectively (see below), were corresponding nucleotide sequences (data not shown). This determined (Fig. 1). The ratio of DR7,8/DRw53p8 cDNA confu-ms two-dimensional protein gel analysis that indicated clones isolated was -2:1. This presumably reflects differ- little if any variation in the DRw53 , chain (32). Since cDNA ences in the steady-state levels of the corresponding mRNA clone MA/316 represented the DRw53 p6-chain gene, we transcripts in this B-cell line. concluded that the other cDNA clone MA,820 was derived The mature proteins encoded by both cDNA clones were from the DR7 ,-chain gene. This was established formally by identical in length (237 amino acids), and both sequences serological analysis of mouse L cells cotransfected with contained a potential glycosylation site (Asn-Gly-Thr) at cDNA clone MAl20 and a DRa cDNA clone. positions 19-21 of the 81 domain (indicated with asterisks in As shown in Fig. 3, the transfected L cells bound the Fig. 1). As shown in this figure, the DRw53p8 sequence DR7-specific monoclonal antibody (mAb) SFR16-DR7M but

FIG. 1 (on opposite page). Nucleotide and amino acid sequences of DR p-chain cDNA clones MA,820 (DR7) and MA,816 (DRw53). The nucleotide and predicted amino acid sequences of cDNA clone MAB16 are compared to those of cDNA clone MA,820. Dashes and dots indicate identical nucleotides and amino acids, respectively. For optimal alignment of 3'-untranslated region sequences, gaps were introduced (indicated by X). Nucleotides are numbered from the 5' end of cDNA clone MA320, and amino acids were numbered from the first residue of the mature DR,8 protein. N-linked glycosylation sites are indicated with asterisks, and the Arg-Phe-Asp-Ser sequence that is conserved in cDNA clone MAB20 is indicated by overlining. Downloaded by guest on September 26, 2021 4932 Genetics: Young et al. Proc. Natl. Acad. Sci. USA 84 (1987) not mAb DA6.164 that binds to all DR serological specific- DR7 8-chain cDNA clone MA,820 (Fig. 2B). The correspond- ities except DR7. The transfectants also bound mAb 17.3.3B ing regions of the other DR/3 sequences demonstrate 3- to that most probably has a dual specificity for DR7 and DRw53 6-base-pair differences from those of DR7 and DRw52a (Fig. molecules (see Discussion). As expected, the transfectants 2B). Similarly, a subregion that extended over a maximum of did not bind the DP-specific mAb B7.21 nor the DQ-specific 55 base pairs, between amino acids 61 and 77 (indicated by mAb Tu22 (Fig. 3). Surprisingly, however, they were also solid boxes, Fig. 2A), was identical between the DR6b negative with mAb DA-2 (Fig. 3), an antibody thought to P-chain gene and the DRw53 ,-chain cDNA clone MA,320. react with all DR types (see Discussion). This homology had been described (4). These data suggest As described (33), there are several regions of extreme that a number of inter-locus and intra-locus sequence ex- variability in the DR p1-domain sequences (amino acids change events may have contributed to the DR7 and DRw53 9-13, 25-32, and 67-77; Fig. 2A). A number of distinct 13-chain sequences. Inter-locus "unequal" gene conversion residues, namely those not found in other DRO sequences, events between murine class II ,-chain loci have been were observed in the DR7 ,1-domain sequence. These were suggested (34). Gly-li, Tyr-13, Lys-14, Gln-25, Leu-30, and Val-78 (Fig. 2A). The difference between the nucleotide sequence of the Similarly, distinct amino acids were found at Ala-il, Cys-13, DR7 p1 domain and that of DR4 (10.6%) is comparable to its Leu-18, Trp-25, Asn-26, Ile-28, Tyr-40, Asn-41, Leu-44, difference from other DRp-related sequences; e.g., DR1 Gln-48, and Tyr-81 in the DRw53p sequence (Fig. 2A). (11%) and DRwS2a (9.7%). However, the 3'-untranslated The remainder of the DR7 and DRw53 p1-domain se- regions of the DR7 and DR4 1B alleles were more highly quences encoded by cDNA clones MA,820 and MA1316, related (differing by only 3.7%, compared with 12.7-14.5% respectively, were a patchwork of all the other DRB-related differences between the DR7,8 sequence and those of other sequences. For example, Glu-28 is in the DR7, DR1, and DRiB alleles), a feature that presumably reflects their more DRw52b p-chain sequences, and Ile-31 is in the DR1 and recent common ancestry (see below). DRw53 p-chain sequences (Fig. 2A). Indeed, a subregion that extended over a maximum of 39 base pairs, between amino DISCUSSION acids 54 and 66 (indicated by dashed boxes in Fig. 2A), was The DR7 haplotypes are serologically interesting as certain identical between the DRw52a 8-chain gene and that of the antibodies bind to all DR specificities except DR7 (22) and in some cases also to DRw9 (19). DA6.164 is an example of such SG465(DP/DQ/DR) DA6.164(DR except DR7) an antibody (Fig. 3). The inability of the DR7 transfectants to bind antibody DA-2 is of interest. Since DA-2 is monomorphic, we assumed this antibody would recognize all allelic Il products of HLA-DR and -DP. The inability of this antibody A~~~~~II to bind to DR molecules on DR7 homozygous B-lymphoblas- m - toid cell lines is presumably obscured by its binding to DP products. In other words the DA-2 antibodies bind to all DR haplotypes but DR7, as do DA6.164 antibodies, but DA-2 SFR16-DR7M(DR7) antibodies are presumably monomorphic to the DP haplo- il DA-2(DP/DR) types. Comparing the DRiB allelic sequences in Fig. 2A with those of DPP alleles (35) revealed that a glutamic acid codon (at amino acid position 14, Fig. 2A) and an arginine codon (at Ii amino acid position 25, Fig. 2A) are shared between alleles of w DP1B and DRiB, except for DR7p8 in which the corresponding codons were for lysine and glutamine, respectively. Therefore, these amino acid residues in DP and DR mole- z cules, either singly or in combination, are most probably -i m critical to the binding specificity ofantibody DA-2. The L-cell wi 17.3.3B(DR7/ DRw53) B7.21(DP) transfectants also bound 17.3.3B, an antibody that appears to have a dual specificity for DR7 and DRw53 products (L. Kennedy and J. G. Bodmer, personal communication). Ifthe dual specificity of antibody 17.3.3B is confirmed by serological analysis of mouse L cells expressing cell surface DRw53 2 .1 i: products, then glutamine-4 (Fig. 2A) may be the critical residue as this is the only residue common to both DR7 and DRw53 p sequences not found in any other DRiB allele. ITu22(DQ) These data clearly emphasize the value of transfection 1 1.4.1(H-2K) experiments in delineating unequivocally monoclonal antibody specificities. In support of the hypothesis that DR4, DR7, and DRw9 haplotypes are derived relatively recently from a common ancestral haplotype (5, 6), we have demonstrated that the DR7 and DR4 haplotypes possess identical DRw53 831- 0 2 5 5 domain sequences at both protein and nucleotide levels. FLUORESCENCE INTENSITY (LOG) Moreover, the 3'-untranslated regions of DRiB alleles from DR4 and DR7 are also highly related. The fact that the 81 FIG. 3. Flow microfluorometric analysis of mouse L-cell domains ofthe DR4 and DR7 transfectants. L-cell transfectants that expressed cell surface HLA- 8-chain genes do not reflect this DR7 products were analyzed by flow microfluorometry with a panel relationship, because they are as unrelated as those of any of monoclonal antibodies. The specificity of each antibody is other DREW allele, illustrates that this DR,81 domain has indicated in parentheses. The negative control in each case is the diversified more rapidly than that of DRw53,8. Such rapid background staining of the transfectants with the fluoresceinated diversification of DR4 and DR7 p1-domain sequences might antibody only. be explained by sequence exchange events, such as those Downloaded by guest on September 26, 2021 Genetics: Young et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4933 illustrated in Fig. 2, coupled with natural selection forcing the 13. Southern, P. J. & Berg, P. (1982) J. Mol. Appl. Genet. 1, new DR type into the population. It is possible, for example, 327-341. that the DR7 ,-chain allele is derived from a DR4,8 allele by 14. Wigler, M., Pellicer, A., Silverstein, S. & Axel, R. (1978) Cell one or more gene conversion events. 14, 725-731. 15. Austin, P., Trowsdale, J., Rudd, C., Bodmer, W., Feldmann, The different levels of polymorphism associated with the M. & Lamb, J. (1985) Nature (London) 313, 61-64. DR7 and DRw53 3-chain loci suggest that evolutionary 16. Goyert, S. M. & Silver, J. (1983) Proc. Natl. Acad. Sci. USA pressures have selected for more variability in DRiB than 80, 5719-5723. DRw53f3. This feature may also reflect functional differences 17. Radka, S. F., Amos, D. B., Quackenbush, L. J. & Cresswell, between the products of these nonallelic loci. Indeed, indi- P. (1984) Immunogenetics 19, 63-76. viduals carrying some DR haplotypes (particularly DR1) do 18. Waters, S. J., Winchester, R. J., Nagase, F., Thorbecke, G. J. not have a DRw53 (or a DRw52) 83 gene. It is interesting, & Bona, C. A. (1984) Proc. NatI. Acad. Sci. USA 81, therefore, that the Arg-Phe-Asp-Ser sequence motif that is 7559-7563. conserved between most DRI3, and 19. Bodmer, J. G., Kennedy, L., Aizawa, M., Dawkins, R. L., DRw52,8, DQ,8, DP/B, Lepage, V., Mazzilli, M. C. & Richiardi, P. (1984) in Histo- major histocompatibility complex class I a alleles, which compatibility Testing 1984, eds. Albert, E. D., Baur, M. P. & might be important for antigen presentation (24), is replaced Mayr, W. R. (Springer, Berlin), pp. 217-236. by Arg-Tyr-Asn-Ser in the DRw53,3 chain. These differences, 20. Oi, V. T., Jones, P. P., Goding, J. W. & Herzenberg, L. A. although conservative in the substitution of tyrosine for (1979) Curr. Top. Microbiol. Immunol. 81, 115-129. phenylalanine, may influence the interaction of this glyco- 21. von Heyningen, V., Guy, K., Newman, R. & Steel, C. M. protein chain with the T-cell antigen receptor and accessory (1982) Immunogenetics 16, 459-469. molecules such as CD4. 22. Brodsky, F. M., Parham, P., Barnstable, C. J., Crumpton, M. J. & Bodmer, W. F. (1979) Immunol. Rev. 47, 3-61. 23. Rudd, C., Bodmer, J. G., Bodmer, W. F. & Crumpton, M. J. Note Added in Proof. Similar DR7 sequences were reported while this (1984) in Histocompatibility Testing 1984, eds. Albert, E. D., manuscript was in preparation (36). Baur, M. P. & Mayr, W. R. (Springer, Berlin), pp. 545-548. 24. Pawelec, G. P., Shaw, S., Ziegler, A., Muller, C. & Wernet, P. We are grateful to J. Morgenstern and H. Ikeda for the generous (1982) J. Immunol. 129, 1070-1075. gifts of plasmid pJ4 and the full-length DRa cDNA clone, respec- 25. Auffray, C. & Novotny, J. (1986) Hum. Immunol. 15, 381-390. tively. We thank S. F. Radka, A. Ziegler, and S. M. Goyert for 26. Bell, J. I., Estess, P., St. John, T., Saiki, R., Watling, D. L., providing antibodies. We also thank our colleagues, especially Julia Erlich, H. A. & McDevitt, H. 0. (1985) Proc. Natl. Acad. Sci. Bodmer, Lorna Kennedy, and Judy Heyes for providing antibodies USA 82, 3405-3409. and for helpful discussions. 27. Kratzin, H., Yang, C., Gotz, H., Pauley, E., Korbel, S., Egert, G., Thinnes, F. P., Wernet, P. & Altevogt, P. (1981) 1. Hardy, D. A., Bell, J. I., Long, E. O., Lindsten, T. & Hoppe-Seyler's Physiol. Chem. 362, 1665-1669. McDevitt, H. 0. (1986) Nature (London) 323, 453-455. 28. Gustaffson, K., Wiman, K., Emmoth, E., Larhammar, D., 2. Spies, T., Sorrentino, R., Boss, J. M., Okada, K. & Stromin- Bohme, J., Hyldig-Nielsen, J. J., Ronne, H., Peterson, P. A. ger, J. L. (1985) Proc. Natl. Acad. Sci. USA 82, 5165-5169. & Rask, L. (1984) EMBO J. 3, 1655-1661. 3. Rollini, P., Mach, B. & Gorski, J. (1985) Proc. Natl. Acad. Sci. 29. Cairns, J. S., Curtsinger, J. M., Dahl, C. A., Freeman, S., USA 82, 7197-7201. Alter, B. J. & Bach, F. H. (1985) Nature (London) 317, 4. Gorski, J. & Mach, B. (1986) Nature (London) 322, 67-70. 166-168. 5. So, A. K. L., Trowsdale, J., Bodmer, J. 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