Evolutionary and Genetic Implications of Sequence Variation In
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Proc. Natl. Acad. Sci. USA Vol. 84, pp. 209-213, January 1987 Immunology Evolutionary and genetic implications of sequence variation in two nonallelic HLA-DR 13-chain cDNA sequences (HLA class II antigens/genetic polymorphism/DNA sequence homology) JULIE MISKIMEN CURTSINGER, JOANNE M. HILDEN, J. SCOTT CAIRNS, AND FRITZ H. BACH Immunobiology Research Center, Department of Laboratory Medicine and Pathology, Box 724 Mayo Building, University of Minnesota, Minneapolis, MN 55455 Communicated by Donald C. Shreffler, August 25, 1986 ABSTRACT Most HLA haplotypes carry two expressed cells that are not homozygous for DR have made it difficult DR 13-chain genes; in the DR4 haplotype, the polymorphic locus to assign a DNA sequence to a particular locus within the DR has been called DR p13 and the apparently nonpolymorphic family and to determine to what degree the variation seen locus has been called DR 12. We have isolated nearly full-length among DR 13 sequences represents allelic variation versus DR 8-chain cDNA clones representing each of these two loci interlocus (isotypic) differences. To address these questions from a cell line homozygous for DR4 and Dw4. The clones have we report here a sequence ofa nearly full-length DR P2 cDNA been sequenced and the sequences compared with published clone derived from a DR4 homozygous cell, which allows DR 13 cDNA sequences derived from other haplotypes. A sequence comparisons to be made with known DR ,1 comparison of our sequences with other published cDNA sequences of DR4 and with DR 13 sequences from other sequences did not allow assignment of these other sequences to haplotypes. either the 81 or 182 locus. Comparison of our DR4 (31 sequence Within the DR4 classification there is a further polymor- with DR p13 sequences isolated from other DR4-positive cells phism, referred to as Dw-subtype polymorphism, which is suggests that the alleles of DR4 181 may have recently diverged detected by T-lymphocyte proliferative responses rather than from a common ancestor. The apparent lack of polymorphism by serological assays (10, 11). The mobility ofa particular DR of DR /2 may in part be a reflection of this recent divergence. P1 protein in isoelectric focusing correlates with the Dw subtype of the cell from which it was derived (12-15). The HLA class II molecules are members of a family of poly- extent of polymorphism of the DR /31 gene and the degree of morphic cell surface proteins that are involved in the regu- conservation of the DR 132 gene of the DR4, DR7, and DR9 lation of immune responses and are found primarily on haplotypes are areas of some uncertainty. Thus, to obtain macrophages, B lymphocytes, and activated T lymphocytes. additional information relevant to DR P1 gene polymorphism They are present on the cell surface as glycosylated hetero- we sequenced a DR Pi cDNA clone derived from the DR4 dimers consisting of an a chain with a molecular weight of Dw4 cell line that was the source of the DR 12 clone. The approximately 34,000 and a 13 chain with molecular weight possible contributions of these sequences to an understand- varying from 25,000 to 29,000. Class II products have been ing of the evolution of the DR4 haplotype are also discussed. subdivided by immunological and biochemical criteria into three families, called DR, DQ, and DP (1-4). MATERIALS AND METHODS In the HLA-DR family, there appears to be only one DR a-chain gene, while the number of DR 1-chain genes varies Construction and Screening of the cDNA Library. A cDNA from one to four among the various serologically defined library was constructed from poly(A)+ RNA isolated from haplotypes (1, 2). A DR4/DRw53 haplotype has been shown the DR4, Dw4-homozygous cell line MJ4 (A2, Bw62, Bw35, by Southern blotting and genomic cloning to have three Cw3, DR4, Dw4, DQw3, DRw53), as described (16). Two complete DR 13 genes and one apparently incomplete DR 13 nearly full-length positive clones were selected for sequenc- gene (5); one of the complete genes is a pseudogene (6). The ing because they had certain restriction enzyme sites char- DR a gene has been shown by biochemical analyses and acteristic of DR 1 genes but also differed slightly from each DNA sequencing to be essentially invariant (1), whereas the other in their restriction site maps. These clones are desig- two expressed DR4 13 genes encode one chain that is variable nated MJ5.4 (1022 base pairs) and MJ8.2 (935 base pairs). and one that is constant, as determined by two-dimensional Sequencing of DR 1-Chain cDNA Clones. The inserts from gel electrophoresis (7). The variable DR 13 chain is here clones MJ5.4 and MJ8.2, or fragments thereof, were sub- designated DR P1i and carries the antigenic determinant(s) cloned into bacteriophage vector M13mp19. Subclones were responsible for the DR4 serological classification (8). The sequenced directly or, alternatively, truncated subclones "nonvariant" chain is here designated DR 132 and carries the were generated by the method of Dale et al. (17). Sequencing DRw53 serological specificity, a so-called "supertypic" was done using the dideoxy chain-termination method (18). specificity also found on cells with the DR7 and DR9 haplotypes (9). RESULTS AND DISCUSSION Molecular cloning and sequencing have shown that overall DNA sequence homology among a or 13 chains within a class Sequence ofcDNA Clones Derived from p3, and /32 Loci ofthe II family is 85% or greater and between families is in the range DR4 Dw4 Haplotype. To obtain additional data about se- of 70% (1). Genes belonging to different families can be quence variation among alleles of DR P1 within the DR4 readily differentiated, especially by sequence differences in haplotype and to obtain a complete sequence known to be DR the 3' untranslated portion of those genes. The existence of 132, we isolated and sequenced two DR ,1 cDNA clones, MJ8.2 multiple DR 13-chain genes and, in some cases, the study of (935 base pairs) and MJ5.4 (1022 base pairs), both generated from RNA isolated from the DR4-homozygous lymphoblas- The publication costs of this article were defrayed in part by page charge toid cell line MJ4. The nucleotide and predicted amino acid payment. This article must therefore be hereby marked "advertisement" sequences for clone MJ5.4 are given in Fig. 1. The sequence in accordance with 18 U.S.C. §1734 solely to indicate this fact. of clone MJ8.2 is identical to the sequence of a clone derived Downloaded by guest on September 24, 2021 209 210 Immunology: Curtsinger et al. Proc. Natl. Acad. Sci. USA 84 (1987) -1 +1 17 32 47 62 77 MJ5.4 Val Leu Ser Ser Pro Leu Ala Leu Ala Gly Asp Thr Gln Pro Arg Phe Leu Glu Gln Ala Lys Cys Glu Cys His Phe Leu (18) CG GTG CTG AGC TCC CCA CTG GCT TTG GCT GGG GAC ACC CAA CCA CGT TTC TTG GAG CAG GCT AAG TGT GAG TGT CAT TTC CTC 92 107 122 137 152 167 Asn Gly Thr Glu Arg Val Trp Asn Leu Ile Arg Tyr Ile Tyr Asn Gln Glu Glu Tyr Ala Arg Tyr Asn Ser Asp Leu Gly Glu Tyr Gln (48) AAT GGG ACG GAG CGA GTG TGG AAC CTG ATC AGA TAC ATC TAT AAC CAA GAG GAG TAC GCG CGC TAC AAC AGT GAC CTG GGG GAG TAC CAG 182 197 212 227 242 257 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu Glu Arg Arg Arg Ala Glu Val Asp Thr Tyr (78) GCG GTG ACG GAG CTG GGG CGG CCT GAC GCT GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CGG AGG CGG GCC GAG GTG GAC ACC TAC 272 287 302 317 332 347 Cys Arg Tyr Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg Val Gln Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gln Pro (108) TGC AGA TAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA GTC CAA CCT AAG GTG ACT GTG TAT CCT TCA AAG ACC CAG CCC 362 377 392 407 422 437 Leu Gln His His Asn Leu Leu Val Cys Ser Val Asn Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg Asn Gly Gln Glu Glu (138) CTG CAG CAC CAC AAC CTC CTG GTC TGC TCT GTG AAT GGT TTC TAT CCA GGC AGC ATT GAA GTC AGG TGG TTC CGG AAC GGC CAG GAA GAG 452 467 482 497 512 527 Lys Ala Gly Val Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro Arg Ser Gly (168) AAG GCT GGG GTG GTG TCC ACA GGC CTG ATC CAG AAT GGA GAC TGG ACC TTC CAG ACC CTG GTG ATG CTG GAA ACA GTT CCT CGG AGT GGA 542 557 572 587 602 617 Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Met Met Ser Pro Leu Thr Val Gln Trp Ser Ala Arg Ser Glu Ser Ala Gln Ser Lys (198) GAG GTT TAC ACC TGC CAA GTG GAG CAT CCA AGC ATG ATG AGC CCT CTC ACG GTG CAA TGG AGT GCA CGG TCT GAA TCT GCA CAG AGC AAG 632 647 662 677 692 707 Met Leu Ser Gly Val Gly Gly Phe Val Leu Gly Leu Leu Phe Leu Gly Thr Gly Leu Phe Ile Tyr Phe Arg Asn Gln Lys Gly His Ser (228) ATG CTG AGT GGA GTC GGG GGC TTT GTG CTG GGC CTG CTC TTC CTT GGG ACA GGG CTG TTC ATC TAC TTC AGG AAT CAG AAA GGA CAC TCT 722 737 753 763 773 783 793 803 Gly Leu Gln Pro Thr Gly Leu Leu Ser * (238) GGA CTT CAG CCA ACA GGA CTC TTG AGC TGA AGTGCAGATG ACCACATTCA AGGAAGAACC TTCTGCCCCA GCTTTGCAAG ATGAAAAGCT 813 823 833 843 853 863 873 883 893 903 TTCCCACTTG GCTCTTATTC TTCCACAAGA GCTTTGTCAG GACCAGGTTG TTACTGGTTC AGCMCTCTG CAGAAAATGT CCTCCCTTGT GGCTTCCTTA 913 923 933 943 953 963 973 983 993 1003 GCTCCTGTTC TTGGCCTGAA GCCTCACAGC TTTGATGGCA GTGCCTCATC TTCAACTTTT GTGCTTCCCT TTACCTAAAC TGTCCTGCCT CCCGTGCATC 1013 1023 TGTACTCCCC TTGTGCCACA FIG.