DOS: a new chain gene ... II antigens, HLA-DP, -DQ and -DR, each consisting of distinct ... plete cDNA clones for the (3 chains of the DR, DQ and DP anti- gens.
The EMBO Journal vol.4 no. I I pp.2839-2847, 1985
DOS:
a new
chain
gene
in HLA-D with
a
distinct regulation of
expression
Cecile Tonnelle, Robert DeMars1 and Eric O.Long Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 5, Room B l-l 1, Bethesda, MD 20205, and 'Laboratory of Genetics 'and Department of Human Oncology, University of Wisconsin, Madison, WI 53706, USA Communicated by B.Dobberstein
The HLA-D region of the human major histocompatibility complex encodes the genes for the a and j3 chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II , chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic ( chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO,B gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO,B mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO,B is not induced by ay-interferon in fibroblast lines. The DO( gene is distinct from the DP,B, DQ,B and DRO genes in its pattern of nucleotide divergence. The independent evolution and expression of DO( suggest that it may be part of a functionally distinct class II molecule. Key words: cDNA clone/histocompatibility/HLA-D/immune response/nucleotide sequence
Introduction The major histocompatibility complex (MHC) encodes antigens which control the initial steps of immune responses. MHC antigens are required in the recognition of foreign antigens by T lymphocytes. The class I antigens of the MHC, which are expressed on virtually all nucleated cells, are primarily involved in antigen presentation to cytotoxic T cells, while class H MHC antigens are generally required in antigen presentation to T cells of the helper/inducer subset (Swain, 1983). Class antigens are expressed in antigen-presenting cells and B lymphocytes. Their expression can also be induced in human T lymphocytes upon antigenic or mitogenic stimulation and in a variety of cells by treatment wtih 7y-interferon. MHC antigens display a very high degree of allelic polymorphism and T cells usually recognize foreign antigens only in the context of a self MHC antigen (Zinkernagel and Doherty, 1979). In fact, MHC antigens themselves elicit strong immune responses in allogeneic hosts as observed in graft rejections and mixed lymphocyte reactions. In addition to the allelic polymorphism there is another level of complexity: multiple isotypic forms of class I and class II genes present in the MHC. In man there are at least three class antigens, HLA-DP, -DQ and -DR, each consisting of distinct and chains, which are encoded by genes in the HLA-D region of the MHC (Auffray and Strominger, 1985). The a and chains associate non-covalently and are both transmembrane glycoproteins with a membrane-proximal immunoglobulin-like domain.
are II a
IRL Press Limited, Oxford, England.
There is evidence that the three class II antigens in man can be involved in antigen presentation to T cells (Qvigstad et al., 1984). The existence of the DR, DQ and DP antigens may serve to diversify the range of immune responses that each individual can mount. An alternative but not exclusive view is that each of these class II antigens plays a distinct role in the generation of immune responses. The molecular analysis of the HLA-D region has revealed multiple genes within each subregion: one a and three ( genes in DR, and two a and two ( genes in both DQ and DP (Auffray and Strominger, 1985). An additional ca gene named DT (Inoko et al., 1984) or DZ (Trowsdale et al., 1985) has been described. Correlations have been made between human and murine class II genes based on their structural similarities. The a and ( chain genes of DR and DQ are the respective counterparts of the I-E and I-A genes of the murine I region. Two additional ( genes named A(32 and A(3 have been found in the I region (Larhammar et al., 1983a; Wake et al., 1985). A(33 appears to be the counterpart of the human DP( gene but no human equivalent for A(2 has been reported yet. It remains to be established how many of these class H genes in man and mouse are functional and whether other undetected genes exist. We have approached these questions in man by constructing a cDNA library from mRNA of a B-cell line which has lost a complete HLA-haplotype (Kavathas et al., 1980). Such a hemizygous mutant containing only non-allelic genes presents an advantage over homozygous cell lines. Even when homozygosity results from consanguinous pairings it is possible that a recombination event, undetectable by serological means, introduces heterozygosity at certain loci (Termijtelen et al., 1983; Robinson et al., 1984). The library was made in a eukaryotic expression vector such that a functional dissection of truly isotypic class II antigens will be possible. Cell surface expression of DR antigens in transfected murine and human cells was obtained from these cDNA clones (Sekaly et al., 1985). We describe here complete cDNA clones for the (3 chains of the DR, DQ and DP antigens. By searching for new (3 chain clones at a low stringency of hybridization, we isolated an additional cDNA clone, designated DO(, which shows good structural similarity with the murine A32 gene. Several properties of DO( distinguish it from the DP, DQ( and DR(3 genes.
Results and Discussion The HLA haplotype-loss mutant 45.1 was derived from the heterozygous B-cell line 721 by 'y-irradiation and immunoselection. The remaining haplotype carries the DRl, DQwl and DPw2 class II specificities. Cytoplasmic poly(A)+ RNA from mutant 45.1 was used to construct a cDNA library by the procedure of Okayama and Berg (1983). 360 000 independent clones were generated and amplified in culture. A plasmid preparation of the entire library was linearized and size-fractionated by agarose gel electrophoresis. Clones with long cDNA inserts of the DR(, DQ(3 and DP( chains were localized by blotting part 2839
C.Tonnelle, R.DeMars and E.O.Long DO DP DQ DR
AACTCATTCTGAAGAGGCTGACGATTTTACTGTCTCAT TTTT--C-GACTGCAGCT--TTTC----G-CA--CTT-
TTTTATTCTTTCTGCCAGGTACATCAGATCCATCAGGTCTGAGCTG-GT-GACT-CC-CTACTTTTCCC--CG-C--AATTAGTTCTCCCTGAGTG-GA-T-G-CTGCTTCT---G-CCC-GGT-CTGTC-TG #C L
N
M
S
L
D
S
---
AGC
---
R
T
Q
T
DP DQ DR
DO DP DQ DR
I V F D E GAA GAT TTT GTG ATT --G A-- -AC C-T T-C --G --- --C --- TAC CC- CG- --C T-- TGG
DO DP DQ DR
N AAC -------
-G- T-- --C A-- ACA --G --- AC-
AL
A Q CAG GCA --- -G--- TTT --- CTT
R V Y E E L TTG GAG GAG TAT GTA CGT CG- --- --- -TC --G --C CGA --- --- --C --G --C CAA --- --- -CC --G --C
---
-CA ---
-TG GTG
E T G N T F Y C D A K AAG GCT GAC TGT TAC TTC ACC AAC GGG ACA GAA --- C-G CG- CAG --A --C --- GCG TTT --T ----- -GC CTG --C --- --- --- --- --- --G --G --- TT- --A --- C-T --- TT- --T --- --G --G
Q F K AAG GTG CAG TTT CGC -T-C CGC -- -G- GGCG- --- -G- --G
V GTG C---C--
Q CAG GCA GCGCC
Y N G L R C H R TGT AGA CAC AAC TAC AGG CTG GGC --_ GA- --- ----C --_ GA- G-- -CG --C --_ G-- G-T --T --C
88 398 393J 441 413
L
H CAC GTATGT-
1 16 482 477 525 497
S M A G V E R E G Q GGG CAG GAG GAG AGA GCT GGG GTC ATG TCC --A --- --- --A -C- --- --- ---G-- -----AT --- --- --- -C- --C --C --T G-- ----C --- --A --- -AG --- --- --G G--
144 566 56 1 609 58 1
T L H V Y P E G E T M L V V M GTC TAC ACC GTG GTG ATG CTA GAA ATG ACT CCT GAA CTT GGA CAT ----- ----G --- --- --C --C C-G -AG --- G-C---G --- --- --- --C C-G -G- --- G-- --- --- --C---G --- -CA GT- --- CGG AG- --- G-G --T --- --C--
172 650 645 693 665
G V T F TTC ACT GTG GGG A-G --C C-- CAGGG -TC C-- CA--- --A --- CA-
R AGA C-C --G C-G
H Q T R L L E P P K E P V Q y v V T CTG CAC CAG AAA GTG CAA CCA GAG GTG ACA GTG TAC CCA GAG AGG ACC CCA CTC -C--G --C GGG -AG -AT---C TCC CG- --C --G --T AG- --- -AT --T -C--- TCC --- --A GAG GC- --C A-- --C -G- --- G-G --C ACA --C A-C -C.ICG--T --T TCA -A- --- -AG -C- --- --G --C G-G --T A-- __- -- T
DO DP DQ DR
D I Y P G G F V T S C TGC TCT GTG ACA GGC TTC TAT CCA GGG GAT ATC --- CAC --- --G -AT --- --C --- --C AGC --T --- --G --- --- -AT --- --- --- A-C C-G ----- --- --- -GT --T --- --- --- --C AGC --T
I
L
R
--_
--_
V--
N
G
D
W
T
F
L W N F K K I AAG ATC AAG TGG TTC CTG AAT --C-A G-- CGA --- --- --- ----A G-- CG- --- --T -GG-A G-- -G- --- --- -G- --C Q
T
DQ DR
T ACT --C --C --A
G GGC AACC---
CCT ATC AGG AAT GGA GAC TGG ACC TTT CAG ACT -TG -- - C-T --- --- --- --- --- --C --- -TC -TC --T --- --C --T --- --- --- --C --- -TC -TG --- CA- --- --- --T --- --- --C --- --C
DO DP DQ DR
C TGC -------
L CTT -AA -AC -AA
GAA TAT TCT GTC GAT CAC TCC AGC CTG CTG AGC CCT GTT TCT GTG GAG TGG AGA GCT CAG TCT --G --G --- A-- --- --- GAT-T --- --C A-C --- --- --- -AG --A --- --- --T -C- G-C --- -C- G-C --G --G --- C-- --- --C -A- --- --C A-C A-C --- --- --- C-G --- --- --- ---C- G-A --G --G --- C-A --T G-- AC- --- --- C-C A-A --- --A --- --- --A -G- ---
DO
DP DQ
DR
DP
0
H
S
S
L
L
S
-CG --A GC- -GG -G--- --- G-- -G- -G--- --A G-C -GG -G-
T
P
V
Q
AGG ACG CAG G-A T-T GCA
DQ
---
F G L I GGG CTA ATC TTC G-G --G --- --C --- A---G-G --G --- --G --G-G --G --C --G C-- ---
-----
H CAT --C --C --C
H AAT --C --C --C
L
CTG CTG --T --- ----C -----
--_ --_
--_
v
s
E
R
W
A
Q
E
S
Y
S
A
A F I A G L L S AGT GGC ATT GCA GCC TTC CTA CTT
R DO
32 230 225 273 245
---
P CCC ---GAG-
G V V D GTG GAT GGG GTC CC- --C A-- A-G -- --C A-- --G --- --C ACC TA-
DO DP DQ DR
V
F I F R V GTC AGA TTC ATC TTT AC -A -AGAAC- --- CAA-G -AA-
P
S
A GCA -GG TAC -AG
A GCC -TG T-G --G
R S R L E D L GAT CTC TTG GAG AGG AGC AGA --C A-- C-- --- GA- GAG C-G --A G-- C-- --- G-- GC- C-G C-- --- CA- --G C-G --C
-
P
A-C -G-
4 146 147 189 161
D
T
G
60 Q E A D P Q G L T K L A M V F D G S D V F CTG GGG CAG CCA GAT GCT GAG CAG 314 TTC GAC AGT GAT GTG GGG ATG TTT GTG GCA TTG ACC AAG --309 --T-C -AG ----T -G--GA- --C CG- --G G-- --G G---- --- --C --C ----TI -T- --C --- T-C 357 -G --- --- --C --C --- --- G-- -AC CG- --- G-- --G CC- -A-G- --T --- --C --- T-C 329 --- --- --C --C --- --- GA- -AC CG- --G G-- --G G-- ---
CTG AAAAAA-
DO DP
77
+1
C-A C-- G-- TTG -CT GGG
R N S W TGG AAC AGC CGG A--- --- --- -A--- --- --- -A-
DO DP DQ DR
-1
-- CAG T-- C-G C-- AAG
V L L A V V W P V TCT CCA CTG CTA GTG AAT CTG ACC CGA CTG GAT TCC TCC ATG ACT CAA GGC ACA GAC A-GTC CCC TGG GTG GTG GCT ----T G-- GTC --G --- -GG -C-C- --- C-- ACA --- --- --- ACG -C- TTA --- -TG GTG --- CTC A-A - --- --- --C -A- -TT C-- --A -CA A-- G-C ACC T-- -TG --- G-G ATC --- AGC --- --AC-- G-- G-G --
G H S TCT GGG TGG GT- TCT GCATC CCC G-A CTC CCT G-A
DO
G -2 5 ATG GGT 6'62
TTCJTCCAGA -jQGTT AA TCT T-T M1GTG
TTT TTT CCT AGC T-C FATGG A-C-CC AGC
L
L L Q G I V I V CTT CTG GTG GGA ATC GTC ATC CAG CTA T-- --G --C AGG TG- GGA --- --C ----- GG- C-T -GT -A--C C-T A---- GG- -CC --G C-G T-- --- T-C T-C ---
W TGG C-CACA-
R AGG
A
Q
GCT
---
AGC
A--
---
L M K R AGA AAG ATG CTG --T --- -CA T---C --C K
CAG AAA
AG- -GAA- ---
-------
200 734
729 777 749
V G Y GGA TAT GTG -TT C-A CGA ---
A6
228 818 813 853 833
C-C TCT
C S Q V A R P S L V L E N G AGA GCT GTT CTG CTC CCT CAG TCA TGC |iGGTCCTCACTAAGCTTGCTCTCTCTGGAGCC --G TTC CT- AG- --- CTG AAA AGA --A T-G TGC -TT AGG AAA AGCAT-TGCTGTGTTT-G-TAG-ATCTG-CTC-A T-T TAA -TA AG- CTG GTTATCA---TTCTGTGA---CTG--TGTCCCTG CTC CTG AGA -G CT- CT- CA-AT --A -A- GAC -A- Ar- --A GG- A-A ACCTTCTG--C-AGCTT-GCAGAATGAAAAGCTT CCA A-A --A TTC CT- AG- 3
M
S
247
ATO TCT GGT AAT GAG GTC TCA
909
117W A-A
904 928
---
CTT
924
DR
G-A
DO
1GAAGTAGTGA0TGAGTAGTCTGGGCCCTGGGTGAGGTAAAGGACATTCATGAGGTCAATfTTCTGGGAATAACTCTCTTCCCTGATCCTTGGAGGAGCCCGAACTGATTC1019 G--cAG-ccTTCA-c-TCCAAATTGGA-ACTGCT-cc--GAAGTTGcTc---A----G-T-cTATcATTcTG----T-GATTcA-AG-AcT-TTTcT-T-AcTGG-cc-CCC--A-T-CCCAGC-GCCTGT-T-AGCTT--CCCCCTG--AT--AAGTCCTACAGTGGC-GTCAC-C-ACCAC-AGG--ATCTCCTT-CATCCCCA---C--GGCGC-G -CCT-CTTG-CA-TTATTCT-CCA-AAGA-AG-GCT-TCTCAGG-CCTGGTT-C-ACTG----G-CA-C-GCAGAAAA-GT-CTCC-T-GT-GCTTC-T-AGCTCCTGC-
DP DQ DR DO
DP DQ
---
1014
1038
TGGAGCTCTGTGTTCTGAGATCATGCATCTCCCACCCATCTGCCCTTCTCCCTTCTACGTGTACATCATTAATCCCCATTGCCAAGGGCATTGTCCAGAAACTCCCCTGA CAACCA-G-TCCC-TCTTCT-AGCA-CA-AAAT-AT--AAAC--AACA-GA--GT-TGT-T-C-T-T
DR
GCTGTGA--C--C-TCCT-CA-TGA-CCAGAG-CA-TGC---TA-A-GG--AGCTGCGTCTACTCAGGCCCCAAGGGGATT-TGTTTCTG--C--TCCTC-GA-TG--CCTTG--CTGAA--C-CAGC--TGATG-CAG-G-CT-ATCT-CAA---T-GTGC--CC-T-TGC-TAA-CCGTATGG-C-CC-GTGCAT-TG-ACT--CCCTG-A-GACA-
DO
GACCTTACTCCTTCCAGCCCCAAATCATTTACTTTTCTGTGGTCCAGCCCTACTCCTATAAGTCATGATCTCCAAAGCTT1
DQ
1129
1081 1148
CTGTCTTCCAACTGCAGTCTCCACAGTCT
AGAGAAG-A-A-GAA-AA-ATT-CCTGAC-T-AGAG--T-TT-A--TAA^..---A-GATCC-G-GTT
DR
ACA-A-TACAT-ATT-AATGTTTC---AAG-TGGAGT-
DO
TCAGAAGACAAATGCTCAGGTAGTCACTGTTTCCTTTTCACTGTTTTTAAAAACCTTTTAITGICAAATAAAATGGAGATACA
1322
al., 1980) 1. Nucleotide sequences of four isotypic ,B chain cDNA clones. The clones were derived from mRNA of the B-cell line 45.1 (Kavathas have been aligned with respect to their reading frame which has a single HLA haplotype with the DRI, DQwl and DPw2 class II specificities. The and identity with DO,B is indicated by hyphens. The presentation in triplets starts with the most upstream initiation codon (in DQf3) and ends with the last boxed and termination codon (in DOf3). The translation of DO: is given with the single-letter amino acid code. Initiation and termination codons The intron-exon defined in DRI3, DQ,B and DP,B addition signals are underlined. Triangles indicate the positions of intron-exon boundaries GT which is organization of the DO,B gene is unknown. The putative exon 6 of DO,B could be short intron because it starts with the # 163, 18 and 73 follows: 10 and 66 in respectively splice donor. The length in bp of the GC-tails and of the AT-tails in the four clones DP,B #003, 6 and 69 in DQ,B #021 and 14 and 126 in DRj3 #008.
Fig.
et
sequences
poly(A)
as
potential
a
were
gel to a nitrocellulose filter and hybridization with previously cloned cDNA fragments. The longest cDNA clones for DR3, DQ,B and DP3 were extracted from slices of the agarose gel by electroelution, introduced into Escherichia coli after circularization with ligase and screened by colony hybridization of replica filters. The same set of filters was subsequently hybridized at a very low stringency and a clone was isolated which had not of the agarose
been positive with cy.
DO,B
as
This
new
DRj3, DQ3
and DP3
probes
clone, designated DOj3, will
DRj,
DQ,B and DP,B clones.
The
chains of HLA-DRJ, -DQwJ and
f
Over 45 clones for
longest, DRj
# 008,
the
DR,(
chain
chosen for
in
at
high
stringen-
be described after
the
DPw2 isolated.
One
analysis
of
the
(Figure
2840
mm.
DO3, a new expressed gene in HLA-D -30 DO DP DQ DR
I
-1+1
-10
-20
I 11 1 MGSGWVPWVVALLVNLTRLDSSMTQGTDSP MVLQVSAA-RT---TAL-MV-LT-VV--RAT-
MSHKKSLRIPGDLRVATVTLM-AI-S--LAE-R--MVCLKLPGGSCMT--T-T-MV-S-PLALAG-TR
EXON 1 10
DO DP DQ DR
S s
70
60
50
40
30
20
S80
90
i I I II EDFVIQAKADCYFTNGTEKVQFVVRFIFNLEEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQAVDGVCRHNYRLGAPFTVGRK -NYLF-GRQE--AF---QR -LE-Y-Y-R--F--------E-R-V-E--R--E-Y---QK-I--EE-AVP-RM-----E--G-M-LQ-R ----Y-F-GL--------R-RG-T-H-Y-R-----------VYR-V-PQ-R-V--Y---QKEV--GA-AS--R------EVAYRGILQ-R
PR-LW-L-FE-H-F----R-RLLE-C-Y-Q--S--------EYR-V-E--R----Y---QK----QR-A---TY-----GV-ES---Q-R
EXON 2 110
100
DO DP DQ DR
S
150
140
130
120
160
170,
180
I I I I ~~- I180II VQPEVTVYPERTPLLHQHNLLHCSVTGFYPGDIKIKWFLNGQEERAGVMSTGPIRNGDWTFQTVVMLEMTPELGHVYTCLVDHSSLLSPVSVEW ---R-N-S-SKKGP-QH----V-H--D----S-QVR--------T---V--NL---------IL-------QQ-D----Q-E-T--D---T---E-T--IS-S--EA-NH----I----D---SQ--VR--R-D---T---V--PL---------IL-------QR-D----H-E-P--Q--IT---E-K-----SK-QP-QH----V---S-----S-EVR--R-----K---V---L-Q--------L----TV-RS-E----Q-E-P-VT--LT---
EXON 3
190
DO DP
DQ DR
I
200
210
220
l l RAQSEYSWRKMLSGIAAFLLGLIFLLVGIVIQLRAQK K---DSARS-T-T-AGG-V----ICG---FMHR-SK-----SAQS-----VGG-V------GL-LI-RQ-SR--R--SAQS-----VGG-V---L--GA-LF-YF-N--
EXON 4
240
230
l
l
GYVRTQMS VQRGSA -HSGL-PT
GNEVSRAVLLPQSC
5
6
-LLH -FLS
Fig. 2. Amino acid sequences deduced from four isotypic (3 chain cDNA clones. Residues identical to DO( are indicated by hyphens. The sequecnes are separated into exons. The first exon encodes the signal sequence and the first four amino acids of the mature ( chains. Exons 2 and 3 encode respectively the first and second extracellular domains. Exon 4 encodes the connecting peptide, the hydrophobic transmembrane region and the first cytoplasmic residues. Exons 5 and 6 encode the rest of the cytoplasmic tail. The intron-exon organization of the DO,B gene is not known. The putative exon 6 of DO,B could be a short intron because it starts with the sequence GT which is a potential splice donor. The coding sequence for DP,B stops in exon 5. The DQ,B genes have lost the use of exon 5 because of a splicing defect (Larhammar et al., 1983b).
1). With 62 bp of 5'-untranslated sequence it is longer than other 1983; Gustafsson et al., 1984; Bell et al., 1985). The first nucleotide of the cDNA insert is 26 bp downstream from the putative TATA box of a sequenced DR3 gene (Larhammar et al., 1985). The translation of this cDNA clone (Figure 2) defines the complete sequence of an HLA-DR1 (3 chain. Alignment with DR( sequences of other specificities (Gotz et al., 1983; Long et al., 1983; Gustafsson et al., 1984) confirms that most of the variable residues are clustered in three regions of the first domain, at positions 9- 13, 26-38 and 70-74 (not shown). The sequence of a DR3 cDNA clone from the DRl B-cell line LG2 was reported (Bell et al., 1985). It differs from our clone #008 by seven arnino acids. However, most of these differences can be accounted for by sequencing or editing errors in the LG2 clone (J.Bell, personal communication). The nucleotide sequence of the longest DQ( cDNA clone, DQi3 #021 (Figure 1), defines the complete sequence of a DQw 1 3 chain (Figure 2). This clone has a very long 5'-untranslated region which starts 19 bp upstream of the first mRNA start mapped by primer-extension (Boss and Strominger, 1984). The 5' end of cDNA clone # 021 is within the putative TATA box of the DQ( gene (Boss and Strominger, 1984). It cannot be due to a cDNAcloning artifact because the additional 19 bp match perfectly the genomic sequence. The serological specificities that define the DQ antigens are in very strong linkage disequilibrium with DR specificities. For instance, DQw1 is almost always associated with the DRI, DR2 and DRw6 specificities. Biochemical studies have shown that some variability in the (3 chains, but not the a chains, existed in the DQw 1 antigens associated with DR 1, DR2 and DRw6 (Shackelford et al., 1983; Bono and Strominger, 1983; Giles et al., 1985). A logical nomenclature would be to call these different DQw 1 antigens respectively DQw 1.1, DQw 1.2 and
DR43 cDNA sequences published to date (Long et al.,
DQw 1.6. The DQw .1 (3 chain sequence deduced from clone #021 is indeed quite different from the amino acid sequence determined for the ( chain of DQw1.2 (Gotz et al.. 1983). Most of the variability is in the first domain where the two sequences share 88 % homology. Two DQ(3 cDNA clones, pII-(-l and pH(-2, from a DR3,w6 - DQw1 ,w2 cell line have been sequenced (Schenning et al., 1984). Their respective allelic specificities have been deduced as DQw2 for pl-,B- 1 by comparison with a DQw2 genomic sequence (Boss and Strominger, 1984) and as DQw1.6 for pII-(-2 because it probably represents the other allele. The amino acid sequence homology of pII-(-l with our DQw .1 (3 sequence is only 77 % in the first domain. On the other hand, pll-(-2 is identical to our DQw 1.1 3sequence, except for a single silent base change. The difficulty in assigning the proper specificity to clones isolated from heterozygous cell lines emphasizes the usefulness of haplotype-loss mutants such as 45.1. The nucleotide sequence of clone DP(3 # 003 (Figure 1) defines the complete sequence of a DPw2 ( chain (Figure 2). The amino acid sequence is identical to a sequence deduced from two overlapping cDNA clones obtained from different cell lines (Kappes et al., 1984). The nucleotide sequences differ by a single silent change. A new class ll ( chain gene in the HLA-D region After identifying DR(3, DQ( and DP( clones in the cDNA library by high stringency hybridizations, we hybridized the same set of filters at a very low stringency. A new clone, (3 # 163, was isolated. The sequence (Figure 1) defines a new ( chain which was given the designation DO. The open reading frame contains 273 codons, flanked by 56 bp of 5'-untranslated region and 447 bp of 3'-untranslated region. The 5'- and 3'-untranslated regions show no significant similarities with their counterparts in the DP, DQ and DR cDNA sequences. The translation of the 2841
C.Tonnelle, R.DeMars and E.O.Long Table 1. Comparison of nucleotide and amino acid sequences of class II a chain genes (31 exon (270 bp) DO DP DQ 67 76
63
DO DP DQ DR A,B2 A,B
53 56 57 54 51
E(
60 65
DR
AO3
E,B
66 76 76
64 72 79 72
63 67 68 79
61 68 59
63 58
32 exon (282 bp) DO DP DQ 71 67 66 66 79 65 54
74
63 64
59
76 73 60 72 69
73 78 71 64 86 69
DR
A32 A,B
E,B
72 77 78
83 71 71 73
70 77 74 83 70 70
64 64 83
59 64
67 76 83 71 66
Transmembrane exon (111 bp) DO DP DQ DR AO3
E,B
70 67 82 73
64 68 70 87
63
64
68 68 78
76 72
43 56 54
59 54
78
54 51
59 56
81 73
71
73 89
67
Numbers represent the percentage of identical positions. Nucleotide comparisons are in the upper right triangle and amino acid comparisons in the lower left triangle. Amino acids encoded by split codons between two exons have been included in the exon which carries two of the three nucleotides (see Figure 2). The murine sequences are from Saito et al. (1983) for E,B and Larhammar et al. (1983a) for A,B and A(2. Only the (2 exon of A,B2 has been sequenced. The numbers for the comparisons of related human and murine genes are underlined. REGION U)
5-
(N Lm
("4r.4 LD
rQ
r cD
C: C)
0
LO t
t
r
f4
