Jan 13, 1986 - ation method according to Maxamand Gilbert (1980). 551 ... N'Guyen,C., Sodoyer,R., Trucy,J., Strachan,T. and Jordan,B.R. (1985) Immuno-.
The EMBO Journal vol.5 no.3 pp.547-552, 1986
Gene conversion-like mechanisms may generate polymorphism in human class I genes
Gerhard H.A.Seemann, Rita S.Rein, Caroline S.Brown and Hidde L.Ploegh The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Communicated by P.Borst
The nucleotide sequences of the human class I major histocompatibility complex genes HLA-B27k and HLA-B27w have been determined. They differ by only four nucleotides over a stretch of 14 bp in exon 2, resulting in three amino acid exchanges at positions 77 (Asp to Asn), 80 (Thr to Ile) and 81 (Leu to Ala). The distribution of these nucleotide substitutions suggests a gene conversion-like event responsible for the generation of these HLA-B27 subtypes. The mechanisms underlying the generation of new polymorphic variants in man are therefore probably identical to the gene conversionlike events postulated in the generation of H-2Kbm class I mutants in the mouse. Key words: class I gene/polymorphism/nucleotide exchanges
Introduction The mechanisms for the generation of polymorphism for products of the major histocompatibility complex (MHC) have been studied extensively for the murine H-2 complex. The examination of mutants of the H-2 loci has strongly suggested the involvement of gene conversion-like events in the generation of polymorphism (Nathenson et al., 1986). In particular, for the H-2kbm mutations, the notion of block-wise exchange of minimal stretches of 538 bp was made plausible by the identification of potential donor genes (Nathenson et al., 1986). No such information is available for any other mammalian MHC. The so-called subtypes at the human MHC loci closely resemble the bm mutations in mice. These subtypic specificities are antigens that cannot be readily distinguished by standard serological techniques, yet are easily recognizable by cytotoxic Tlymphocytes (CTLs) and by biochemical methods (Molders et al., 1982; Breuning et al., 1983). We report here the DNA sequence of two HLA-B27 genes, 5corresponding to the B27w and B27k subtypes as shown by transfection experiments. The nucleotide sequence of these genes differ by only 4 bp over a stretch of 14 nucleotides. Thus, HLA-B27w and HLA-B27k are no more different from each other than is the Kb gene and many of its mutant alleles, indicating that the mechanisms underlying the generation of new polymorphic variants are probably identical for mouse and man. Results and Discussion The HLA-B27w and HLA-B27k genes were isolated from a genomic library constructed from the BRUG cell line (HLA type: A3, Al 1; B27w, B27k; Cwl, Cw2) in the lambda vector EMBL 3. Screening of this library with a probe reported to be B locusspecific (Koller et al., 1984) resulted in the isolation of four inde-
© IRL Press Limited, Oxford, England
pendent recombinants containing complete class I genes, two of which (I 10 and I 6) were subsequently identified as indeed containing B locus genes. Their restriction maps were identical but for the presence of an additional XbaI restriction site in the 3'-flanking region of I 6 (Figure 1). Identification of I 10 and I 6 as corresponding to the HLAB27w and HLA-B27k genes, respectively, was based on the following criteria. An HLA-B27 amino acid sequence, corresponding to residues 1-271, was available (Ezquerra et al., 1985) for comparison with the amino acid sequences deduced from the genomic clones (Figure 2), and established the identity of the I 10 sequence with the HLA-B27 protein sequence at all positions available for comparison except position 242, where a Gln residue was identified in I 10 instead of Glu in the protein sequence. This is most likely attributable to a protein sequencing error, since Gln residues are often misidentified as Glu residues. Secondly, since the HLA-B27w and HLA-B27k heavy chains are readily distinguishable by isoelectric focusing (IEF) (Molders et al., 1982), DNA-mediated gene transfer and biochemical characterization of the gene products in the transfected cells should allow the identification of the genes encoding the subtype specificities. We introduced the I 10 and 1 6 clones together with the neomycinresistance gene into mouse 3T3 cells (Southern and Berg, 1982). After selection for neomycin-resistant cells, detergent extracts were prepared from them and analysed by IEF and immunoblotting (M6lders et al., 1982. Neefjes et al., 1986). The results, shown in Figure 3, unequivocally demonstrate that the products encoded by I 10 and I 6 are indistinguishable from the HLAB27w and HLA-B27k heavy chains as produced by the donor cell-line BRUG. Therefore I 10 and I 6 correspond to the HLAB27w and HLA-B27k genes, respectively, as indicated by sequence comparison and transfection experiments. The available HLA-B27 protein sequence does not extend beI6/110 Sst1
S.tl
Sstl Bgl 11 Xbai Pst1 Pst1 EcoRISst1 Xbal Ia ,Bg911 is Kpn11 aI ~~~a o3 ~ 41 TUT 1_-~ ~ ~ ~ ~ ~ ~ ~~~~~111
Ssti
Pst1
o
511
C2
C3
6
Xbal e16} 100bp
Fig. 1. Restriction map and sequencing strategy for the HLA-B27w and HLA-B27k genes. The two genes have identical restriction maps for the enzymes indicated. Only one polymorphic XbaI site was found, overlapping SstI the SstI site (TCTAGAGCTC), about 250 bp 3' of the poly-A addition site. XbaI This site is present only in the I 6 (HLA-B27k) and is indicated in the restriction map. The open bars represent the exons (S: signal peptide; a1, Ca2 and (3: the three extracellular domains; TM: transmembrane region; C1, C2 and C3: cytoplasmic domain exons; 3' UT: 3' untranslated region). The arrows indicate the sequencing strategy.
547
G.H.A.Seemann et al.
B2 7w
-24
gccaatcagtgtcgccggggtcccagttctaaagtccccacgcacccaccMggactcagaatRVtcc
-20
gcgagATGCGGGTCACGGCGCCCCGAA M
R
V
T
A
P
100
R
B2 7k -10 B2 7w CCCTCCTCCTGCTGCTCTGGGGGGCAGTGGCCCTGACCGAGACCTGGGCTGgtgagtgcggggtgggcagggaaatggcctctgtggggaggagcgaggg T
L
L
L
L
W
L
G
A
V
A
L
T
E
T
W
200
A
B27k 1 B27w gaccgcaggcggggcgcaggaacccggggagccgcgccgggaggagggtcgggcggctctctgcccctcctcgccccagGCTCCCACTCCATGAGGTATT G
S
H
S
R
M
300
Y
B27k -------------------------------------------------------------------------__--__---------------------
20 10 30 40 B27w TCCACACCTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGC rCATCACCGTGGGCTACGTGGACGACACGCTGTTCGTGAGGTTCGACAGCGACGCCGC H
F
T
S
V
S
R
G
P
R
G
P
E
R
I
F
T
G
V
Y
V
D
T
D
F
L
V
R
F
S
D
D
400
A
A
B2 7k 50 60 70 B27w GAGTCCGAGAGAGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCGGGAGACACAGATCTGCAAGGCCAAGGCACAGACTGAC S P R E E P R A P W I E QE G P E Y W D R E T I C K A K A Q T D B27k 8 B27w CGAGA GACTGCGGACCC JC R E D L R T L L N - - I B27k
_____.---------- T-GC
500
-090
0
GCTACTACAACCAGAGCGAGGCCGgtgagtgaccccggcccggggcgcaggtcacgctccccatcccccacgtac R
Y
Q
N
Y
S
E
600
A
-------------------------------------------------------------------------------
B27w ggcccgggtcgccccgagtctccgggtccgagatccgcscccgaggccgcggggctcgctcagccctcgccggcgagagtcccaagcgcgtttacccggt B27k
700
ttcattttcagttgaggccaaaatccccgcgggttggtcggggcggggcggggctcggggggacggggctgaccgcggggggacggggccagGGTCTCAC
800
------------------------------------------------------__---------------------__---------------------
B27w
G
S
H
B27k
B27w
100 110 120 ACCCTCCAGAATATGQATGGCTGCGACGTGGGGCCGGACGGGCGCCTCCTCCGCGGGTACCACCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGA T
QN
L
M
Y
G
C
G
V
D
P
G
D
R
L
L
R
G
Y
H
QD
Y
A
G
D
K
Y
D
A
I
900
L
B27k 130
B27w
140
150
16
, AA uuU ACGAGGACCTGAGCTCCTGGACCGCCGCGGACACGGCGGCTCAGATCACCCAGCGCAAGTGGGAGGCGGCCCGTGTGGCGGAGCAGCTGAGAGCCTACCT .u1 E
N
D
S
L
S
W
T
A
A
D
T
A
A
TQ R K W E A A R V A E Q L
I
R
A
Y
L
B27k 0
170
180
GGAGGGCGAGTGCGTGGAGTGGCTCCGCAGATACCTTGAGAACGGGAAGGAGACGCTGCAGCGCGCGG2taccaggggcagtggggagccttccccatct E G E C V E W L R R Y L E N G K E T L R A
1100
B2 7w B27k
cctataggtcgccggggatggcctcccacgagaagaggaaaatggggtcagcgctgaatgtcgccctcccttgaatggagaatggcatgagttttcctga
1200
B2 7w
gtttcctctgagggccccctcttctctctaggcaattaagg
B27w
Q
B2 7k
190 atcagACCCCCCAAAGACACACGTGACCCACCACCCCAT 1300
380bp
gap
D
P
P
K
V
H
T
T
H
H
P
I
B2 7k
200 B2 7w B27k
548
210
220
CTCTGACCATGAGGCCACCCTGAGGTCTGGGCCCTAGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGCGAGGACCAAACTCAGGAC 1400 S
D
H
E
A
T
L
R
C
W
A
L
G
F
Y
P
A
E
I
T
L
T
W
QR
D
G
E
D
QT
QD
Polymorphism generation in human class I genes
B27k
B27w
260
250
240
230 B27w
ACTGAGC'17GTGGAGACCAGACCAGCGGGAGATAGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAAGAGCAGAGATACACATGCCATG 1500 E
T
L
V
E
T
R
P
A
G
R
D
T
F
Q
K
W
A
A
V
V
V
P
S
G
E
E
Q
R
Y
T
C
H
--------------------------------------------------------------------------------------------------
270 TACAGCATGAGGGGCTGCCGAAGCCCCTCACCCTGAGAT V
Q
H
G
E
L
P
P
K
L
T
L
R
aaggagggggatgaggggtcatatctcttctcagggaagcaggagcctcagcagg
1600
W
B27k
B2 7w
280 290 gtcagggcccctcatcttcccttcctttcccagAGCCGTCTTCCCAGTCCACCGTCCCCATCGTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGT 1700 E P S S Q S T V P I V G I V A G L A V L A V V
B27k
-
- -
-
-
-
300 B27w
-
-
-
-
- - -
-
- -
-
_- _-
-
-
310
GGTCATCGGAGCTGTGGTCGCTGCTGTGATGTGTAGGAGGAAGAGCTCAGtagggaaggggtgagtggtggggtctgagttttcttgtcccactggggg 1800 V
I
G
V
A
V
A
A
V
M
C
R
R
K
S
S
B27k
B27w B27k
1900
B27w B27k
tttgtgcggcacatgtgacaatgaaggacggatgtatcaccttggtggttgtggtgttggggtcctgattccagcattcatgagtcaggggaaggtccct
B27w B27k
gctaaggacagaccttaggagggcagttggtccaggacccacacttgctttcctcgtgtttcctgatcctgccttgggtctgtagtcatacttctggaaa
B27w B27k
2000
----------------------------------------------------------------------------------------------------
2100
------------------------------------------------------__---------------------__---------------------
ttccttttgggtccaagacgaggaggttcctctaagatctcatggccctgcttcctcccagtcccctcacagggcattttcttcccacagGTGGAAAAGG G
G
K
2200
G
-------------------------------------------------------------------------__--__--------------G----G320
B27w
AGGGAGCTACTCTCAGGCTGCGTgtaagtgatgggggtgggagtgtggaggagctcacccaccccctaattcctcctgtcccacgtctcctgcgggctct G
S
Y
QA
S
2300
A
B27k
B27w
B27k
B27 B27k
330 340 gaccaggtcctgtttttgttctactccagGCAGCGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGAAAAGgtgagattcttggggtctagagt -
-
-
C
S
-
-
D - -
S
A
Q
G
S
D -
V -
S
L
T -
2400
A * -
--_-
- -
-
- -
-
gggtggggtggcaggtctgggggtgggtggggcagtggggaaaggcctgggtaatggagattctttgattgggatgtttcgcgtgtgtggtgggctgttt
2500
agactgtcatcacttaccatgactaaccagaatttgttcatgactgttgttttctgtagCCTGAGACAGCTGTCTTGTGAGGGACTGAGATGCAGGA7w
2600
----------------------------------------------------------------------------------------------------
B27w B27k
CTTCGCGCCTCCCCTTTGTGACTTCAAGAGCCTCTGGCTCTCTTTCTGCAAAGGCACCTGAATGTGTCTGCGTCCCTlTTAGCATAATGTGAGGAGGTGG 2700
B27w B27k
AGAGCCAGCCCACCCCCGTGTCCACTGTGACCCCTGTTCCCATGCTGACCTGTGTTTCCTCCCCAGTCATCTTTCCTGTTCCAGAGAGGTGGAACTGGAT
2800
B27w B27k
GTCTCCATCTCTGTCTCAACTTTATGTGCACTGAGCTGCAACTTCTTACTTCCCTACTGAAAATAAGACTCTGAATATAAATTTGTTCTCAAATATTT
2900
----------------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------
Fig. 2. DNA sequence of HLA-B27w and HLA-B27k. The nucleotide sequence and the deduced amino acid sequences are compared. Lower case letters represent intron and flanking, upper case letters exon sequences. A CAAT and TATA box, splice donor and acceptor sites, the translation termination and the polyadenylated signals are underlined. Three asterisks represent the translation termination codon. The amino acids are given in the single-letter code and have been numbered according to the amino acid positions in the antigen in its mature form. For HLA-B27k only the nucleotides and amino acids different from HLA-B27w have been indicated. The region where the differences occur is boxed. 549
G.H.A.Seemann et al.
N%.1
s%.
S/
F-P -tr
0l
B27k B27w-
antibody HC10 Fig. 3. Expression of the HLA-B27w and HLA-B27k genes. Onedimensional IEF gel analysis of the gene products encoded by I 6 and I 10. After electrophoretic transfer, blots were developed with the HC 10 antibody. Lane 1: the donor cell-line BRUG showing the presence of the two HLA-B27 subtypes, as indicated. Lane 2: 3T3 cells transfected with the neomycin-resistance gene alone. Lanes 3 and 4: extracts of 3T3 clones transfected with I 6 and I 10, respectively showing co-migration of their products with HLA-B27k and HLA-B27w, respectively.
yond the alpha-3 domain (Ezquerra et al., 1985). From I 10 and I 6, the remainder (transmembrane and cytoplasmic portion) of the amino acid sequence may be deduced, and no features remarkably different from any of the class I sequences determined to date (Jordan et al., 1985; Sood et al., 1985) are apparent. The translation termination codon is located at the end of exon 7, at the same position as was found for HLA-B7 (Sood et al., 1985). Peculiarities of the amino acid sequence for the first three extracellular domains have been commented on previously (Ezquerra et al., 1985). Inspection of the nucleotide sequences of I 10 and I 6 reveals several interesting features. Only four nucleotide exchanges are seen in close juxtaposition, each of which would lead to an amino acid replacement. Two of these exchanges have taken place in a single codon (codon 80) and therefore the net result is a predicted difference of three amino acids at positions 77, 80 and 550
81, changing them from Asp to Asn, Thr to Ile and Leu to Ala, respectively (Figure 2). Since one of the predicted amino acid replacements involves an Asp to Asn substitution, the I 6 product will have one acidic charge less than the I 10 product. This is in agreement with the results of IEF, which show the HLA-B27k product to be approximately one charge unit more basic than the HLA-B27w product (Molders et al., 1982; Figure 3). It is of interest to note that this very same region of exon 2 constitutes a mutational site in the H-2kbm mutants bm3, bml 1 and bm23 (Nathenson et al., 1986), directly implicating this portion of the class I chain in T-cell recognition in man and mouse. We further note that, as far as determined, no other nucleotide exchanges are seen between I 10 and I 6 for the remainder of the sequence. Thus it is likely that, in evolutionary terms, the separation of HLA-B27w and HLA-B27k is a recent event. The difference between the genes encoding HLA-B27w and HLA-B27k is strikingly reminiscent of those observed between the H-2kb gene and its spontaneously derived bm mutants. In both cases, the only nucleotide exchanges present are clustered, which has been explained in the mouse by block-wise exchange of DNA in a gene conversion-like event. Therefore very similar mechanisms are likely to be operative to generate polymorphism in class I genes in mouse and man. In the mouse, potential donor genes could be identified at a considerable distance from the H-2K locus in the H-2b haplotype. In the study of HLA class I genes, such efforts would undoubtedly suffer from the fact that we do not know in which individual, or when, such events may have taken place. In addition, subsequent recombination events may have amalgamated potential donor genes with the HLA gene pool of the population. The nucleotide sequence of HLA-A24 (N'Guyen et al., 1985) contains a 92-bp segment (Figure 4, boxed) spanning the region which differs between HLA-B27w and HLA-B27k with only a single base pair mismatch in codon 77 between HLA-B27k and HLA-A24, and of lower homology up- and downstream from this segment. Stretches of identity straddling the blocks of sequence that were exchanged in gene conversion-like events have been found in the H-2kbm mutants and their putative donor genes. The most frequently encountered bm mutations are found associated with the longest stretch of homology (Nathenson et al., 1986). Such extended segments of sequence identity are thought to promote gene conversion. Although it cannot be proved that HLA-A24 was actually involved in a gene conversion-like event to generate HLA-B27k from HLA-B27w, the HLA-A24 sequence does demonstrate that class I genes that could have participated in this event do exist. The single (silent) base pair exchange in codon 77 might then have occurred subsequent to the presumed gene conversion. Our observations suggest that not only do gene conversionlike events take place in a manner similar to that postulated for the H-2kbm mutants, but that for the first time it is clear that variants thus generated can spread successfully in the population. Such genetic exchanges might take place in the population multiple times, and independently, involving the same or similar sequences (Nathenson et al., 1986). This would help to explain the lack of sequence divergence between 10 and I 6, which are both members of an extremely polymorphic gene family. Even though in Caucasians the frequency of HLA-B27k is 10% of the HLA-B27 positive population, we do not know whether all HLA-B27k individuals carry the identical genes derived from a single mutational event and transmitted in Mendelian fashion. To resolve this issue, multiple sequences for independently iso-
Polymorphism generation in human class I genes
B27w B27k A24 A2 A3 Cw3
B27w B27k A24 A2 A3 Cw3
B27w B27k A24 A2 A3 Cw3
30 20 10 agGC TCC CAC TCC ATG AGG TAT TTC CAC ACC TCC GTG TCC CGG CCC GGC CGC GGG GAG CCC CGC TTC ATC ACC GTG GGC TAC GTG GAC GAC
--T -
-
---__
_-
_-
_-
---_
_-
----
--- --- --- --- --- ---
----
--- --- --- --- --
---
-- TC--- TT--- TT--- TG-
--A ----A ----A ----- G--
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___ __ __
_-
---__
_-
-~~~~~~G
--_
---__ --
---__ --_
--- --- --- --- --- --- --- --- --- --- --- ---
__
--- --- --- --- --- --- --- --- --- --- --- --- ---
60 50 40 ACG CTG TTC GTG AGG TTC GAC AGC GAC GCC GCG AGT CCG AGA GAG GAG CCG CGG GCG CCG TGG ATA GAG CAG GAG GGG CCG GAG TAT TGG -GC -A-A-- - -A --- -A ---
--- --- C-
-
_
--- - -- C-
-
_
--- --- C-
-
_
--- --- C-
GAC CGG GAG ACA CAG __ GA- --- --- GG---G-A-G---
--- --- --- --- ---
---
---_
---_
-A- --G AT--C -A- --G AT--- --- --C -A- --G AT-A- -A- --- --- --- -G--- --- ---
--- --- --- ---
--- ---
--- --- --- ---
_-
-------- --
T
--- --- --- ---
--- --- --- ---
--- --- --- --- --- ---
G-G --- -G- A--
--- --- --- --- ---
90 80 70 ATC TGC AAG GCC AAG GCA CAG ACT GAC CGA GAG GAC CTG CGG ACC CTG CTC CGC TAC TAC AAC CAG AGC GAG GCC --- --- --- --- --- --- A-- --- --- - T- GC - --- --- --- --- --- --- --- --- ---AA GTG --- --- C-C T-- --- --- --- --- --- A-G --- --- -T- GC- --- --- --- --- --- --- --- --- ---AA GTGC-C T-- --- --- C-- --- -T- --- --- G-- --- --- -G- G-- --- --- --- --- --- --- ---AT GTG --- --- C-- T-- --- --- --- --- -T- --- --- G-- --- --- -G- G-- --- --- --- --- --- --- ---- -T- AG-AG -A- --- C----A- --- -G- G-- --- --- --- --- --- --- --C
B27w Ggtgagtgaccccggcccggggcgcaggtcac tccccatcccccacgtac B27k A24 __ c --t- g-g ___________________ _ A2 .-------------------------------.ac-ti ----------9-t A3 g -----------------g-------------g-ac--- t-----_ ------Cw3 tcct g
-------------------------tacc-------------tg--
Fig. 4. Comparison of the HLA-B27 subtype sequences with HLA-A24, HLA-A2, HLA-A3 and HLA-Cw3. Exon 2 and part of intron 2 are compared. Upper case letters represent exon and lower case letters intron sequences. Splice donor and acceptor sites are underlined. Sequence differences with HLAB27w are indicated in the HLA-B27k and HLA-A24 sequences. The 92-bp segment, showing almost complete identity between HLA-A24 and HLA-B27k, discussed in the text, is boxed.
lated HLA-B27w and HLA-B27k determined.
genes
will have to be
Materials and methods Cell lines The human B-cell line BRUG (HLA-A3, HLA-AI 1; HLA-B27k, HLA-B27w; HLA-Cwl, HLA-Cw2) and the mouse 3T3 cell line were grown in DMEM supplemented with 2 mM L-glutamine and 10% fetal calf serum. The cell line BRUG was kindly provided by Dr Pavol Ivanyi, Central Laboratory of the Blood Transfusion Service, Amsterdam, The Netherlands. Enzymes and reagents Restriction endonucleases, T4 DNA ligase, large-fragment DNA polymerase and alkaline phosphatase were purchased from BRL and Boehringer Mannheim. Radiochemicals were obtained from Amersham International. Construction of the genomic library High mol. wt DNA was extracted from BRUG cells following standard procedures (Maniatis et al., 1982). After partial digestion with MboI and phosphatase treatment, the genomic DNA was ligated into the BamHI sites of the EMBL 3 lambda vector (Frischauf et al., 1983). The recombinant phage were packaged in vitro (Maniatis et al., 1982) and used to infect Escherichia coli NM 539. The library contained about I x 106 independent recombinants and was screened without further amplification as described below.
as
Isolation of HLA-B27 genes The genomic library in EMBL 3 was screened by hybridization of filters at high stringency (68°C; 0.1 x SSC) with the HLA-B locus-specific probe pHLAl-l (Koller et al., 1984). Phage plaques hybridizing to this probe were plaque-purified three times. Phage DNA was prepared by standard methods (Maniatis et al., 1982). DNA prepared in this way was used to construct restriction maps following standard protocols and DNA probes specific for different regions of a human class I gene.
DNAn-mediated gene transfer and biochemical characterization ofthe gene products Transfection of mouse 3T3 cells with I 10 and I 6 DNA was carried out using DNA-mediated gene transfer and co-transfection with the neomycin-resistance gene (Southern and Berg, 1982). After selection for neomycin-resistant clones, detergent extracts were prepared, digested with neuraminidase (Molders et al., 1982) and analysed on IEF gels (Neefjes et al., 1986). Proteins were transferred to nitrocellulose by electroblotting (Towbin et al., 1979). The blots were developed with the monoclonal antibody HC 10 raised against a mixture of purified denatured HLA-B7 and HLA-B40 heavy chains. On blots HC 10 reacts preferentially with B locus products (Stam, Neefjes and Ploegh, unpublished results). Blots were developed using standard procedures (Towbin et al., 1979). DNA sequencing The recombinant phage clones were subcloned in the pUC 18 plasmid vector using the restriction endonucleases PstI and SstI. Sequencing was performed using the 3' end-labelling protocol (Maniatis et al., 1982), followed by the chemical degradation method according to Maxam and Gilbert (1980). 551
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Acknowledgements This research was supported in part by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Immunogenetik) and the Nierstichting Nederland.
References Breuning,M.H., Breur,B.S., Engelsma,M.Y., Huis,B. and Ivanyi,P. (1983) Tissue Antigens, 22, 267-282. Ezquerra,A., Bragado,R., Vega,M.A., Strominger,J.L., Woody,J.N. and Lopez de Castro,J.A. (1985) Biochemistry, 24, 1733-1741. Frischauf,A.M., Lehrach,H., Poustka,A. and Murray,N. (1983) J. Mol. Biol., 170, 827-842. Jordan,B.R., Caillol,D., Damotte,M., Delovitch,T., Ferrier,P., Kahn-Perles,B., Kourilsky,F., Layet,C., Le Bouteiller,P., Lemonnier,F.A., Malissen,M., N'Guyen,C., Sire,J., Sodoyer,R., Strachan,T. and Trucy,J. (1985) Immunol. Rev., 84, 73-92. Koller,B.H., Sidwell,B., DeMars,R. and Orr,H.T. (1984) Proc. Natl. Acad. Sci. USA, 81, 5175-5178. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY. Maxam,A.M. and Gilbert,W. (1980) Methods Enzymol., 65, 499-560. Molders,H.H., Breuning,M.H., Ivanyi,P. and Ploegh,H.L. (1982) Hum. Immunol., 6, 111-117. Nathenson,S.G., Geliebter,J., Pfaffenbach,G.M. and Zeff,R.A. (1986) Annu. Rev. Immunol., in press. Neefjes,J.J., Breur-Vriesendorp,B.S., van Seventer,G.A., Ivanyi,P. and Ploegh, H.L. (1986) Hum. Immunol., in press. N'Guyen,C., Sodoyer,R., Trucy,J., Strachan,T. and Jordan,B.R. (1985) Immunogenetics, 21, 479-489. Sood,A.K., Pan,J., Biro,P.A., Pereira,D., Srivastava,R., Reddy,V.B., Duceman, B.W. and Weissman,S.M. (1985) Immunogenetics, 22, 101-121. Southern,P.J. and Berg,P. (1982) J. Mol. Appl. Genet., 1, 327-341. Towbin,H., Staehlin,T. and Gordon,J. (1979) Proc. Natl. Acad. Sci. USA, 76, 4350-4354.
Received on 16 December 1985; revised on 13 January 1986
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