one of the cosmid clones containing both SBa and SB,B genes. Expression of the a and ( genes was detected byfluor- escene-activated cell sorting (FACS) and ...
The EMBO Journal vol.4 no.3 pp.739-748, 1985
SB subregion of the human major histocompatibility complex: gene organization, alielic polymorphism and expression in transformed cells
Kiyotaka Okada', Holly L.Prentice, Jeremy M.Boss, Daniel J.Levy, Dietmar Kappes, Thomas Spies, Rajgopal Raghupathy, Rosemarie A.Mengler, Charles Auffray and Jack L.Strominger Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, USA 1Present address: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan Communicated by A.Rich
The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and X phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two a genes and two (3 genes are encoded in the 100 kb long SB region in the order SBaSB(3-SXa-SX(3. The SBa and SB(3 genes encode the a and ( subunits of the SB subset of class II MHC molecules. Both the SXa and the SX,B genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB,B and SXY3 genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SBa and SB,B genes. Expression of the a and ( genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies. Key words: HLA complex/major histocompatibility complex/ transfection/polymorphism/gene structure
Introduction The MHC class II molecules are heterodimers of transmembrane glycoproteins, consisting of a heavy or a subunit and a light or (3 subunit, and are located on the surface of B cells, activated T cells and macrophages. The molecules are a key element in the control of the immune response to self and non-self antigens functioning in cell-cell interactions and antigen presentation to regulatory T lymphocytes (reviewed in Benacerraf, 1981; Shackelford et al., 1982; Hurley et al., 1983; Steinmetz and Hood, 1983; Kaufman et al., 1984; Melchers and Andersson, 1984). In man, three distinct groups of class II molecules, DR, DC and SB, have been identified by immunological and biochemical studies, and mapped to the HLA-D region on chromosome 6. In mouse, two class II antigens, I-A and I-E, have been mapped to the H-2 I region on chromosome 17. The molecules are highly polymorphic. The polymorphisms detected serologically and functionally are mainly due to amino acid sequence changes in the (31 domain of the (3 subunits although the a subunit of DC is also polymorphic. Several genomic DNA clones encoding ai and ( genes of the three subgroups of human class H molecules (as well as some corresponding cDNA clones) have been reported (Korman et al., 1982; Das et al., 1983a, 1983b; © IRL Press Limited, Oxford, England.
Lee et al., 1982; Auffray et al., 1993a, 1984; Larhammar et al., 1983; Schamboeck et al., 1983; Trowsdale et al., 1983, 1984; Schenning et al., 1984; Boss and Strominger, 1984; Kappes et al., 1984; Gorski et al., 1984; Inoko et al., 1984). Analyses of the clones and Southern blot hybridizations with isolated cDNA clones as probes show that the HLA-D region has many genes, at least six a genes and seven ( genes (Wake et al., 1982; Arnot et al., 1983; Auffray et al., 1983a, 1983b; Bohme et al., 1983; Erlich et al., 1983; Owerbach et al., 1983; Cohen et al., 1984; Spielman et al., 1984; Inoko et al., 1984), as compared with only two a and two to four (3 genes in the mouse (Hood et al., 1983). The organization of the genes is, however, not well understood, since the number of clones is limited and the clones are derived from different cell strains. In order to investigate the genetic structure of the HLA-D region and to study the nature of the polymorphism, overlapping clones from a genomic library must be collected and linked, if necessary, by the chromosome-walking method. A genomic library from the typed B-lymphoblastoid cell line, Priess (DR4/4, DC4/4, SB3/4) (Thomsen et aL., 1975; Hurley et al., 1982) was constructed and screened with class H cDNA probes. Many overlapping clones which cover the HLA-D region were isolated. This paper presents two physical maps of the complete SB subregions which correspond to the SB3 and SB4 haplotypes of this cell line. The entire 100-kb region derived from 11 cosmid and seven phage clones contains SBa and SB,B genes as well as additional a and (3 genes termed SXca and SX,B. Some sequence information on each of these genes has been obtained. A partial linkage map covering 60 kb of this region including three clones from two different haplotypes has recently been reported (Trowsdale et al., 1984); several cosmid clones containing these genes have also been detected in other laboratories without linkage of the second SX( gene (Gorski et al., 1984; Inoko et al., 1984). Restriction site polymorphisms between SB3 and SB4 DNAs have been observed and localized. Moreover, one of the clones carrying the SBca and SB(3 genes was transfected into a mouse macrophage cell line, and expression of the genes on the cell surface was detected.
Results Isolation of cosmid clones carrying SBax and SB(3 genes Cosmid libraries of 106 clones were screened by colony-filter hybridization with nick-translated cDNA fragments of SBa and SB(. Positive clones were isolated and analyzed by blot hybridization using a set of cDNA clones derived from ca and (3 genes of the three known subtypes (DR, DC and SB). Those clones which showed strongest hybridization with SB cDNA fragments under stringent washing conditions were tentatively considered to be SB clones. Finally, 11 cosmid clones of this type were isolated and analyzed further. The other clones which hybridized weakly with SB cDNA fragments and strongly with DR or DC fragments were studied separately and identified later as belonging to DR or DC subtypes, respectively. The SB clones 739
K.Okada et al. Okb
10
20
5I
I
SB. 4-
3'UTITM
30
40
60
50
I
I
SB1
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a2E1SS/5'UT 5'UJT/SS 013
SX@
70
80
90
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I
I
100
SXp
4 .2l1t3mSU -
52TMs3SJTj3ja2sjSS/5'UT
5'UT/SSPg
PzTM3UT i
>o
polynwpic reio
polymorphic region
61II BamHI' II I* I I II I I I§ *1 4'"t' ' ClO I I EcoRI' * I I IH II I1 ',* 1 11111 1,,,1 1 111 * * . EcoRI~~~~~~~~~~E EcoRV ^ I I I II , 1 1 1 I. KpnI '''I I I I SoIl t * SstI I 11111 , ,I Ii 1p ' XbaI I I 1 1I I It i * ' I XhoI j = Da r= E B0 C G17A
HIA VIIA
L GOA BIBA e
B9J
L
A206' X208 X202 'X203 X211 X205 X204A -
TIOB S28
I
H14D '
-
84A
Fig. 1. Molecular maps of SB subregion for SB3 and SB4 haplotypes. The length of the region (kb) is shown on the top line. On the second line, four genes mapped. Solid boxes indicate exons. For SBa and SX,B genes, approximate sites for exons are shown by open boxes. Arrows indicate direction of transcription 5'-3'. Restriction sites for nine enzymes (BamHI, ClaI, EcoRI, EcoRV, KpnI, Sall, SstI, XbaI and X1oI) are shown by vertical bars. Bars above horizontal lines indicate restriction sites in SB3 DNA and those below in SB4 DNA. Polymorphic regions which show different restriction sites between SB3 and SB4 DNAs are indicated. The ends of the cloned region are indicated by e; DNA fragments indicated as * and ** have slightly different sizes as described in Table I. At the DNA region marked +, five small XbaI-digested fragments of 1.1 kb, 0.85 kb, 0.55 kb, 0.4 kb and 0.3 kb are mapped. Their order is unknown. Small open boxes A-E indicate DNA fragments used as hybridization probes. Fragment A (1.6 kb) is derived from EcoRV and PstI digest of TIOB clone, fragment B (0.6 kb) and fragment C (1.2 kb) from Kpnl and XbaI digest of TIOB clone, fragment D (0.4 kb) from KnpI and Sall digest of G8A clone, and fragment E (4.5 kb) from XhoI and Sall digest of H14D clone. Sll enzyme cleaves the genomic DNA fragment from vector DNA. These fragments do not contain repetitive sequences. Overlapping cosmid and X phage clones are given below the restriction maps. Seven cosmid clones shown at the upper part represent SB3 DNA, and four cosmid clones in the lower part, SB4 DNA. The seven X phage clones shown in the middle are not identified by SB type, because they do not cover polymorphic regions. are
were separated into two groups from restriction mapping and blotting data. One group of eight clones (K18A, G17A, G8A, B18A, B9J, T1OB, S2B and B4A) spans a DNA stretch of 50 kb and encodes two a genes and one gene (Figure 1). The other group of three clones (H14D, HIA and V1 lA) covers a 50 kb length and has one gene as described below. Linking the whole region Since the two groups of cosmid clones did not overlap, the chromosome walking technique was applied to link them. H14D cloned DNA was digested with XhoI and Sail restriction enzymes. A 4.5-kb fragment which is located at the left end of the insert in H14D (shown as open box E in Figure 1) was isolated and used as a probe to screen the cosmid libraries, but no positive clones were found. A genomic library in X phage vector constructed from the same cell line (Priess) used to make the cosmid libraries was then screened. Seven clones (X202, X203, X204, X205, X206, X208 and X21 1) were isolated. These clones contain DNA stretches overlapping not only with the left end of the H14D clone, but also with the right end of clones of the first group (Figure 1). The total length of the region covered with 740
the 11 cosmid and seven X phage clones is 100 kb. Two of the cosmid clones, B4A and H14D, abut or nearly abut each other. -
Identification
and sequencing
of two
chain genes, SB13 and SX1
The cloned region has two genes. The following results indicate that the gene on the left side as drawn encodes the subunit of the SB determinant. The other gene on the right is closely related to the SB,B gene in structure and was named SX1. The SB1 gene hybridizes more strongly with the SB13 cDNA fragment than does the SX13 gene. Figure 2 shows blot hybridization patterns of TIOB (SB1) and H14D (SXO) clones, as well as several clones identified as DR,8 and DC,B clones (Okada et al., unpublished results). Two HindII fragments of 7.4 kb and 1.8 kb from the TIOB clone and a 5.7-kb fragment from the H14D clone hybridized with the probe, whereas DR,B and DC: clones hybridized very weakly under stringent washing conditions. Nucleotide sequences of each of the two genes, SB1 from the G17A clone and SX1 from the H14D clone, were analyzed and compared with SB1 cDNA sequences (Roux-Dosseto et al., 1983; Kappes et al., 1984). The entire coding sequences of the
SB subregion of the human MHC
SB( gene on the G17A clone have been determined, as well as the promoter region and the 5'- and 3'-untranslated regions. This gene encompasses 11 kb and consists of six exons, corresponding to the 5'UT/SS, (31, (2, TM, CY and 3'UT sequences located as shown in Figure 1. The first and second introns (4.4 kb and 6 kb) are unusually large for a class II ( chain gene. The amino acid sequence deduced from the nucleotide sequence of the G17A clone is identical to the limited N-terminal amino acid sequence of the SB( molecule of the SB3 haplotype (Figure 3). This result strongly suggests that the SB(3 gene on the G17A clone carries the SB3 phenotype. The nucleotide sequences of the SB(3 genomic clone from the SB3 haplotype and SB(3 cDNA clone from a SB2 haplotype show 36 base differences out of 1079 bases compared (Kappes et al., 1984). The signal sequence peptide, transmembrane and cytoplasmic domains are identical. The 3'UT region contains 16 differences, while (31 and (32 domains have 13 and seven, respectively (although five out of seven in (32 are silent while all 13 in (1 produce amino acid differences). Therefore, the amino acid sequences are quite similar throughout all protein domains except (31. These changes occur because the clones encode alleles of the SB( molecules, i.e., the genomic DNA
1
2
4
3
4.W
5.7
1.8
_rn
is SB3( and the cDNA is SB2(. The SX(3 gene appears to be very large (up to 16 kb) and is located as shown in Figure 1. A 6-kb BamHI fragment located at the center of the SX( gene region does not hybridize with either the 5' half (5' UT to (31 sequence) or the 3' half ((32 to 3'UT sequence) of the SB,B cDNA probe, indicating an extraordinarily large intron between the (31 and (32 exons. Nucleotide sequences of the (2 domain exon show higher homology with the SB(3 gene than with DR(3 or DC,B genes (Kappes et al., 1984). The SX( gene differs from the SB( gene at 19 out of 285 nucleotides encoding the (2 domain, resulting in 13 amino acid substitutions. However, the SX(3 gene on the H14D clone seems to be nonfunctional since it has a one-base deletion which causes a frameshift and results in a stop codon within the (32 domain in the new reading frame (Kappes et al., 1984). Moreover, even ignoring the base deletion, one of the cysteine residues needed to form a disulfide loop has been mutated. The SB(3 and SX( genes show the same direction of transcription from left to right as shown in Figure 1. The SB(3 gene is transcribed and expressed in lymphoblastoid cells, but transcription of SX( has not yet been detected. Identification and sequencing of two a chain genes, SBai and SXa Two a genes as well as two (3 genes were mapped in the cloned region. One gene located at the left end of the region encodes the a chain of the SB molecule. The other gene located at the center, named SXa, is closely related to the SBca gene from blot hybridization and DNA sequence analyses. Figure 4 shows an example of blot hybridization of EcoRV-digested clones with a SBat cDNA fragment as the probe. A 12-kb fragment of the K18A, T1OB and G17A clones which contains the SBca gene hybridizes more strongly with the probe than with the 8-kb fragment of G17A, S2B and G8A clones which contains the SXa gene. The 8-kb fragment is truncated in the TIOB clone, and not present in the K18A clone. The sizes of the fragments encoding the SBa gene in the S2B and G8A clones are increased by fusion with vector DNA. Both SBa and SXa genes have the same direction of transcription, from right to left in Figure 1. The SBa gene encompasses 7 kb and has a large intron of 3 kb between the a2 and 3' UT exons. As for the SXa gene, four exons corresponding to the al, a2, TM and 3'UT domains are encoded in 3 kb of DNA. DNA sequences homologous to the highly conserved class II upstream promoter sequences are 5S kb upstream from the a 1 domain. The c2 domains of SBa and SXa genes from the G8A clone were sequenced and compared with the SBa cDNA sequence (Auffray et al., 1984). The SBa gene is identical to SBa cDNA sequences (Figure 5). SXoa has 55 nucleotide differences -
-
Fig. 2. Blot hybridization of class II ,3 gene-containing cosmid clones. Clones were digested with HindlI and hybridized with SB,B cDNA fragment containing (32 to 3'UT domain sequences. The filter was washed with 0.5 x SSc, 0.05% SDS at 65°C. Lanes: (1) H14D clone which has the SX(3 gene; (2) T8B (DR,8 gene); (3) T28L (DC( gene); (4) TIOB (SBI3 gene). Fragment sizes are shown in kilobases (kb).
Residue # 7 8 9 11 16 18 24 28 30 36 55 56 57 65 76 84 85 86 8 7 - Y _ F F Y Y FYn V Y D D L D V Y Y E V E A F F L V
'
-
-
-
-
-
-
-
L F G Y F F Y Y A A A E I M G G P M L F G Y F F Y Y A A A E I M G G P M L IFj G Y F F YY A A A E I M G G P M
SB type 3 3 4 4 4
References protein (Hurley et al, 1982) genomic DNA (G17A clone)
genomic DNA (TIOB clone) cDNA (Gustafsson et al, 1984) genomic DNA (Gorski et al, 1984)
Fig. 3. Comparison of amino acid residues. The first lane shows tyrosine (Y) and phenylalanine (F) residues in the SB,B subunit of SB3 typed molecules. Unassigned residues are indicated by -. The second to the fifth lines show amino acid sequences deduced from nucleotide sequences. Tyrosine and phenylalanine residues are boxed. 741
K.Okada et al.
(Figure 5) with the SBa cDNA resulting in 28 amino acid changes (Figure 6). A comparison between the SBa cDNA amino acid sequence and the SXcx, DRa and the DCca sequences show that each of these genes are as different from SBax as they are from each other, having 28, 28 and 31 differences, respectively. The entire sequence of the SXa gene from the G8A clone has been completed (J.Boss, in preparation) and several abnormalities were found which suggest that SXa, like SX,B, is a pseudogene. These abnormalities include frameshift mutations in the eatl and transmembrane exon as compared with SBct which would cause an aberrant protein to be made. Additonally, a splice donor mutation is present at the end of the adl exon. It should be pointed out, however, that both the SXa! and SX,3 genes sequenced are from a single haplotype (SB3, see below), leaving open the possibility that SXa and/or SX3 may be intact expressed genes in other haplotypes. Allelic polymorphism of the SB region Since the Priess cell line has been typed to be heterozygous for
1
2
3
4
5
SB in its two haplotypes, i.e., SB 3/4 (Hurley et al., 1982), it is expected to have DNA fragment size polymorphism in the SB region on allelic chromosomes. Genomic blot analysis of HindI digested cellular DNA with a probe of SB,B cDNA containing 32 to 3'UT sequences (pHAfl) hybridized with four bands (8.3 kb, 6 kb, 5.6 kb and 1.9 kb) in Priess and three bands (8.3 kb, 6 kb and 1.9 kb) in three different SB4 homozygous cell strains, indicating that the 5.6-kb Hindl fragment is associated with the SB3 haplotype and the 6.0-kb fragment with the SB2 and SB4 haplotypes (Roux-Dosseto et al., 1983). (The sizes of the HindIf fragments previously reported, 8.3 kb, 6 kb, 5.6 kb and 1.9 kb, have been corrected here to 7.4 kb, 5.7 kb, 5.3 kb and 1.8 kb, respectively). Similar observations were also made in a population study using peripheral blood cells from SBtyped individuals (Roux-Dosseto et al., 1983; see also Gorski et al., 1984; Robinson et al., 1984). As shown below, however, analyses of the cosmid clones showed that the 5.3- and 5.7-kb HindIll polymorphic bands are derived from SX(3, the pseudogene in this cell line, while the 7.4-kb and 1.8-kb bands, nonpolymorphic with Hindu, are derived from the gene associated with the polymorphic determinant observed in cellular typing. Among the cosmid clones, the H1A and V llA clones have a 5.3-kb HindIll fragment (derived from SXO) which hybridizes with the same SB3 cDNA probe that was used for genomic DNA hybridization, whereas H14D has a 5.7-kb fragment (Figure 7a). It is reasonable to conclude, therefore, that the H1 A and VI1 A clones derived from chromosomes of the SB3 haplotype, and H14D from the SB4 haplotype. In addition to HindIll, restriction site polymorphisms for BamHI, EcoRI, KpnI, SstI and XbaI were seen at the SX,B gene locus (see Figure 1 and Table IB). All the detected polymorphic sites are focused in a small region of 11 kb from 3 kb upstream of the fl2 exon to -4 kb downstream of the 3' UT exon. No restriction site polymorphisms were found in the 5' part of the gene. Restriction site polymorphisms were also found in the SB,B gene region. For example, when digested with the EcoRV enzyme, three clones (TIOB, S2B and B4A) generated 7-kb and 3.5-kb fragments which hybridize with the SB,B probe, whereas four clones (G17A, G8A, B18A and B9J) generated 10.5-kb fragments. The 10.5-kb fragment of the K18A clone is truncated to 9 kb (Figure 7b). Other enzymes such as BamHI, EcoRI, SstI and XbaI also show restriction site polymorphisms between the -
Fig. 4. Blot hybridization of clones containing SBa and SXa genes. Clones were digested with EcoRV. The SBa cDNA fragment having 5'UT to al domains was used as a probe. Lanes: (1) K18A. (2) TIOB; (3) G17A; (4) S2B; (5) G8A.
-
AT CCC CCT GAG GTG ACC GTG TTT CCC kAG GAG CCT GTG GAG CTG GGC CAG CCC kAC ACC CTC ATC TGC CAC ATT GAC AAG TTC TTC CCA A) SB cDNA B) SB genohio -T G------------C) SX genomic CC -A-C -- A -G- -C -------------T--- ------------G--
A)
CCA GTG CTC AAC GTC ACG TGG CTG TGC AAC GGG GAG CTG GTC ACT GAG GGT GTC GCT GAG AGC CTC TTC CTG CCC AGA ACA GAT TAC AGC TTC CAC
B) C)
-----G---A- -------T------CA----TT -----
A)
AAG TTC CAT TAC CTG ACC TTT GTG CCC TCA GCA GAG GAC TTC TAT GAC TGC AGG GTG GAG CAC TGG GGC TTG GAC CAG CCG CTC CTC AAG CAC TGG G
A-T --A
---------
-C
-AG A-ACT- -A
---
---
B) C)
-G- ---C T--- --
C-C -T --- ATG --C
AC- -G-
CT- CA- -G-
C- C-
-T
C-
Fig. 5. Comparison of nucleotide sequences of the SBa-like genes. Nucleotide sequences of the a2 domains are compared. SBa cDNA was derived from LB cells (Auffray et al., 1984). SBa genomic and SXa genomic DNAs are derived from the G8A clone (see text). Identical sequences are indicated by -. SB cDNA SB genomic SX genomic DR DC-1 eDNA
DPPEVTVFPKEPVELGQPNTLICHIDKFFPPVLNVTWLCNGELVTEGVAESLFLPRTDYSFHKFHYLTFVPSAEDFYDCRVEHWGLDQPLLKHW
I------P-I--I--TI---SKKLR--R-----L--M--TCDLQG----H---R-R -T--S- - -D ------ V--V- V- -LTNS---RE--V--F---T--V----R--KP--T--S-TV----E-HL-R-----P-L--T--V ---- E- -__ WU__
-U--T_R__RT_.RRR-T--T-nT _T ----T___l -
-------
Fig. 6. Comparison of the amino acid residues of the a chains. a2 domain sequences deduced from cDNA and genomic DNA sequences were compared. References are: SB cDNA (Auffray et al., 1984), DRca (Schamboeck et al., 1983), DC-Ila (Auffray et al., 1982), SBa and SXa (G8A clone, see text).
742
~~
SB subregion of the human MHC
1
2
_
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3
4
1
3
2
~~~~~~~~~~ i
p
5-.3
5
4
.-10.5
4111
.ur.
Immmoomppl-
7.0
3.5
-1.8
A
B
Fig. 7. Blot hybridization of SB( and SX(3 cosmid clones. The probe is a SB(3 cDNA fragment containing (32 to 3'UT domains. (A) Clones were digested with HindIII. Lanes: (1) G17A. (2) TIOB; (3) HIA; (4) H14D. (B) Clones were digested with EcoRV. Lanes: (1) S2B; (2) G8A; (3) G17A; (4) TlOB; (5) K18A.
Table 1. Restriction site polymorphism between SB3 and SB4 clones Restriction enzyme BamHI EcoRI
(A) SB,B gene region SB3 SB4 4.7 kb 1.6 kb
4.8 kb 1.65 kb
Covering $2-TM Covering the intron between 5'UT/SS and (1
HindIII KpnI
7.0 kb An additional site in (2 3.5 kb 7.3 kb 7.4 kb Covering 02-3'UT No polymorphism observed
SstI
16 kb
XbaI
2.3 0.7 0.4 0.3
EcoRV
(B) SX,B gene region SB3 SB4
Location
10.5 kb
kb kb kb kb
10 kb 3.8 kb 2.4 kb 2.35 kb 0.7 kb
Two additional sites in
02-3'UT
Covering (32-TM
9.0 kb 9.2 kb Covering (32-3'UT 7.1 kb 7.5 kb Covering (32 2.7 kb 4.0 kb 0.85 kb No polymorphism observed kb kb kb kb kb
5.7 kb 30.5 kba
Covering (32-3'UT An additional site downstream of 3'UT
11 kb
An additional site downstream of 3'UT
2.7 kb 1.8 kb
2.9 kb 1.6 kb 0.3 kb
5.3 26.5 6.5 9.5 4.5
Approximate sizes of polymorphic fragments on SB3- and SB4-typed clones are listed. aFragments truncated on cosmid H14D (see Figure 1). SB3- typed clones are K18A, G17A, G8A, B18A, B9J, S2B, B4A, H14D.
two groups of cosmid clones (see Figure 1 and Table IA). In the case of HindIII, detailed analysis shows that the 7.4-kb fragment of the TIOB clone is slightly larger than that of the G17A clone (Figure 7a and Table IA); their 1.8-kb fragments appear to be exactly the same size. All of the polymorphic sites are restricted to one section of the gene, namely, from the intron between the signal peptide and the , 1 domain exons to the exon
encoding the 3'-untranslated region. As mentioned above, the amino acid sequence deduced from the nucleotide sequence of the SB1 gene in the G17A clone is identical to the amino acid sequence of the SB1 molecule isolated from Priess cells (Hurley et al., 1982). Since the monoclonal antibody used for immunopurification of the SB molecule (ILRI) is specific for the SB3 molecule and does not recognize SB4,
Location
Covering (32
HIA, VIIA.
SB4 typed clones are
TIOB,
the haplotype of the SB1 gene of the G17A clone, and clones of the same group (K18A, B18A and B9J), should be the SB3 allele, and that on clones of the other group (TlOB, S2B and B4A), should be the SB4 allele. The complete nucleotide sequence of the SB1 gene from the TIOB clone has been determined (Kappes et al., in preparation). The deduced amino acid sequence in the 131 domain is different at position 9 from the SB3 protein sequence and the sequence deduced from the G17A clone (TyrPhe) (Figure 3). From the nucleotide sequence, 12 other positions (Figure 3) are also different from those of the SB31 allele in the G17A clone but identical to 13 chain sequences of the SB4 type (Gustafsson et al., 1984; Gorski et al., 1984). Are there restriction site polymorphisms in the SBa and SXa gene regions or in the intergenic regions? Restriction site maps 743
_ =. r-.
K.Okada et al.
1
3
2
1
4
2
4
3
2
1
3
.
1
4
2
3
4
-1.2
-0.8
-r &i
-o0.25
A
B
-0.6 - 0.4
-0.2 -0.1
C
D
Fig. 8. Blot hybridization of SB cosmid clones with genomic probes isolated from intergenic spaces. Clones were digested with Sau3A to completion. Small genomic DNA probes (A-D) were isolated from cosmid clones as described in the legend of Figure 1. Probes are: (A) fragment A; (B) fragment B; (C) fragment C; (D) fragment D. Lanes: (1) S2B (SB4 haplotype); (2) G8A (SB3); (3) TIOB (SB4); (4) K18A (SB3).
A
r wTu 39 COntrol r r r ::=
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Fig. 9. FACS analysis of SB expression in a transfected mouse macrophage line. In panels A and B, the staining of a transfected macrophage line with fluorescein-conjugated goat anti-mouse IgG is shown for cells which either have (Tu39) or have not (control) been incubated with the anti-SB monoclonal antibody Tu39. Panel A: 1 month after the initial transformation. The brightest 10% of these cells were sorted out and grown. Panel B: the sorted 10% after 3 weeks growth. Cell sorting and analysis were performed on a Coulter Epics V sorter. The untransformed parental cell line gave identical FACS profiles with or without pre-incubation with Tu39 (not shown).
744
SB subregion of the human MHC
- SDS- PAGE AW
L
_] L
I
I
ULLJ I*
u
ft
U
I%' L:
"W
.II
+ B Fig. 10. Two-dimensional gel of SB antigens immunoprecipitated from a macrophage line transfected with the cosmid clone TIOB. Antigens were precipitated from a transfected macrophage line with two anti-SB monoclonal antibodies. Panel A: MHM4 (Makgoba et al., 1983), panel B: Tu39 (Pawelec et al., 1982) and panel C: untransfected cell line immunoprecipitated with Tu39. H: heavy or ca chains; L: light or chains; I: invariant or -y chains; U: unidentified.
with the nine enzymes shown in Figure 1 were identical in these regions for both SB3 and SB4 haplotype clones. The seven X clones, located in the center of Figure 1, have identical restriction sites with those of the overlapping cosmid clones. It might be difficult, however, to detect small size differences by comparing large DNA fragments digested with six-base recognizing restriction enzymes. Therefore, the four-base recognizing enzymes, Sau3A and TaqI, which produce very small DNA fragments were tested. Clones from the SB3 and SB4 haplotypes digested with these enzymes were blotted and hybridized with either an SBa cDNA probe or genomic DNA probes from intergenic regions A - D, shown in Figure 1, which detect unique sequences. Cosmid clones of the SB3 and SB4 haplotypes showed the same hybridizing patterns with all of these probes. Figure 8 shows blot hybridization patterns of the Sau3A-digested clones. These results indicate that the cloned sequences in the SB region from both SB3 and SB4 chromosomes from Priess cells have conserved sequences, except for the 3' part of the SB3 and SX,B gene regions. Obviously, the nucleotide sequences in the /31 domains of SB33 and SB4/ are also polymorphic, but a restriction enzyme site within this region was not found. Transformation of an SB clone into a murine macrophage line The macrophage line, P3M, was readily transformed by the TIOB cosmid and fluorescence-activated cell sorter (FACS) analysis demonstrated 40 % of the G418-resistant transformants to be weakly SB positive (Figure 9A). Several lines showed more than a single peak of fluorescence in the sorter indicating that either they were not isoclonal or that the introducing sequences had undergone rearrangement after transfection. Lines which were enriched for high expression by FACS showed correspondingly elevated expression levels after several weeks growth, so the varying levels of expression seen in the transformants was a stable -
property and not due to transient regulatory phenomena (Figure
9B). Transformants were initially tested with the monoclonal antibody Tu39. They were also immunoreactive with another SBspecific monoclonal, B7/21 (FA), and with IVA12, a monoclonal antibody recognizing all human class II dimers. The parental P3M line was negative to all of these. Two-dimensional gel analysis of immunoprecipitated material from the two lines analyzed demonstrated the presence of proteins migrating at positions expected for the authentic SBa and chains (Figure 10). Multiple species were noted, particularly in the ca chain region. The origin of this heterogeneity is not clear (see Discussion). An invariant chain spot, presumably the mouse invariant chain, was also precipitated by many of the anti-SB antibodies examined, suggesting association of the mouse invariant chain with the SB antigen similar to that found with its human equivalent. The transformation of this and other cell lines with SB cosmids will be described in detail elsewhere (Levy et al., in preparation). Discussion Genes for a and ,B subunits of SB molecules as well as two closely related pseudogenes, SXa and SX/, have been mapped to a DNA stretch of 100 kb. The cloned region seems to cover the whole SB locus determined genetically and immunologically, since (i) all of the genomic DNA fragments hybridizing strongly with SBoe and SB/3 cDNA probes are mapped in this region, and all of the genes appear to be complete on at least one cosmid, and (ii) SBot and SB/ genes mapped in this region have exactly the same amino acid sequences with those isolated from cells using monoclonal antibodies against SB molecules. The SB region has been mapped by immunogenetics between HLA-DR/DC and GLO-1 on 745
K.Okada et al.
chromosome 6 (Shaw et al., 1981; Kavathas et al., 1981; Termijtelen et al., 1983). The order of the genes is SBa-SB(-SXa-SX(3. The transcriptional directions of the genes are inverted with respect to neighboring genes, i.e., SBa (-), SB(3 (-), SXa (-), SX,B (-). Although the sequences of the exons are relatively conserved, the lengths of the introns are different when comparing SBa and SXa genes or SB,B and SX(3 genes; the introns in the SXa and , genes are much larger; the space between the SXa and SX( genes is larger than that between the SBa and SB(3. These observations suggest that: (i) one a and one (3 gene facing 5' to 5' make a structural unit; (ii) this unit has duplicated; (iii) one unit composed of SBa and SB( genes has remained functional; (iv) the other unit of SXca and SX,B genes accumulated mutations in its exons and both became pseudogenes; and (v) mutations have also accumulated in intergenic spaces and in introns, so that the two units differ in the length of their introns and the space between their a and (3 genes. About half of the SB region has also been cloned from other cell lines, HHK (Gorski et al., 1984) and AKIBA (Inoko et al., 1984). In both cells, three genes, SBa, SB(3 and SXa, are located in the same order and at approximately the same spacings found in the Priess cell line shown here. An SB region of 60 kb has also been cloned using a composite of clones from human placenta and lung carcinoma and shows a similar organization of the two SB-related a genes and the two SB-related (3 genes (Trowsdale et al., 1984). The orientation of transcription of the a and ( gene unit facing 5' to 5' (head to head) is unique in class II MHC genes. In mouse, I-A a and (3 genes as well as I-E a and ( genes face 3' to 3' (tail to tail) (Steinmetz et al., 1982). In man, other class II genes, DCca and DC(3 genes as well as the DXa and DX,B genes are closely linked in the tail-to-tail manner (Okada et al., in preparation). The structure of the ancestral unit of class II a and (3 genes changed from a tail-to-tail orientation to a head-to-head orientation when the SB loci generated. The clones were separated into two groups, one corresponding to the SB3 and the other to the SB4 haplotype. From comparison of the restriction maps for SB3 and SB3 DNAs, it was concluded that (i) restriction site polymorphisms are located in regions of -7 kb encoding the (31 to 3'UT exons of the SB,B gene and of 11 kb encoding the (32 to 3'UT exons of SX(, and (ii) polymorphisms were not seen in other regions, including the 5' ends of the SB( genes, the SBa and SXa genes, and the intergenic spaces so far tested with > 10 restriction enzymes. The first point is consistent with the observations that the structural polymorphism in class II MHC molecules is mainly due to amino acid changes in the (3 subunit. Why are the polymorphic sites restricted to small regions of the (3 genes, while other regions seem to be non-polymorphic? Possibly, the nucleotide sequences of the polymorphic ( gene region may have arisen from corresponding ( gene regions of other loci by a copy repair mechanism similar to gene conversion, or alternatively some structural features in the ( gene region on the chromosome might be recognized by a mutator-like molecular complex with accumulation of mutations. That the genes we have isolated encode the a and ( chains of the SB locus is indicated not only by the sequence data presented but also by the results of transfecting these sequences into a mouse macrophage line. When the cosmid clone TIOB was introduced into P3M cells by the calcium phosphate coprecipitation technique these cells produced surface proteins, recognized by anti-SB monoclonal antibodies, whose mobility on two-dimensional gel analysis was that of the authentic SB -
-
746
product. It is of note that the pattern of the a chain spots seen in the 2-D gel system was of greater complexity than that seen with cultured human lymphocytes. At least two minor species appear to be present (Figure 10). This pattern is not likely to be due to minor differences in the glycosylation of this chain (e.g., sialic acid variations) but major glycosylation differences are not excluded (for example, species containing only one of the two glycans found on ax chains). However, these results might also be due to the expression of genes which suffered truncations or underwent recombination during the integration process. Although the SXca gene may be a pseudogene, a recombination event involving this gene and/or the SBca gene is possible (Goodenow et al., 1982), as is expression of a mouse a chain gene in combination with the human SB(3 gene in the TIOB cosmid. It is also of note that a spot corresponding to the murine invariant chain appears on these gels (and on gels of immunoprecipitated material from other transfectants). This protein is presumed to be associated with the SB dimer since it does not bind to any of the sera tested and is not seen on gels of the untransformed parental line. Whether the murine invariant chain is required for surface expression of the SB antigen, as has been suggested for the human invariant chain, or whether its association is coincidental cannot be ascertained. The SB genes have also been introduced into mouse Ltk- cells and the presence of the invariant chain was again noted on 2-D gels of the transformants (Levy et al., in preparation). This may be particularly significant since L cells do not express murine class II genes. Several laboratories have previously studied the introduction of human DRa and (3 or murine I-AO and/or I-Aa genes into murine and hamster cells (Rabourdin-Combe and Mach, 1983; Ben-Nun et al., 1984; Germain et al., 1984). In one of these cases (Malissen et al., 1984), the association of endogenous mouse or hamster invariant chain with the class II product of the transfected genes was also observed. Materials and methods Construction of cosmid and X phage libraries High mol. wt. DNA was prepared from Priess cells, partially digested with MboI and fractionated on sucrose density gradients (Maniatis et al., 1982). DNA fractions of 35-45 kb were collected and ligated with cosmid vectors pTCF and pGNC (Grosveld et al., 1982) which had been digested with BamHI and treated with alkaline-phosphatase. The ligated mixture was packaged in vitro and transfected into Escherichia coli strain 1046 (Maniatis et al., 1982). DNA fractions of 15-25 kb were also collected, ligated with X phage vector EMBL3B (Maniatis et al., 1982) which had been digested with BamHI and EcoRI and treated with phosphatase and packaged in vitro. 106 cosmid clones and 106 X phage clones were obtained. Cosmid clones were spread on nitrocellulose filters (Millipore HATF) and replicated (Hanahan and Messelson, 1980). Screening of libraries cDNA or genomic DNA fragments were labelled with 32P by nick-translation (Rigby et al., 1977). Usually 3 x 107 c.p.m. of labelled fragment (> 10' c.p.m.//Lg DNA) were used to screen 20 filters of the cosmid library. In the case of screening the X phage library, a smaller amount of the probe (5 x 106 c.p.m.) was used. Hybridization was performed in 5 x Denhardt's, 10% dextran sulfate, 6 x SSC, 0.1% NaSO4 SDS, 100 tg/ml sonicated salmon sperm DNA at 65°C for 18 h. Filters were washed in 3 x SSC and 0.5 x SSC solution containing 0.05% SDS at 65°C and exposed for 24 h using Kodak XAR-5 film at -70°C with Dupont Lighting Plus intensifying screen. cDNA clones used as probes SBa cDNA clone (LB14) (Auffray et al., 1984) and SB,B cDNA clone (pSB,B) (Roux-Dosseto et al., 1983) isolated from the homozygous human B cell line LB (DRw6, DC 1, SB2) were used. For the purpose of exon mapping, small DNA fragments encoding one or more exon sequences were obtained from digestion of the cDNa clones with appropriate restriction enzymes. Blot hybridization DNA fragments were separated on agarose gels and transferred to nitrocellulose filter according to the method of Southern (1975). Filters were baked and hybrid-
SB subregion of the human MHC ized with nick-translated DNA probe and washed in 0. 1 x SSC and 0.05 % SDS
at
65°C.
Restriction site mapping Restriction maps were made by digestion of cosmid and X phage clones with one or a combination of restriction enzymes, and by blot hybridization of the digested fragments with cDNA or genomic DNA probes Nucleotide sequence analysis DNA fragments were subcloned into the vectors of pUC9 or pUC 12 (Vieira and Messing, 1982) and sequenced according to the method of Maxam and Gilbert (1980).
Transfection assay The murine macrophage line, P3M, (a gift of Avi Ben-Nun, Harvard Medical School) was grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 Ag/ml streptomycin and 100 it/ml penicillin. Transformations were performed as described by Wigler et al. (1983). Briefly, cells were seeded in fresh medium the day before transformation at a density of - 5 x 106 per 10 cm culture dish. A calcium phosphate co-precipitate was prepared containing 2 jig/ml cosmid TIOB DNA and 18 ug/ml murine DNA as carrier. One ml of this coprecipitate was added directly to the medium of each dish and left on the cells for 8 h. Following 2 days recovery in fresh medium the cells were split, at various dilutions, into 48-well microtitre clusters and selected in medium containing 0.8 mg/ml of the neomycin derivative G418 (Gibco). (Note that the original cosmid vector for TIOB, pTCF, encodes a selectable gene conferring resistance to G418.) Colonies surviving after selection for 3 weeks in G418 were picked and grown in 10 cm dishes for analysis by FACS and 2-dimensional gel electrophoresis. For FACS, 2 x 106 cells were stained by indirect immunofluorescence using Tu39 (kindly given by Dr Andreas Ziegler), a monoclonal antibody (MAb) specific for the SB and DR molecules (Paewlec et al., 1982; 1:50 dilution of hybridoma culture supernatant used in a 60 iLl incubation mix), and fluorescein-conjugated goat anti-mouse IgG serum as the second antibody (Cooper Biomedical, 1:30 dilution used). Cells were analyzed with a Coulter FACS II. Fluorescence levels were compared with control stainings in which the same cell line was incubated with second antibody alone. Cell populations containing Tu39-positive cells were sorted for high expressors. The top 5% of the cells were collected, grown in culture for several weeks and then labeled with [35S]methionine (1 14Ci per 10 x 106 cells) and analyzed by 2-dimensional polyacrylamide gel electrophoresis and autoradiography. Electrophoresis was performed after the method of Singer et al. (1978) as modified by Shackelford and Strominger (1980) with SDS electrophoresis as the first dimension and isoelectric focusing as the second. Immunoprecipitations were carried out as described by Shackelford and Strominger (1980) using Tu39 and another SB specific monoclonal antibody MHM4, kindly given by Dr A.McMichael (Makgoba et al., 1983).
Acknowledgements We thank M.Steinmetz for helpful advice in making and screening cosmid libraries. We thank Daphne Haas, Cheryl Ellis, Hugo Giamberella and Mike Greenburg for technical assistance, and Alice Furumoto-Dawson for typing the manuscript. This research was supported by research grants from the NIH (AM-30241 and AM-13230) to J.L.S. K.O. is a recipient of a long-term overseas research fund from the Ministry of Education of Japan. J.B. is supported by Damon RunyonWalter Winchell and NRSA (5 F32 AI06860) fellowship grants. D.L. is supported by an American Cancer Society fellowship and T.S. by the Deutsche Forschungsgemeinschaft.
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Received on 18 December 1984
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