Sequence Analysis of the Human Major HistocompatibilityGene SXa
JEREMY M. BOSS, ROSEMARIE MENGLER, KIYOTAKA OKADA,t CHARLES AUFFRAY,t ANDJACK L. STROMINGER*
Department ofBiochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138
Received 20 May 1985/Accepted 25 July 1985
The DP subregion of the human major histocompatibility complex contains two closely linked gene pairs,DPa, DPOi and SXa, SX13. The exon-intron organization and the complete DNA sequence of the SXa gene arereported here. There are several mutations within the SXot gene which strongly suggest that it is a pseudogene.These include two frameshift mutations, one in the al domain and the other in the cytoplasmic domain. A 5'splice site mutation at the end of the aLl exon also exists. DNA sequence homology between DPoa and SXotsuggests that these genes arose through a gene duplication event.
The class II genes of the major histocompatibility complex(MHC) encode cell surface glycoproteins that function in theregulation of the immune response by association with"processed" foreign antigen to provide a recognition unit forhelper T cells as well as for class TI-directed cytotoxic Tcells. Class II MHC proteins are heterodimers (an a chain of33 kilodaltons and a 1 chain of 29 kilodaltons [21]) and inhumans are found on the surfaces of B cells, macrophages,and activated T cells. In humans the class II genes arelocated within the HLA-D region of chromosome 6. Thisregion is homologous to the Ia region of chromosome 17 inmice. To date six human a-chain genes and seven 13-chaingenes have been cloned as cDNAs or as genomic clones (forreviews see reference 8 and A. J. Korman, J. M. Boss, T:Spies, R. Sorrentino, K. Okada, and J. L. Strominger,Immunol. Rev., in press). The cloning and subsequentsequence analyses of some of these genes have helpedorganize the genes into three families. The DR regioncontains one a- and three 13-chain genes (22a); the DQ(formally DC) region contains the DQa and DQ,B genes aswell as a homologous pair of DQ-like genes called DXa andDX1 (16a). The DR and DQ families have analogous coun-terparts in mice, namely, IE and IA, respectively. The thirdfamily, DP (formally SB), has been shown to contain fourgenes which are closely linked, i.e., within 120 kilobases (kb)of DNA (17, 20, 24). This cluster contains the genes encod-ing the DPa and DP1 proteins as well as two homologousgenes which we have termed SXa and SX1.(These genes arealso referred to as DPa2 and DP,B2 by some groups. Thenomenclature DPal [for SBa], DPa2, DP131 [for SB,B], andDP132 introduces confusion both with the numerical namingof alleles and with the naming of the domains of the chains asal or ,13 and a2 or 12. We therefore prefer to retain SXa andSX1 until these genes have been named officially.) In addi-tion to these families, an a-chain cDNA clone termedDO/DZa has been isolated by several groups (22; H. Inoko,A. Ando, H. Kimura, S. Ogata, and K. Tsuji, in Histocom-patibility Testing 1984, Springer-Verlag, in press). DO/DZahas equal homology to DRa, DQa, and DPa and appears to
* Corresponding author.t Present address: Department of Biophysics and Biochemistry,
Facultys Science, University of Tokyo, Tokoy 113, Japan.t Present address: Institut d'Embryologie, 94130 Nogent sur
Mamne, France.
be in a family by itself. It is not presently known whetherthere is a 13 chain associated with or linked to this gene.Although many class II genes are encoded in the HLA-D
region, it is not certain that all these genes are expressed atthe level of either mRNA or protein on the cell surface. Oneof the DR,B genes has been shown to be a pseudogene owingto two in-frame stop codons and several splice site mutations(10). Likewise, the SX1 gene may also be a pseudogene forthe same reason as the DR1 gene metioned above (7). In thispaper we report the structure of the SXa gene from twodifferent DP haplotypes, DP3 and DP4, and present evidencewhich suggests that SXa is a pseudogene in both of thesehaplotypes. These observations are further supported by thepreviously reported sequence of an SXa gene (termed DPa2)from an untyped individual (20).
MATERIALS AND METHODS
Isolation and subcloning. Clones were obtained byscreening a cosmid library constructed from the humanlymphoblastoid cell line Priess (DR4,4; DQ3,3; DP3,4) withDPa and DP1 cDNA clones as probes as previously describedby Okada et al. (17). Two of the many positive clones, cG8A(from the DP3 haplotype) and cS2B (from the DP4 haplotype),were found to have two a-chain genes, DPa and SXa,surrounding a 13-chain gene, DP1. Subclones pSX17 (fromcG8A) and pS2B2 (from cS2B) containing a 5-kb EcoRIfragment which encodes three exons of the SXa gene wereinserted into pUC13 to facilitate detailed restriction fragmentmapping and sequence analysis. An additional EcoRIsubclone, pSX20, containing the 1.8-kb fragment adjacent toand upstream of pSX17 was also cloned. An ordered set ofdeletions were constructed from pSX17 by the DNase Imethod as previously described (5) to allow rapid sequencingof part of the gene.
Sequencing. DNA fragments were prepared for sequencingby 3' end labeling with either the Klenow fragment of DNApolymerase (Bethesda Research Laboratories) with a32P_labeled deoxynucleoside triphosphates or by terminal trans-ferase [a-32P]ddATP, using (Amersham Corp.). Labeledfragments were digested with a second restriction enzymeand isolated from low-meling-point agarose by extractionwith phenol and ether. Sequence reactions were carried outby the method of Maxam and Gilbert (14).
2677
2678 BOSS ET AL.
kSXZSX& D8WVP
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I<Zz I w
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A
B
pSX17pS2B2
FIG. 1. Restriction map and DNA sequence strategy. The arrangement and orientation (arrows) of the genes in the DP subregion areshown in the top line, along with cosmid clones G8A and S2B. (A) Maxam and Gilbert (14) sequence strategy for clones pSX17 and pSX20.Arrows with open circles indicate sequences of clones obtained from deletions. (B) Sequence strategy for clone pS2B2.
RESULTS
Cloning, subcloning, and sequencing. The cosmid clonescG8A and cTlOB were isolated from a cosmid library con-structed from the human lymphoblastoid cell line Priess andwere previously characterized by Okada et al. (17). Each ofthese cosmids as shown in Fig. 1 contains three class IIgenes, DPa, DPO, and SXa. Southern blot experiments withDPa cDNA as a probe showed that the SXa gene was highlyhomologous, but not identical, to the DPa gene, since ithybridized less strongly. Polymorphic restriction fragmentmapping and comparison of a partial DP,B amino acid se-
quence with the DNA sequence of the DPI gene separatedthe DP3 haplotype on cG8A from the DP4 haplotype on
cS2B.The EcoRI fragments containing the SXa gene were
subcloned into pUC13 for sequence analysis. Figure 1 showsa restriction enzyme map of clones pSX17 (SX3) and pS2B2(SX4) as well as pSX20, the subclone adjacent to pSX17.Sequencing strategies for these clones are shown in Fig. 1.A comparison of the nucleotide sequence and amino acid
sequence of SX3a with the DPa cDNA sequence (1) showedthat the SX3a gene contains a total of three nucleotidedeletions, two 1-base-pair (bp) deletions (3' to nucleotides4764 and 5905) and one 3-bp deletion (3' to nucleotide 5891).Maximum homology with the DPa amino acid and cDNAsequence was obtained by placing gaps, represented byasterisks, in the sequence to correct the reading frame at thedeletions (Fig. 2). Individual deletions and their possibleeffects are discussed below.The exon-intron arrangement of the SXa gene. The exon-
intron arrangement of SXa was determined first by hybrid-ization to various restriction fragments from DPa cDNA andlater by direct amino acid and nucleotide sequence compar-ison with the DPa cDNA sequence (1). Exon 1 (nucleotides4550 to 4794), exon 2 (nucleotides 5189 to 5670), and exon 3(nucleotides 5786 to 5949) corresponded to the al domain
(amino acids 5 through 94), the a2 domain (amino acids 95through 178), and the connecting peptide-transmembrane(TM) domain-cytoplasmic domain (amino acids 179 through232) of the DPa gene, respectively. The 3' end of the TMexon was placed at the first 5' splice site after the terminationcodon, as is the case with DPa, DQa, and DRa (1, 19). Thethree exons were spaced at similar distances, as are theexons in the DQa and DRa genes. For example, the dis-tances between the al and a2 domains of SXa, DQa, andDRa were 395, 491, and 354 nucleotides, respectively.Promoter and signal sequence region. Sequencing the 5'
end of the DNA on clone pSX17 revealed little homology tothe DP signal sequence and to the highly conserved class IIupstream promoter sequence (UPS) (13, 18; Okada et al., inpress). Additional sequencing of the upstream EcoRI frag-ment in pSX20 revealed a region with a high degree ofhomology to the UPS (Fig. 3) and suggested that this is thepromoter for the SXa gene. The distance between this UPSand the xl exon (3 to 4 kb) was similar to that of the DRa(19), DQa, and DXa genes (Okada et al., in press). As withother a-chain genes, typical TATA and CCAAT sequenceswere not found downstream of the UPS. The first ATGfollowing the UPS was 73 bp 3' to the end of the conservedregion and is probably too close, since for DRa (19) and DQP(2) the distance is about 138 bp. The next ATG was locatedabout 197 bp downstream of the UPS and may represent thebeginning of a signal sequence which is 18 amino acids longbefore it would have to splice out to avoid a terminationcodon. This "signal sequence" (Fig. 2) or the other transla-tion reading frames in this region do not share homologywith the DPa signal sequence (H. Erlich, personal commu-nication).A region homologous to the 3'-untranslated sequence of
DPa was not found in the DNA fragment (SX17) sequenced,either by direct sequence comparison or through hybridiza-tion on Southern blots with a 3' UT DPa probe. In addition,a similar hybridization analysis on the 3' adjacent EcoRI
MOL. CELL. BIOL.
GAGATTTCTTICTTAGGTTTGGATCTATTCGCTGGGTCCAGTGTGGGGGTAOGGGATGA0GA6AATAGACTCTG OAGAATCTTGTTTAGATAGAFTTAGTGTGTFGTTTCTCJTGAATTGTTGTAATOTCCTGAGTOGTGGCFTCCAGCCAGGTGTGT~GGTGT CAAAGCACACCGCTACAATAGTAGAAGGTGATATAAGCTTCCCTAAGTTGCCAAGATAA~* 3 G V I K L P R V XV F CD W L P 'VATTCAGTTTCTCAGGCA ATG AGT GGOT GTC ATA fAAG -CTC CCA AGA OTT TAC GTC -TTT TGT GAT ToG -CTA CCA G CTOGGOTAGAGAAATOCCCTCAGTGT.T
II,CTCTGTTTCCCTACAACCATCTccTGTCCTCACGTC'CCGoc
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ATTOAATOCTAACTAGFTAAFTTAFTAAFT
TAFT
VA D H V S T V A R F V Q T H R P S 0 K Y H F KE ETTCATGCAG CA GAC CAT OTG TCA ACA TAT 000 AGO TTT GTG CAG ACGO CAC AGA CCC TCT 000 GAO TAT ATG TTT GAA TTT GAT GAO GAO GAG
-G-T - A.T-A-T - A-AT--F.D-T
Q F V V N L D E K E H V V P L P K F I H T F D F G A Q0R01CAG TTC TAC GTG AAC CTG OAT GAG ALAG GAO ATG GTC TOG CCT CTA CCA GAG TTT ATT CAC ACC TTT GAC TTT GOT*T CGAGO GOT ATTAT---T-G-T---CA-C C -- A--CC O-A---G GAG O GC --AOG-- TC- --AG,' =HM D - - K - - T - - H E - G- Q A - E - G0 L
IVIA 0 I V H A R K H L N T R I ? V S K Q TVW A T NVOCT GGC ATC GTC AVG GCA AGG AAG CAC TTG AAC ACC COG ATC *AA TGG TCC AAA CAG ACT TOG 0CC ACA AAT 0 GCACTGCCTATAGCTG-AA- -TGCT -A TTG -AC -C--CA--T-TT--C-GOC-T--C --C ---CA- -C --C -
N - A I L N N N - - - L QOR - N H - Q- - -
IVA P T K V S V F P K K P V D L 0 Q P N T L V C H V D K F F PCC CCC ACC GAG GTA AGC OTC TTT CCC LAG GAG CCT GTG OAT CTG GOC CAG CCC AAC ACC CTC GTC TGC CAT OTT GAC AAG TTC TTC CCAAT-T-- -C -G--GA-- ---CA--
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F H R F H V L T L V PHM A K D T C D L 00 K H V 0 L H Q P LTTC CAC AGO TTC CAC TAT CTG ACC CTC OTT CCC ATG GCC GAG GAC ACC TOT GAC CTC CAG 000 GAG CAC TOG GGC CTG CAC CAG CCT CTC
-A--T--C -T --G TCA-A --- ---TT- TG- AG--T---T--G-----0-G-- K.F - - S - - - F VY C R V-D - - -
L R H RCTC AGO CAC CG0 0 GTATGGAGCGCCCTCCCTCTGCCCTCACGGCCTTGGCACCACCTTTATTTCCTGGGCCCATCGCCCCTCAG'CACCTOCCTTCCTCAATCCCAGTGTTTTA
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GGC ACC ATT GTC TCA fL*AG ACC fLAG CGA TCT *GA CAG CAT CCC COGG GTC CAG GGG CTC CTA TGA GTCATCCTATAGGTGTATTAGGGACAGAGTG0 5%96--G-C C--ATC ATA -T-T Cr--T-G-C -TG-C .----- A-_-- V L I I - S L GOH D - A T 229
FIG.- 2. DNA sequence of the SXa-chain gene. The SXaL DNA and protein se-quences are aligned and compared with DPa (1) in the codingregions. The top two lines are the SXa amino acid and nucleotide sequences, respectively. The bottom two lines are the DPaL nucleotide andamino acid sequence, respectively. Asterisks represent deletion mutations in the SXoa sequence. Dotted underlines show the putative signalsequence; dotted arrows point to sdml and a putative cryptic splice site; solid arrows point to intron-exon boundaries. Boxed nucleotides atpositions 132, 171, and 6519 highlight the two conserved class II upstream promoter sequences and a putative polyadenylation site. Bracketedsequence 4709 to 4879 and 4880 to 4990 highlight a 110-bp tandem segment. Dashes indicate sequence homology.
bp 5 ' to the initiation of transcription of DRaFIG. 3. Upstream promoter sequence. The upstream promoter sequences for class II a-chain genes are compared. Homology with a
consensus sequence is indicated by dashes, and gaps are placed to maximize the homology. DRa (19), DQa, DXa (Okada et al., in press);I-Ea (11), DPa, and DZa (9). R, purine; Y, pyrimidine.
fragment to pSX17, which contains the 3' end of the DP,chain gene in the opposite orientation, also gave a negativeresult even under relaxed hybridization conditions. Thisresult was not surprising because 3' untranslated regions cantypically be used as probes to distinguish closely relatedgenes. In the DRot chain gene the distance from thetransmembrane domain to the 3'UT is approximately 1,100bp. A scan of the DNA sequence 3' to the TM exon revealeda single polyadenylation site ATTAAA, at nucleotides 6528through 6533, which is 589 bp 3' to the end of thetransmembrane region, suggesting the possibility that this isthe end of a putative 3'UT. Although AATAAApolyadenylation sites are more frequent, ATTAAA has beenfound in several MHC genes, such as DC, (2) and DR, (12).
Mutations. For the purposes of presenting and discussingthe mutations in the SXa gene and their effects on thestructure of a class II a chain, it will be assumed that SXacan be transcribed, processed, and translated, althoughthere is no evidence to support even transcription. In fact, itappears that SXa cannot be properly processed if tran-scribed. Deletions are referred to by a A followed by thenumber of the previous 5' bp. For example, A4764 is in theal domain and is 3' to nucleotide 4764 (Fig. 2, representedby an asterisk).
Mutation A4764 located in the al exon altered the readingframe of only the last 10 amino acids of that domain such thatthere was no amino acid sequence homology to DPa (one of
nine residues; SX* in Fig. 4). These altered residues alsoshowed little homology to other class II a chains.The next mutation is the 5' splice site 3' to the al domain
(smal). The canonical dinucleotide at 5' splice sites, GT(16), was changed to GC. A few examnples of GC 5' splicesites have been reported. Both the duck and chicken aD_globin genes normally have a GC 5' splice site in intron 2,(3,4) and are thought to be expressed properly. Other 5' splicesite mutations have been found in some of the human ,Bthalassemias. In these cases, splicing from the mutated 5'splice sites is reduced or abolished and cryptic 5' splice sitesare activated (for a review, see reference 23). None of the 1Bthalassemia splice site mutations are the same as SXa (i.e.,GT to GC). However, in vitro mutagenesis of the rabbit1-globin IVS2 5' splice site to a GC was shown to activatecryptic splice sites (28). It remains to be determined whetherthe GC dinucleotide in SXa can function as an efficient 5'splice site or whether normal splicing is abolished, with orwithout activation of cryptic splice sites. Since all 5' splicesites are GT in the closely related gene DPa, the splicingapparatus may not recognize the GC in SXa.
Utilization of the putative GC 5' splice site would result instop codons in the a2 domain, since the reading frame in therest of the gene would be changed owing to frameshiftA4764. If sdml resulted in inactivation of this splice site andthe activation of cryptic splice sites, then a structurallyhomologous class II protein may be possible. For example,
FIG. 4. Comparison of class II a chains with DPa. Dashes indicate homology with the DPa sequence. The SX3 amino acid sequence iscorrected for the frameshift mutation, while SX* is not. DPa (1); DRa (19); DQa (1).
MOL. CELL. BIOL.
HUMAN MAJOR HISTOCOMPATIBILITY GENE SXa 2681
TABLE 1. Comparison of haplotypesa
Nucleotide(s) Amino acidsLocation Nucleotide(s)
SX3 SX4 SX3 SX4
5' to al 4479 A G5' to al 4517 A Gal 4572, 4573 AG GA R Eal-a2 intron 5036 C _ba2 5402 T C C Ra2 5470 C A F La2 5567 C T R Wa2-TM intron 5663 G ba2-TM intron 5671 C Ta2-TM intron 5758 C TTM exon 5843 G C L L3' to TM exon 6037 - GCATTGAc3' to TM exon 6124 G T3' to TM exon 6198 G A
a Differences at both the amino acid and nucleotide levels are shown.Nucleotide positions are for SX3 (Fig. 2).
b Deletion in SX4.c Insertion in SX4.
if splicing occurred after A4764 at bp 4769 (open arrow inFig. 2), the domain would be shortened by only eightresidues and would not incorporate any of the frameshiftedresidues into the protein. The effects of the frameshift wouldthen be nullified. Alternatively, other putative cryptic splicesites could be used, such as the one located 19 nucleotidesdownstream of smal, the use of which would incorporateseven additional amino acids into the peptide.One additional point involves the occurrence of a 110-bp
tandem repeat (nucleotides 4609 through 4719 and 4720through 4830; underlined in Fig. 2) spanning the al exon-intron region. Each repeat contained half of the al domain,the frameshift mutations, A4764, and smial. The secondrepeat was in frame with the coding sequence, so that ifcryptic splicing did not occur within the first copy of therepeat, as discussed above, it probably would not occur inthe second, resulting in an even larger transcript and protein.There are many GT dinucleotides 3' to the second repeat,and it is difficult to assess which, if any, of these could beused as a splice site.The rest of the deletions occurred in exon 4, which
encodes three structural domains, the connecting peptide(nucleotides 5887 through 5824), the transmembrane domain(nucleotides 5825 through 5893), and the cytoplasmic domain(nucleotides 5894 through 5936). The connecting peptide wasintact. The transmembrane domain contained three deletionsin both haplotypes. Deletion A5890, which occurred at theend of the transmembrane domain, was a 3-bp deletion (Fig.2) and did not affect the reading frame; therefore, bothcoding sequences were intact within this domain. The lastdeletion, A5905, occurred in the cytoplasmic domain of bothhaplotypes and altered the reading frame. This mutationframeshifted the normal stop codon so that translation wouldcontinue and the next termination codon would be read 123bp downstream, with the result that 45 amino acid residueswould be added to the cytoplasmic domain. Considering theabove mutations in this gene, the abnormally long cytoplas-mic tail is least likely to have an effect. It has been shown byexon deletion and shuffling experiments that the cytoplasmictail is not necessary for class I MHC molecules to functionproperly in the systems assayed (29).Another interesting difference between the DPa and SXa
chains involves the cysteine residues which would form a
disulfide loop in the a2 domain. However, one of the Cysresidues (DPa position 159) was changed to a Leu in SXa,and a reciprocal mutation changing a Tyr to a Cys at position157 occurred. The size of the putative disulfide loop wouldtherefore be decreased by two amino acids. The SX4 allelealso shared these changes but only had two Cys residues inthis domain owing to a Cys-to-Arg change at position 123(see below). It is not clear whether changing the size of thedisulfide loop or removing the extra Cys residue would makea difference in the function of this a chain.
Haplotypes and homology to other class II genes. Themutations listed above have serious effects which, as dis-cussed below, raise questions whether SX3a would be anexpressed gene. To see whether these mutations existed inanother haplotype, a second haplotype was subcloned andthe SX4a chain gene was partially sequenced. The regionsequenced as compared with SX3a covered nucleotides 4400to 6220 in Fig. 2, including the al, a2, and TM exons andtheir introns. There were 21 differences beween these al-leles, none of which involved any of the deletions or thesplice site mutation, i.e., the same deletions were present inSX4a as in SX3a. Of the 21 changes, 4 affected amino acidcoding sequences, and the rest were found within theintrons. All differences and their effects are listed in Table 1.A partial sequence of the SXa gene has recently been
published (20). This sequence most closely matches theSX4a sequence presented here and is also suggested to be apseudogene by the authors. There are, however, severaldifferences between the two sequences that are worth men-tioning. The two frameshift mutations, A4764 and A5905, areplaced 6 and 3 bp downstream, respectively, of those foundhere. For A4764, their placement results in three amino aciddifferences with the DPa (1) sequence instead of two. Theplacement of A5905 has no effect on the homology compar-ison. A nucleotide substitution at position 5402 (T to C)changes the Cys residue shared with DPa to an Arg residue,thus removing the extra Cys residue in this domain. Thesubstitution of a C by a T in the TM exon at nucleotide 5923causes a stop codon to form, but only when aligned with theDPa reading frame. This mutation has no effect on thepresent reading frame of SXa, owing to the frameshifts inboth the al and TM exons.
If the deletions above are ignored by inserting gaps in thesequence, a comparison between SX3a and DPa can becarried out at the DNA or at the protein level (Fig. 2 and 4;Table 2). The al, a2, and transmembrane exon domainsbetween these genes have 72, 82, and 70% nucleotidehomology and 63, 70, and 52% amino acid homology,respectively. The P-chain genes in the DP-region DPPi andSXP genes are much more closely related, having 93%nucleotide and 85% amino acid homology in their codingregions (7). This high degree of homology, as well as the
TABLE 2. Amino acid homologya% Homology between ax chairs for:
a References are DPax, DXca, and DQ4a (1) and DRa (19).
VOL. 5, 1985
2682 BOSS ET AL.
proximity of these genes to one another (50 kb) suggests thatone of these two pairs of genes arose by a gene duplicationevent. This observation has been previously noted (7, 9, 17,20, 24). For comparison, the amino acid homology betweenDQa and DXa is 89% and that between DQi (1) and DXP inthe polymorphic p1 domain (the only sequence available inDXP; Okada et al., in press), is 73%. The DQ/DX genes,although located farther apart than the DP/SX genes, haveprobably arisen by an analogous gene pair duplication event.Homology between the different a chains can be seen in Fig.4 and is shown in Table 2. DPa and SXa are much moreclosely related to each other than either is to the DRa andDQa genes. There are, however, regions which have re-mained conserved between all these genes. Most of theseregigns occur in the a2 domain and may determine basicstruttu'ral features in the skeleton of a class II a chain.
DISCUSSIONThe SXa gene probably represents a pseudogene, that is,
a gene whose product is no longer made or functional in thecell. This is supported by the following line of evidence fromthe DNA sequence data reported here. First, there are two1-bp deletions which alter the reading frame. The first ofthese deletions occurs after 80 amino acids of the al domainand radically changes the last 10 amino acids. Second, the 5'splice site at the end of what would be the al domain isaberrant; this would probably result in improper splicing ofthe mRNA precursor by activation of cryptic sites or greatlyreduce proper splicing at this site. A further complication inthis region involves the occurrence of a 110-bp tandemduplication which includes the first two mutations (i.e., theframeshift A4764 and smial). It is difficult to predict whatrole the repeat may play if this exon were expressed. Ifcryptic splicing occurs before the end of the exon, asdiscussed above, the repeat would play no role in expres-sion. On the other hand, if splicing at a site within the repeator 3' to it occurs, a partial duplication of the al domain ofthis protein would result. Additional reading frame problemswould also arise if splicing occurred at the normal site or anyother site which did not place the a2 domain in frame. Third,the frameshift in the cytoplasmic domain significantly altersthe length of this domain. Because it is not clear whathappens before the cytoplasmic domain, it is difficult topredict which reading frame would be used if the mRNAwere translated, but if the proper reading frame was en-coded, an additional 43 amino acids would be encoded as aresult of A5906. The effect of additional amino acids in thecytoplasmic tail of class II molecules is unknown, butdeletion experiments involving cytoplasmic domains ofother MHC antigens suggest that it is not needed for manyfunctions (29). Overall, the combined mutations would resultin a frameshifted, improperly spliced mRNA which, if trans-lated, would result in a protein resembling a class II a chainfor only the first 80 amino acids, unless, as discussed, aproximal alternative splice site was used, shortening the aldomain by eight amino acids. Interestingly, SXI is also apseudogene, although it could also be corrected by translat-ing a shortened ,2 domain with a putative cryptic splice site(7).The SXa gene sequenced by Servenius et al.(20) is closely
related to the SX4 allele presented here. Some of thedifferences involve our placement of the deletions, as dis-cussed above, while others represent actual nucleotide dif-ferences. Conservation of DNA sequence information be-tween these alleles is remarkable. A comparison of the SX3aand SX4a genes shows that there are only 21 nucleotide
differences over 1,820 bp, of which 6 are within the codingregion (Table 1). This gives a level of homology ofjust under99%. The homology among any of the DR,B or DQP alleles ismuch lower. This observation may be interpreted in manyways. For example, the expressed SXa gene may have beensimilar to the DRa gene in that polymorphism betweenhaplotypes is extremely limited or even suppressed. Thepolymorphism seen today in SXa would then represent therecent decay of an inactive gene. Alternatively, the conser-vation of DNA sequences within pseudogenes may reflectthe need for expression and selection to generatepolymorphisms.To investigate the expression of this gene at the level of
transcription, Northern blots were carried out on B cell andfibroblast RNA by using a genomic probe spanning the endof the a2 and TM exons. The results of this experiment aswell as the screening of a large B-cell cDNA library werenegative. These results imply that the SXa gene is nottranscribed in B cells and fibroblasts. On the other hand, thegene could be developmentally regulated or even unstableowing to splicing difficulties. However, the accumulateddata presented here suggest that in these two haplotypes theSXa gene is a pseudogene.To date, there are three known pseudogenes in the class II
region of the MHC, i.e., one of the DRI genes (10), the SX,gene (7), and the SXa gene presented here and in reference20. In the mouse Ia region, the A,3 gene is also a pseudo-gene (G. Widera and R. A. Flavell, submitted for publica-tion). The DXa and DX, genes could also be pseudogenes,because their transcripts and protein products have not asyet been detected, although they are structurally intact,including intact promoter regions and, at least for DXa, ademonstration of normal splicing (A. Korman, personalcommunication).Why do pseudogenes occur in the human MHC? This
genetic region has undergone a moderate expansion relativeto that of the mouse and may be as much as three times aslarge. It includes a minimum of six a- and seven 3-chaingenes (compared with two a- and five P-chain genes in mice),of which eight are known to be expressed (four in mice).What could be the function of the remaining genes, and whyhave they persisted? One possibility is that some of thesegenes are expressed only in a tissue-specific or developmen-tally specific manner. Another possibility is that they serveas reservoirs of gene sequences for the generation of poly-morphism within this family of genes. The class I and classII MHC genes are the most polymorphic known to occur inhumans. One mechanism for generation of this polymor-phism is the copy repair mechanism analogous to geneconversion, which can occur in multigene families and whichhas been shown to operate on both class I and class II MHCantigens (15, 26, 27). Thus the pseudogenes in the class IIregion may simply serve as reservoirs of genetic informa-tion. Lastly, the genes in this region may have duplicated inresponse to some ancient environmental pressure, and then,in the absence of continued pressure, the nonessential genesmay have degenerated. Pseudogenes have also been shownto occur in other gene families, including the class I MHCgenes (25), the immunoblogulin genes (6), and the humanP-globin genes (11).
ACKNOWLEDGMENTS
We thank James Sonner for technical assistance and A. Korman,D. Dialynas, G. Blanck, and A. Krainer for helpful discussions.
This research was supported by Public Health Service researchgrant AM-30241 to J.L.S. from the National Institutes of Health.
MOL. CELL. BIOL.
HUMAN MAJOR HISTOCOMPATIBILITY GENE SXa 2683
K.O. is a recipient of a long-term overseas research fund from theMinistry of Education of Japan. J.M.B. is supported by DamonRunyon-Walter Winchell and National Research Service Award (5F32 A106860) fellowship grants.
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