genetic makeup of the dr region in rhesus macaques: gene

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of March 17, 2018. This information is current as Pseudogenes Macaques: Gene Content, Transcripts, and Region in Rhesus DR Genetic Makeup of the and Ronald E. Bontrop Nel Otting, Corrine Heijmans, Annemiek J. M. Rouweler Nanine de Groot, Gaby G. Doxiadis, Natasja G. de Groot, http://www.jimmunol.org/content/172/10/6152 doi: 10.4049/jimmunol.172.10.6152 2004; 172:6152-6157; ; J Immunol References http://www.jimmunol.org/content/172/10/6152.full#ref-list-1 , 15 of which you can access for free at: cites 45 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 2004 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on March 17, 2018 http://www.jimmunol.org/ Downloaded from by guest on March 17, 2018 http://www.jimmunol.org/ Downloaded from

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of March 17, 2018.This information is current as

PseudogenesMacaques: Gene Content, Transcripts, and

Region in RhesusDRGenetic Makeup of the

and Ronald E. BontropNel Otting, Corrine Heijmans, Annemiek J. M. Rouweler Nanine de Groot, Gaby G. Doxiadis, Natasja G. de Groot,

http://www.jimmunol.org/content/172/10/6152doi: 10.4049/jimmunol.172.10.6152

2004; 172:6152-6157; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/172/10/6152.full#ref-list-1

, 15 of which you can access for free at: cites 45 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Genetic Makeup of the DR Region in Rhesus Macaques: GeneContent, Transcripts, and Pseudogenes1

Nanine de Groot,2 Gaby G. Doxiadis, Natasja G. de Groot, Nel Otting, Corrine Heijmans,Annemiek J. M. Rouweler, and Ronald E. Bontrop

In the human population, five major HLA-DRB haplotypes have been identified, whereas the situation in rhesus macaques (Macacamulatta) is radically different. At least 30 Mamu-DRB region configurations, displaying polymorphism with regard to number andcombination of DRB loci present per haplotype, have been characterized. Until now, Mamu-DRB region genes have been studiedmainly by genomic sequencing of polymorphic exon 2 segments. However, relatively little is known about the expression status of thesegenes. To understand which exon 2 segments may represent functional genes, full-length cDNA analyses of -DRA and -DRB wereinitiated. In the course of the study, 11 cDRA alleles were identified, representing four distinct gene products. Amino acid replacementsare confined to the leader peptide and cytoplasmatic tail, whereas residues of the �1 domain involved in peptide binding, are conservedbetween humans, chimpanzees, and rhesus macaques. Furthermore, from the 11 Mamu-DRB region configurations present in this panel,28 cDRB alleles were isolated, constituting 12 distinct cDRA/cDRB configurations. Evidence is presented that a single configurationexpresses maximally up to three -DRB genes. For some exon 2 DRB sequences, the corresponding transcripts could not be detected,rendering such alleles as probable pseudogenes. The full-length cDRA and cDRB sequences are necessary to construct Mhc class IItetramers, as well as transfectant cell lines. As the rhesus macaque is an important animal model in AIDS vaccine studies, the infor-mation provided in this communication is essential to define restriction elements and to monitor immune responses in SIV/simian humanimmunodeficiency virus-infected rhesus macaques. The Journal of Immunology, 2004, 172: 6152–6157.

T he rhesus monkey provides a valuable model in preclin-ical studies of infectious and chronic diseases as well asfor tissue and organ transplantation (1–11). The applica-

tion of macaques in immunological research necessitates an ex-tensive characterization of the MHC region, because the high de-gree of polymorphism of most of its genes is not only a maincharacteristic in humans, but also in nonhuman primates (12). Cellsurface glycoproteins of the MHC, divided into class I and class IIgene products, present peptides to effector T cells, and thereforeplay an important role in adaptive immunology. As one wouldexpect, several susceptibility or resistance traits have been mappedto particularMhc alleles in humans and rhesus macaques (13–22).

The polymorphic MHC class II genes of the rhesus macaque(MhcMamu) map to theDP, DQ, and DR regions. One expectsthat, as is found in humans, the actual polymorphism is mostlyconfined to exon 2 of theMamu-DPB1, -DQA1, -DQB1, and-DRBloci encoding the contact residues of the peptide binding site. Asa consequence,Mamu class II sequencing has been mainly focusedon the determination of allelic variation at exon 2 segments.

TheMamu-DRA locus encoding the DR�-chain is thought to bemonomorphic and highly conserved through primate evolution, asit shows only limited variation in comparison toHLA-DRA (23).

The organization of theMamu-DRB region is complex and it dis-plays variation at the population level with regard to numberand/or combination of loci present per configuration (24–28). Inhumans, the number of -DRBloci present per configuration differsfrom one to four, whereas in the rhesus macaque, one to eight locican be observed (12, 28). Only fiveHLA-DRB region configura-tions are known, and all of them display a high degree of poly-morphism, mainly at the-DRB1 locus (29, 30). The situation inrhesus macaques is radically different, as�30 Mamu-DRB regionconfigurations have been described so far (28). Although the totalnumber of apparentMamu-DRB alleles is comparable to those oftheHLA-DRB1 locus, theMamu-DRB region configurations them-selves show only a limited degree of allelic variation (25, 26).

The absence or lack of allelic polymorphism atMamu-DRB re-gion configurations can be explained in several ways. One inter-pretation is that these configurations are relatively young and didnot have time to accumulate variation. Alternatively, it is conceiv-able that these configurations experience conservative selectionand have been maintained over longer evolutionary time spans. Inboth cases, rhesus macaquesused a radically different strategy thanhumans to initiate Th cell responses to combat infections. While thehuman population invested mainly in generating a high degree ofallelic variation at the variousDRB loci, the rhesus macaque popula-tion primarily generated a large number of singular combinations ofDRB loci. We published evidence recently thatMamu-DR/DQ con-figurations appear to be unique for a given population living at distinctgeographic locations (31). This implies thatMamu-DR/DQ regionconfigurations originated after the separation of eastern and westernrhesus macaque populations, which is thought to have been caused bya glacial ice barrier during the Pleistocene era (32).

Most of theMamu-DRB alleles belong to lineages or loci thatare shared between humans and macaques. In addition, present inthe rhesus macaque are loci/lineages for which no human equiv-alent is known (-DRBW). Some of theMamu-DRB loci appear to

Department of Comparative Genetics and Refinement, Biomedical Primate ResearchCentre, Rijswijk, The Netherlands

Received for publication November 14, 2003. Accepted for publication February27, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported in part by the European Union Project IMGT-QLG2-CT2000-01287 and the National Institutes of Health Project 1-R24-RR16038-01 (Cata-log of Federal Domestic Assistance 93.306).2 Address correspondence and reprint requests to Dr. Nanine de Groot, BiomedicalPrimate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands.E-mail address: [email protected]

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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have been duplicated and can be present twice, or even three times,on the same configuration (26).

One needs to realize that many pseudogenes have been identi-fied in the various HLA-DR regions. However, little is knownabout the expression of the various Mamu-DRB loci, lineages, oralleles. For example, the Mamu-DRB6 locus, although it may betranscribed (33, 34), does not seem to code for a functional class IIgene product, because its exon 2 sequences show various character-istics such as inserts, stop codons, and deletions that would render ita pseudogene (24). Only for some alleles of the Mamu-DRB1*03,-DRB1*10, -DRB1*04, -DRB*W3, DRB*W4, and -DRB*W5 lin-eages, immunoprecipitation studies suggested that these particular al-leles code for a class II molecule (12, 35). Restriction element studiesrevealed that certain alleles of the -DRB1*03 lineage, as well as-DRB1*0406 and -DRB*W201, encode gene products which are ableto present peptides to CD4� Th cells (1, 36, 37). Only for these lasttwo alleles, and one particular -DRB1*03 allele, has the completecDRB sequence been published (23). Mamu-DRB1*0406, and espe-cially -DRB*W201, seem to be significant restriction elements in cel-lular response to conserved regions of SIV/simian human immuno-deficiency virus (38, 39).

To assign a full-scale analysis of the Mamu-DR region geneticmakeup and the transcription of its genes, we analyzed cDRA andcDRB alleles present on the most prominent -DR region configu-rations in our pedigreed colony of rhesus macaques.

Materials and MethodsAnimals and cells

The rhesus macaques (Macaca mulatta) were serologically typed for theirMHC class I (Mamu-A and -B) and class II (Mamu-DR) Ags. In the Bio-medical Primate Research Center breeding colony (Rijswijk, The Nether-lands), 253 MHC haplotypes have been defined based on the segregation of13 Mamu-A, 14 Mamu-B, and 9 Mamu-DR serotypes. Peripheral blood lym-

phocytes or immortalized B cell lines used in this study originate from 14pedigreed animals in Biomedical Primate Research Center’s self-sustainingcolony (Indian origin). Of these rhesus macaques, five were homozygous andderived from consanguineous origin, two were homozygous, and seven wereheterozygous for their Mhc regions. This panel covers 11 of the most frequentMamu-DRB region configurations present in our colony, as well as some ex-amples of DRB region configurations displaying allelic polymorphism (12, 26,28, 31).

RNA extraction, cDNA synthesis, and amplification

RNA was extracted from immortalized B cell lines of rhesus macaquesusing the RNeasy mini kit (Qiagen, Hilden, Germany) according to themanufacturer’s recommendations. cDNA was then synthesized fromfreshly extracted mRNA using the Universal RiboClone cDNA SynthesisSystem (Promega, Madison, WI) according to the manufacturer’s recom-mendations. Full-length Mamu-DRA sequences were amplified by PCRfrom cDNA using primers specific for human DRA 5� and 3� untranslatedsequences (1): 5�DRA-SalI, 5�-TCC CGT CGA CCG CCC AAG AAGAAA ATG GCC-3�, and 3�DRA-BamHI, 5�-CAT TGG ATC CGA AGTTTC TTC AGT GAT CTT-3�.

Likewise, Mamu-DRB sequences were amplified by PCR using primersspecific for human 5�- and 3�-untranslated sequences (1): 5�DRB-SalI, 5�-GCC CGT CGA CCT GTC CTG TTC TCC AGC ATG-3�, and 3�DRB-BamHI, 5�-GGC GGG ATC CCT TTT CAT CCT GCA AAG CTG-3�.Primers were synthesized by Invitrogen (Paisley, U.K.). PCR was performedin a 100 �l reaction volume containing 5 U of Taq polymerase (kindly donatedby M. Morl, Max-Planck-Institut, Saarbrucken, Germany) with 0.5 �M ofeach primer, 1.5 mM MgCl2, 250 �M dNTPs, 1� PCR buffer II (AppliedBiosystems, Foster City, CA) , and 10 �l of DNA. The cycling parameterswere a 2 min at 94°C initial denaturation step, followed by 25 cycles of a 2 minat 94°C denaturation step, a 2 min at 60°C annealing step, and a 2 min at 72°Cextension step. A final extension step was performed for 7 min at 72°C.

Cloning and sequencing

PCR products were digested with the restriction enzymes SalI and BamHI(Invitrogen). The 5� SalI and 3� BamHI restriction sites facilitated sticky-endedligations into the multiple cloning site of the sequencing vector M13mp18(Qbiogene, Montreal, Canada). The M13mp18 vector includes a M13 se-quence before the cloning site, which is used for the sequencing of the product.

FIGURE 1. Polymorphic sites of the full-length coding Mamu-DRA nucleotide sequences (A) and deduced amino acid sequences (B) in comparison toHLA-DRA and Patr-DRA. Only polymorphic sites are shown using small and capital letters, which indicates synonymous and nonsynonymous mutations,respectively. Identity to the consensus is illustrated by a dash. The Mamu-DRA*0101 sequence published earlier is boxed (23). The two shadowed boxesrepresent two distinct lineages. 4 Patr, Pan troglodytes; 5 LP, leader peptide; 6 TD, transmembrane domain; 7 CD, cytoplasmatic domain.

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The purified cDNA was sequenced on the ABI 3100 genetic analyzer (AppliedBiosystems, Foster City, CA) using 0.2 �M M13 primer, 1 �l of BigDyeTerminator (Applied Biosystems), and 2 �l of 5� dilution buffer (400 mMTris-HCl, 10 mM MgCl2) in a total of 10 �l. The resulting sequences wereanalyzed using the Sequence Navigator program (Applied Biosystems). All

sequences have been deposited in the databank (cDRA accession numbers:AJ586874–AJ586884, and cDRB accession numbers: AJ601348–AJ601351,AJ601354–AJ601362, and AJ601364–AJ601372) and are also available viathe IMGT/MHC database (www.ebi.ac.uk/ipd/mhc/nhp; European Bioinfor-matics Institute, Cambridge, U.K.).

Phylogenetic analysis

Phylogenetic analysis of the cDRA and cDRB sequences was performedusing PAUP, version 4.0b.10 (40). Pairwise distances were calculated us-ing the Kimura-2 parameter, and the neighbor-joining method was used tocreate a phylogram. Confidence estimates of the groupings were calculatedaccording to the bootstrap method generated from 1000 replicates.

Results and DiscussionMamu-cDRA alleles

As in humans, the Mamu-DRA gene encoding the DR �-chain isthought to be monomorphic. However, only one study on a full-length cDRA sequence has been published (23) and to our knowl-edge, population analyses have not been conducted. To investigatethe existence of Mamu-DRA polymorphism, full-length cDRA al-leles of 17 selected B cell lines have been amplified and se-quenced. This enterprise resulted in the detection of 11 unpub-lished cDRA alleles. Based on deduced amino acid sequences, fourdistinct Mamu-cDRA transcripts could be distinguished in ourpanel named Mamu-DRA*0102 to -DRA*0105 (Fig. 1, A and B).The existence of the previously reported Mamu-DRA*0101 allelecould not be confirmed in our panel of Indian monkeys, but itssequence is nevertheless included in the alignments. Most of thealleles have point mutations with a synonymous character, as isreflected in the names of the alleles: Mamu-DRA*01021 to-DRA*01027 (30). The end of exon 3 of the Mamu-DRA alleles ischaracterized by the presence of two different motifs (Fig. 1A).Phylogenetic analysis demonstrates that, based on the presence of

FIGURE 2. Phylogenetic tree of HLA-, Patr-, and Mamu-cDRA alleles.As in Fig. 1, the shadowed boxes represent two distinct lineages. The treeis rooted with Patr-DRA as the outgroup. 4 Patr, Pan troglodytes.

FIGURE 3. Deduced amino acid sequences of full-length Mamu-cDRB alleles. The HLA-DRB1*0101 sequence is chosen as reference. Identity to theconsensus is illustrated by dashes. The three Mamu-DRB alleles published earlier are boxed (23). The underlined and shadowed amino acid sequences ofthe �1 domain, represent the motifs differentiating the lineages. CP, Connecting peptide; TD, transmembrane domain; CD, cytoplasmatic domain.

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these motifs, the Mamu-DRA alleles cluster into two distinct lin-eages (Fig. 2). Based on the generally accepted divergence time ofrhesus monkeys and humans of 35 million years, the mean evo-lutionary rate of HLA-DRA and Mamu-DRA alleles can be calcu-lated to be �0.31 � 10�9 substitutions per site, per year (41). Thedivergence time of the two Mamu-DRA lineages is then calculatedto be �10 million years. Thus, the Mamu-DRA lineages seem to berelatively old and, as a consequence, they may also be present in othermacaque and Old World monkey species. The Mamu-DRA*01041and -DRA*01042 alleles code for the same protein (Fig. 1B). How-ever, they do group into different lineages (Figs. 1A and 2). Thisexample illustrates that the boxed polymorphic motif probably hasbeen exchanged in a recombination-like event (Fig. 1A). Hence, at thisstage we have decided not to implement a nomenclature protocol thatdiscriminates between the two Mamu-DRA lineages.

Whereas the synonymous mutations are randomly distributedover the entire cDRA sequence, the nonsynonymous mutations ob-served in the rhesus macaque panel result in amino acid replace-ments restricted to the leader peptide or the cytoplasmic domain(Fig. 1, A and B). If one takes the HLA-DR �-chain as a reference,the chimpanzee orthologue displays only two polymorphic resi-dues, whereas in total, 16 aa replacements have been observed inthe Mamu-DR �-chains (Fig. 1B). Humans, chimpanzees, and rhe-sus macaques shared a common ancestor �35 million years ago.Because all the anchor residues of the �1 domain have been con-served in all three primate species studied (Fig. 1B), this observa-tion underlines the importance of these specific amino acids forpeptide binding. The question left to answer is why do rhesusmacaques display polymorphism at the Mamu-DRA gene, and is asimilar phenomenon apparently not the case for HLA-DRA gene inthe human population? One argument could be that not many hu-man subjects have been studied for HLA-DRA polymorphism. Amore plausible explanation takes the differential divergence timesof both species into account, which is thought to be 250,000 yearsfor modern humans and �700,000 years for rhesus monkeys (32,42, 43). In brief, the time period needed to accumulate point mu-tations was approximately three times longer for rhesus macaques.On top of that, and as will be discussed later in detail, the Mamu-DRA alleles appear to be more or less Mamu-DR configuration-specific. This suggests that some of these DR configurations mayhave been stable entities over a relatively long evolutionarytime span.

Mamu-cDRB alleles

To date, 134 Mamu-DRB exon 2 sequences have been identified(30). Most of these alleles belong to loci/lineages that are sharedbetween humans and rhesus macaques, whereas for the DRB*Walleles, no human equivalents have been described. Little is knownabout the gene products (35) and only a meager number of full-length cDRB sequences have been published so far (23, 37). Tolearn more about the peptide-binding profiles of rhesus macaqueclass II molecules it is, in the first place, essential to know if theclass II genes are actually expressed. An example is provided bythe Mamu-DRB*W201 molecule, which plays an important role inthe peptide binding of conserved epitopes of the simian humanimmunodeficiency virus (39, 44).

To obtain more fundamental insight into the genetics of theMamu-DR region and the expression status of the various genes,cDRB genes were analyzed in a panel of 15 animals with thor-oughly defined DRB configurations. From these animals, 28 cDRBalleles could be isolated, and the deduced amino acid sequenceshave been determined (Fig. 3). Moderate heterogeneity is observedwithin the �2 domain, whereas relatively little variation is noticedin the leader peptide, connecting peptide, transmembrane domain,

and cytoplasmatic domain. As expected, the �1 domain encodedby exon 2 represents the most polymorphic part of the -DRB gene.Most of the residues that are known from the HLA-DRB1 moleculeto contribute to the peptide binding are variable in the rhesus ma-caque (45). Phylogenetic analysis shows that alleles of the samelineage within one species, for example, members of the Mamu-DRB*W6 lineage, cluster tightly together. A similar observationcan be made for rhesus macaque, chimpanzee, and human -DRB5lineage (Fig. 4). If sequences fall apart in the phylogenetic anal-ysis, an identical or similar motif constituted by aa 9–13, has beenthe decisive factor for lineage designation. (Figs. 3 and 4).

Mamu-DR configurations

Although Mamu-DRB sequences show a high degree of variability,the main feature is its unprecedented -DRB region configurationpolymorphism, which is defined by a variable number and contentof -DRB genes per haplotype (28). Animals selected for this studypossess the 11 most prominent Indian DRB region configurationsin our colony and each configuration harbors 2–4 -DRB genes (28,31). In addition, nearly every Mamu-DRB configuration contains1–3 DRB6 genes, most of them characterized by a 62 bp deletion(24, 26). The choice of animals, which are mainly homozygous fortheir MHC, allowed us to define the DRA/DRB gene combinationssegregating on one haplotype (Fig. 5). As can be seen, the 11Mamu-DRB region configurations analyzed previously could bedivided into 12 different DRA/DRB combinations. Only one -DRB

FIGURE 4. Phylogenetic tree of HLA-, Patr-, and Mamu-cDRB alleles.Some selected HLA-DRB and Patr- alleles, representing different lineages,have been added to the phylogenetic analysis. The tree was rooted with the-DRB orthologue, Sanguinus oedipus -DRB*02, of the cotton-top tamarinas the outgroup. 9 Saoe, Sanguinus oedipus.

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region configuration displayed limited allelic variation at the DRBloci (Fig. 5; 1a, 1b, 2a, and 2b), which can be separated into twoDRA/DRB configurations according to different accompanying-DRA alleles. The DRB combinations 1a/1b and 2a/2b differ onlyfor one nucleotide in their DRB1*03 or DRB1*10 lineage alleles,respectively. This is most likely an example of a polymorphismthat was generated after the DRB region configurations themselveswere established. We have postulated that the differential numbersof Mamu-DR genes and their order is due to rearrangements byunequal crossing-over events (12, 26). In this light, the followingobservation is highly indicative. In one rhesus macaque of Bur-mese origin, three different -DRA alleles have been detected, andthe presence of two -DRA alleles on one haplotype was proven bysegregation analysis (data not shown). This unique configuration isprobably caused by an unequal crossing-over event, once againillustrating the great plasticity of the Mamu-DR region. Futureplans to initiate the sequencing of the whole genome of the rhesusmacaque will elucidate the order of the genes and shed light on therecombination hotspots and physical distances between the differ-ent loci. On average, one can conclude, however, that most of theDRB configurations segregate in combination with a uniqueMamu-DRA allele (Fig. 5).

DR region: transcripts and pseudogenes

Some of the -DRB exon 2 sequences detected by amplifyinggenomic DNA could not be recovered at the transcript level. In thecase of the configuration n 4, the Mamu-DRB6*0101 and-DRB1*0309 genes are not transcribed in such a way that theyresult in a functional �-chain (Fig. 5). This result was confirmed bythe analysis of cDRB of a Burmese-origin rhesus macaque family,in which exon 2 of the DRB1*0309 allele can also be detected onthe genomic, but not on the cDNA level. These results have beenvalidated by dot-blot experiments (results not shown). In the caseof the Mamu-DRB6 gene, this observation is not surprising be-cause this gene is a pseudogene in humans and chimpanzees (33).Because the separation of eastern (Burmese and Chinese) andwestern (Pakistan and Indian) macaque populations is speculated

to be due to a glacial barrier during the Pleistocene era, the prob-able inactivation of a formerly active gene took place before thattime point (32). In the case of the MHC, it has been shown thatpseudogenes are maintained over long evolutionary time spans.This may be due to a “piggy back” effect (one gene is linked toanother that is experiencing strong conservative or positive selec-tion). It has also been hypothesized that pseudogenes are a reser-voir for gene segments that can be exchanged between relatedgenes by recombination-like processes. In contrast, some pseudo-genes contain premature stop codons and may be partially trans-lated into proteins. In such a case, a pseudogene may encode pep-tides that are important for thymic education/selection.

In five other -DRA/DRB conformations (Fig. 5; n � 3, 6, 9, 11,and 12), there is one -DRB allele of which the exon 2 sequence ofgenomic DNA was sequenced, but a transcript has not been detected.The results have been confirmed by analysis of a second animal withthe same region configuration and whenever necessary, they havebeen repeated (data not shown). These untranscribed alleles belong tovarious loci/lineages that have most likely been inactivated and nowcan be considered as pseudogenes. This phenomenon is known fromthe human situation where DRB6 as well as other DRB loci, for ex-ample DRB2, are rendered as pseudogenes.

However, in rhesus macaques, a certain -DRB locus or even alineage may harbor transcribed, as well as untranscribed, alleles(pseudogenes); examples are given for -DRB1*03, -DRB3, andMamu-DRB*W6 (Fig. 5; n � 3, 4, 6, 9, and 11). In relation to HLA,the Mamu-DRB5 locus appears to be coding, whereas none of the-DRB6 alleles was completely transcribed, which is an indicationthat this locus is a pseudogene in the rhesus macaque as well. Thenumber of Mamu-DRB genes that are transcribed into RNA canvary from 1 to 3 per haplotype.

The results of this study provide a detailed answer to the questionof which -DRB alleles are transcribed, and they unravel the complex-ity caused by the large number of Mamu-DRB region configurations.In conclusion, the data presented here will be invaluable in preclinicalstudies in which a detailed knowledge of the rhesus macaque DRregion makeup is essential.

FIGURE 5. Overview of the genecontent of Mamu-DRA/DRB configu-rations. The different configurationsare numbered arbitrarily in Arabic nu-merals; same configurations with al-lelic variations are indicated by A andB, respectively. Expressed loci/allelesare depicted in black, whereas pseudo-genes are boxed. aOnly DRB6 alleleswithout the 62-bp deletion are listed.bMHC homozygous animals from con-sanguineous matings. cMHC homozy-gous animals. dDRA allele belonging tothis confirmation was detected by se-quencing analysis of genomic DNA.eThe serological Mamu-DR nomencla-ture is described by Bontrop et al. (46).

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AcknowledgmentsWe thank Donna Devine for assistance in editing the manuscript, andHenk van Westbroek for preparing the figures.

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