The MHC class I genes of the rhesus monkey: Different evolutionary histories of MHC class I and II genes in primates

The MHC Class I Genes of the Rhesus Monkey

Different Evolutionary Histories of MHC Class I and II Genes in Primates'

Jonathan E. Boyson,* Clare Shufflebotham,* Luis F. Cadavid,* Julie A. Urvater,* Leslie A. Knapp,* Austin L. Hughes,+ and David 1. Watkins2*

Homologues of the human HLA-A and -B MHC class I loci have been found in great apes and Old World primates suggesting that these two loci have existed for at least 30 million years. The C locus, however, shows some sequence similarity to the B locus and has been found only in gorillas, chimpanzees, and humans. To determine the age of the MHC class I C locus and to examine the evolution of the A and B loci we have cloned, sequenced, and in vitro translated 16 MHC class I cDNAs from two unrelated rhesus monkeys (Macaca mulatta) using both cDNA library screening and PCR amplification. Analyses of these sequences suggest that the C locus is not present in the rhesus monkey, indicating that this locus may be of recent origin in gorillas, chimpanzees, and humans. The rhesus monkey's complement of MHC class I genes includes the products of at least one expressed A locus and at least two expressed 6 loci, indicating that a duplication of the B locus has taken place in the lineage leading to these Old World primates. Comparison of rhesus monkey MHC class I cDNAs to their primate counterparts reveals fundamental differences between MHC class I and class I1 evolution in primates. Although MHC class II allelic lineages are shared between humans and Old World primates, no such trans-species sharing of allelic lineages is seen at the MHC class I loci. The lournal of Immunology, 1996, 156: 4656-4665.

M HC class I3 molecules play a crucial role in the im- mune response to both viral and bacterial pathogens (1). They bind peptides derived from these pathogens

and present them to CD8+ CTLs, which mediate destruction of the infected target cell, thereby preventing widespread dissemination of the pathogen (2-6). The human MHC class I loci, HLA-A, -B, and -C, are not orthologous to their mouse H2-D, -K, and -L coun- terparts. That is, it is difficult to trace the mouse and the human MHC class I genes back to a common shared ancestor. In contrast, MHC class I1 genes of the mouse and human appear to be ortholo- gous. This suggests that there is a certain amount of plasticity at the MHC class I loci that is not present at the MHC class I1 loci. We examined the MHC class I loci of the rhesus monkey to shed further light on the mechanisms of evolution of the primate MHC class I loci.

Unlike the MHC class I loci, the MHC class I1 loci and alleles of the rhesus monkey have been extensively studied. MHC class I1 genes and allelic lineages are remarkably well preserved during

*University of Wisconsin, Madison, WI 5371 5 ; and +Perm State University, Uni- versity Park, PA 16802

Received for publication December 26, 1995. Accepted for publication March 14, 1996.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by grants from the National Institutes of Health (RR00167 and A132426 to D.I.W. and CM43940 to A.L.H.) and a Biomedical Science Grant from the Arthritis Foundation.

Address correspondence and reprint requests to Dr. David I. Watkins, Wiscon- sin Regional Primate Research Center and Department of Pathology, University of Wisconsin, 1220 Capitol Court, Madison, WI 5371 5.

posited with CenBank with accession numbers: Mamu-A'03, U41379; Mamu- Data deposition: The sequences described in this manuscript have been de-

A*04, U41380; Mamu-A*05, U41831; Marnu-A*06, U41834; Mamu-A*07, U41832; Mamu-6'02, U41833; Mamu-B*03, U41825; Mamu-B*04, U41826; Mamu-B*05, U41827; Mamu-6'06, U41828; Mamu-B*07, U41829; Mamu- 6'08, U41830; Mamu-6'09, U41835; Mamu-6'1 I, U41838; and Mamu-E*05, U41837.

Copyright 0 1996 by The American Association of immunologists

evolution of the great apes and Old World primates (7). Although there is evidence that diversity at the DRB loci in primates has been generated by recombination, HLA-DRB and Patr-DRB alleles of the same lineage cluster together in phylogenetic trees (8, 9). Indeed, Patr-DRB4*014 and HLA-DRB4*0104 differ at only three residues in the a 1 domain, and the a 2 domains of DRB molecules are conserved between chimpanzees and humans (IO). This shar- ing of trans-species DRB sequence lineages is observed even be- tween humans and rhesus macaques ( I l). Furthermore, a rhesus monkey Mamu-DRB1*03 molecule can present a peptide from Mycobacterium tuberculosis to a human T cell clone restricted by HLA-DR17 (12). These data therefore imply that the homologues of HL4-DRA (IO), -DRB (8 , 9), -DQA (13), and -DQB (14, 15) alleles in great apes and Old World primates are stable and evolve in a trans-specific fashion (16).

MHC class I cDNAs have been cloned from several different species of great apes and Old World primates. These include chim- panzees (17-21), pygmy chimpanzees (20-22), gorillas (23, 24), orangutans (25), gibbons (25), and three MHC class I sequences have been isolated from the rhesus monkey (26-28). In these stud- ies, it has been shown that the HLA-A and -B homologues are present in all of these Old World primates and great apes. Indeed, the HLA-A alleles of the chimpanzee and pygmy chimpanzee are very similar to their human counterparts in that an allelic lineage is preserved between these two species (17-20, 22). Similarly, exon 2, encoding the a 1 domain of the HLA-B locus is conserved between chimpanzees and humans (21). However, in exon 3 en- coding the a 2 domain there is very little sequence similarity be- tween these two species. This dissimilarity is exaggerated in go- rillas, orangutans, gibbons, and rhesus monkeys. Additionally, it has been suggested that the orangutan has two HLA-B locus ho- mologues (25). No C genes or cDNAs from the C locus have as yet been cloned from the Old World primates, orangutans and gib- bons. In contrast, C locus cDNAs are easily isolated from chim- panzees and gorillas (17, 23). This has led to speculation that the C locus is a recent duplication of the B locus in great apes and

0022-1 767/96/$02.00

The Journal of Immunology 4657

Table I. Primers used for the amplification and sequencing o f MHC class I cDNAs from the rhesus monkey

Amplification Primers

Primer Binding region and position Sequence ~

5' BETA 3 XHO" 3' BETA 2 H3" 3' ALOC H3' 3' CLOC H3' 5' MAS",' 3' MASc 5' MBS' 3' MBS' NA1 STARTd A2 END Hl l l A3 MID T7

Sense 5'UTR ( -27-7) A l l Loci Antisense 3'UTR (Approx. 170-205) 6 Locus Antisense 3'UTR (Approx. 170-205) A LOCUS Antisense 3'UTR (Approx. 170-205) c Locus Sense exon 1 (10-34) A Locus Antisense exon 8 (2)-3'UTR (1 8) A LOCUS Sense exon 1 (10-34) 6 Locus Antisense exon 8 (2)-3'UTR (1 8) 6 Locus Sense exon 2 (1-23) All Loci Antisense exon 3 (248-272) All Loci Antisense exon 4 (85-1 04) Al l Loci T7 Promoter-pSP72 (99-1 18)

5'-CGCTCGAGGACTCAGAATCTCCCCAGACGCCGAG-3' 5'-CGAAGCTTGGAGGAAACACAGGTCAGCATGGGAAC-3' 5'-CCGCAAGCTTTTGGGGAGGGAGCACAGGTCAGCGTGGGAAG-3' 5'-CCGCAAGCTTTCGGGGAGGGAACACAGGTCAGTGTGGGGAC-3' 5'-GCCTCGAGAATTCATGGCGCCCCGAACCCTCCTCCTGG-3' 5'-GCAAGCTTCTAGACCACACAAGGCGGCTGTCTCAC-3' 5'-GCCTCGAGAATTCATGGCGCCCCGAACCCTCCTCCTGC-3' 5'-GCAAGCTTCTAGACCACACAAGACAGTTGTCTCAG-3' 5'-GCGAATTCGCTCYCACTCCWTGAFlGTATTTC-3' 5'-GCAAGCTTGCGCTGCAGCGTCTCCTTCCCGTTC-3' 5'-CCAGGTCAGTGTGATCTCCG-3' 5'-TAATACGACTCACTATAGGG-3'

Sequencing Primers

Primer Binding region Sequence

T7 T7 Promoter-pSP72 (99-1 18) 5'-TAATACGACTCACTATAGGG-3' SP6 SP6 Promoter-pSP72 (2445-2462) 5'-GATTTAGGTGACACTATAG-3' PJ1+ SP6 Promoter-pSP72 (2438-2462) 5'-CACATACGATTTAGGTGACACTATAG-3' 812 + Sense exon 3 (83-1 01 ) 5'-CGACGGCAAGGATTACATC-3' c11 + Sense exon 3 (235-254) 5'-GCAGATACCTGGAGAACGGG-3' IV Sense exon 4 (1 68-1 86) 5'-GGAACCTTCCAGAAGTGGG-3' El1 - Antisense 3'UTR (12-30) 5'-TGCATCTCAGTCCCACACA-3' F/1 - Antisense exon 4 (85-1 04) 5'-CCAGGTCAGTGTGATCTCCG-3' GI2 - Antisense exon 2 (249-268) 5'-GCCTCGCTCTGGTTGTAGTA-3' 3'UTR Sense exon 7 (1 5-38) 5'-CAGGGCTCTGATGTGTCTCTCACG-3'

a Based on Blocus specific primers modified from HLA-5P2 and HLA-3PBloc. (23). From Lawlor et al. (23). Rheusus macaque locus-specific primers that were designed in our laboratory, based on Dr. Ronald Bontrop's and our own unpublished rhesus macaque cDNAs. Mixed bases in the above primers are as follows: Y = C or T, R = A or C, and W = A or T.

humans. Since the ancestors of the rhesus monkey diverged from the lineage leading to humans approximately 35 million years ago, we compared the MHC class I genes of this species to their non- human primate and human counterparts. This has enabled us to determine the constitution of an MHC class I haplotype in the rhesus monkey and to gain insight into how the MHC class I loci may have evolved in primates.

Materials and Methods Animals

Whole blood was obtained by venipuncture from rhesus monkeys (Mucuca muluttu) from the Wisconsin Regional Primate Research Center and the New Iberia Research Center.

Cell culture

PBLs were separated from whole blood using FicolVdiatrizoate gradient centrifugation. These cells were transformed with Herpesvirus pupio by culturing PBLs with supernatants of the S-594 cell line. PBLs were also cultured with Con A (5 pg/ml; Sigma Chemical Co., St. Louis, MO) and rIL-2 (20 U h l ; a gift from Roche, Nutley, NJ). Transformed and Con A-activated lymphocytes were cultured at lo6 cellslml in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Sterile Systems, Inc., Logan, UT), 2 mM L-glutamine, 5 X IO-' M 2-ME, 20 mM HEPES, 50 U/ml penicillin, and 50 pg/ml streptomycin.

RNA extraction, cDNA synthesis, and polymerase chain reaction from rhesus monkey 84557

PCR amplification of cDNA was conducted on H. pupio-transformed cells from rhesus monkey 84557. Total cellular RNA was extracted from 7 X IO6 H. pupio-transformed lymphocytes using RNAzol (Tel-Test Inc., Friendswood, TX). One microgram of this RNA was then used to synthe-

size cDNA with random hexamers and Moloney murine leukemia virus reverse transcriptase (Gene Amp-Perkin Elmer, Foster City, CA). The cDNA synthesis reaction mixture contained 50 mM Tris, pH 8.3, 5 mM MgCl,, 1 mM each of dATP, dGTP, dCTP, and dTTP (Gene Amp-Perkin Elmer). Random hexamers (25 pg/ml), 50 U of Moloney murine leukemia virus reverse transcriptase and 20 U of RNAsin were added to give a final volume of 20 pl. cDNA was then synthesized at room temperature for 10 min, 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min.

PCR was then conducted using the primers listed in Table I, each at a final concentration of 250 pM. The PCR mixture contained 2 mM MgCI,, 50 mM Tris, pH 8.3 and 2.5 U AmpZiTuq DNA Polymerase (Gene Amp- Perkin Elmer). Each reaction contained 20 pl of cDNA and the final vol- ume was 100 PI. The reactions were heated to 94°C for 2 min, and then amplification was conducted for 30 cycles as follows: denature for 1 min at 94T , anneal for 1 min at 60°C. and extend for 1 min 30 s at 72OC. A final extension was then conducted for 10 min at 72°C.

Subcloning and sequencing

After amplification, the PCR product was ligated into pSW2 (Promega, Madison, WI), and double-stranded sequence was obtained from LO MHC class I-specific sequencing primers using an AB1 373 automated sequenc- ing machine (ABI, Foster City, CA). At least three copies of each cDNA (except for Marnu-AWS) were sequenced to avoid PCR-generated artifacts (29). Only two copies of Mumu-A*05 were obtained from rhesus monkey 84557. These were identical with the exception of one nucleotide (position 112 in exon 4). One of the substitutions was found to be conserved among all other MHC class I cDNAs. Thus, we concluded that the correct nucle- otide was the conserved one. Sequence was analyzed with the use of soft- ware from IBI (New Haven, CT) and ABI.

cDNA library synthesis and screening from rhesus monkey 88090

mRNA was extracted from 220 X lo6 4-day-old Con A blasts from rhesus monkey 88090 using TRIzol and oligo dT cellulose, as per the manufac- turer's protocols (Life Technologies, Gaithersburg, MD). mRNA (1.8 mg) was used to construct a cDNA library in pSPORTl using the Superscript

4658 THE MHC CLASS I LOCI OF THE RHESUS MONKEY

A3MIDLIB 5" CSGAGATCAYRBTGACVTGGC -3'

85 105

HLA-A'OIOZ HLA-B*2703 .....................

HLA-E*0102 HLA-C*0102

HLA-G ..................... HLA-F ..................... HLA-H .....................

I I . . ...................

.....................

.....................

Paga-A+04 Paga-B'O1 Patr-A*O1 Patr-B*Ol Patr-C'O1 Patr-F*Ol ~090-~*0201 Gogo-B*OlOl Gogo-C*OlOl Pogy-A*Ol PoRY-B'O~ Pogy-E+O1 Hyla-A*01

Mamu-A*Ol Hyla-B*Ol

Mamu-B*Ol Mamu-F'01

Mafa-E*Ol Mafa-A*Ol

Saoe-G*Ol Saoe-F*Ol

EXON: 1 2 3 4 5 6 7 8 3 UTR

FIGURE 1. Oligonucleotide design used for cDNA library screening. The oligonucleotide A3 MIDLIB was designed to screen the rhesus monkey cDNA library for MHC class I cDNAs by binding to a con- served region of exon 4 (spanning nucleotides 85 to 105). Shown is an alignment of the A3 MIDLIB primer with MHC class I sequences from different loci of a variety of primate species. Identical bases are repre- sented by a period (.), and the mixed bases in the oligonucleotide are as follows: Y = C or T; R = A or G; B = C, G, or T; S = C or C; and V = G, A, or C.

Plasmid System (Life Technologies). The library was used to transform competent DHIOBTM cells (Life Technologies) using electroporation giv- ing 1.2 X IO6 primary colonies. After amplification, the library was screened using the Gene TrapperTM cDNA positive selection system (Life Technologies). The only modification to the manufacturer's instructions was that a QIA filter and QIAGEN plasmid Midi kit (Chatsworth, CA) was used to prepare double-stranded plasmid DNA from the amplified library.

The library was screened using the A3 MIDLIB oligonucleotide (Fig. I ) , which was designed to hybridize to a conserved region in exon 4 of all primate MHC class I cDNAs. Recovered colonies were screened using colony PCR with PCR primer pairs NAlSTART and A2END and T7 and A3 MID (Table I). Sixty of 160 colonies resulted in positive PCR reactions and these were sequenced with the NAlSTART primer after shrimp alka- line phosphatase and exonuclease I purification of the PCR product (30). Full length sequencing of eight unique MHC class I cDNAs was performed as described for analysis of the PCR products from 84557 except that only one clone was sequenced for each cDNA.

Gene tree analysis of the MHC class I loci of the rhesus monkey

Gene trees were constructed by the neighbor joining method (31) on the basis of the number of nucleotide substitutions per site (d ) , estimated by Jukes and Cantor's method (32). In estimation of d in pairwise comparisons between sequences, sites were excluded from the analysis at which the alignment indicated a gap. The reliability of clustering patterns in the trees was tested by the standard error test for internal branches (33).

Results PCR amplification from rhesus monkey 84557 results in the isolation of eight cDNAs

Initial amplification of full-length MHC class I cDNAs from rhe- sus monkey 84557 using PCR primers specific for cDNAs of the human A, B, and C loci resulted in the isolation of six different cDNAs (Table 11). There was no concordance between the rhesus monkey locus amplified and the human locus-specific primers set used. We then designed two new primer sets (MAS and MBS; Table I) based on unpublished MHC class I cDNAs from our lab- oratory and from Dr. Ronald Bontrop's laboratory (TNO Primate Center, The Netherlands). These primers proved to be locus-spe- cific in the rhesus monkey and resulted in the isolation of two additional cDNAs that were not amplified by the human locus- specific primers. These new primers also amplified the original six cDNAs. Therefore, PCR amplification using five different sets of PCR primers resulted in the isolation of three A locus and five B locus alleles from animal 84557 (Table 11). Analysis of the pre- dicted amino acid sequence enabled us to determine that these alleles were products of the A and B locus (Fig. 2).

Table II. Summary o f a l l M H C class I cDNAs isolated from rhesus monkeys 84557 and 88090

Allele Number of

copies Animal Primer paids

Mamu A*05 2 84557 5'/3' MAS Mamu A*06a 18 84557 5'/3' MAS AND 5' BETA 3 XH0/3' CLOC H3 Mamu A*07 21 84557 5'/3' MAS, 5' BETA 3 XHO/ 3' CLOC H3 AND 5' BETA 3 XH0/3' ALOC H3 Mamu B*01 6 84557 5'/3' MBS Mamu 5*06 6 84557 5'/3' MBS AND 5' BETA 3 XHO/ 3'BETA 2 H3 Mamu 5*07 24 84557 5'/3' MBS, 5' BETA 3 XHO/ 3'BETA 2 H3 AND 5' BETA 3 XHO/ 3' ALOC H3 Mamu B*08 16 84557 5'/3' MBS, 5' BETA 3 XHO/ 3'BETA 2 H3, 5' BETA 3 XHOI 3' CLOC H3 AND

Mamu 5*09 5 84557 5'/3' MBS, 5' BETA 3 XHO/ 3'BETA 2 H3 AND 5' BETA 3 XHO/ 3' CLOC H3 Mamu A*03 7 88090 cDNA library Mamu A*04 2 88090 cDNA library Mamu B*02 8 88090 cDNA library Mamu B*03 2 88090 cDNA library Mamu B*04 3 88090 cDNA library Mamu 5*05 5 88090 cDNA library Mamu 5*7 1 2 88090 cDNA library Mamu E*05 16 88090 cDNA library

Bold typing indicates MHC class I cDNAs encoding proteins with isoelectric focusing points identical to immunoprecipitated MHC class I

5' BETA 3 XHO/ 3' ALOC H3

molecules from PBLs (data not shown).

The Journal of Immunology 4659

Leader Peptide -20 -10

FIGURE 2. Rhesus monkey MHC class I cDNAs are derived from the A and B loci. Predicted amino acid sequences of the coding region of the rhesus MHC class I cDNAs compared with the human se- quence HLA-A*0101 and selected primate class I sequences are shown. Identity with HLA-A'0101 is indicated with periods (.). Gaps introduced to maximize alignment are indicated by dashes (--). Amino acids 2-71 of the alpha 1 domain are boxed to illustrate the relationship among alleles of the primate A, B, and H loci.

HLA-A'0101 Mamu A*Ol Mamu-A*02 Mamu-A'O4 Mamu-R'O3 Mamu-A*05 Mamu-A'O6 Mamu-A'07 Gogo-OXO HLA-H HLA-B'2702 HLA-B'7901 Popy-B'O1 Mamu-B*Ol Mamu-8.02 Mamu-B'O3 Mamu-B'O4 Mamu-B"O5 Mamu-B*06 Mamu-B'O7 M~~u-B'O~ Mamu-8.09 Mamu-B'11 HLA-C'OlOl HLA-E'O101 Mamu-E'O5

Alpha 1

Mamu A.01 HLA-A'0101

Mamu-A.02 Mamu-A'O4 Mamu-A'O3 Mamu-A'OS Mamu-A'O6 Mamu-A'07 Gogo-OK0 HLA-H HLA-8.2702 MLA-B'7901

Mamu-B'O1 Mamu-8.02 Mamu-0'03 Mamu-B'O4 Mamu-8'05 M m - B * 0 6 Mamu-B*O? Mamu-B'O8 Mamu-B'O9 Mamu-8.11 HLA-C.0101

Mamu-E'OS HLP-E.0101

Popy-B.01

Alpha 2

HLA-A.0101 Mamu A'Ol Mamu-A.02 Mamu-A'O4 Mamu-R'O3 Mamu-A'OS Mamu-A'06 M m - A * 0 7 Gogo-OK0 HLA-H HLA-B.2702 HLA-B.7901 Popy-B"O1 Mamu-8-01 Mamu-B'OZ Mamu-8-03 Mamu-8'04 Mamu-8'05 Mamu-B'06 Mamu-8.01 Mamu-8.08

Mamu-8.11 Mamu-B"O9

HLA-C'0101 HLA-E'0101 M~~u-E'OS

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.V .........RN.. IC .NT..Y.ES.RN.LR..

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. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . LK..H . . . . . . . . . . . . . . S.... . . . . . . . N. ..PR V.. .M....S....R ..SARDTA lF.V . . R . . . . . . . . . . A . . . LK . . H . . . . . . . . . . . . . . s . . . . . . . . . . . . . . . Ph . . . . . . V . . . . . . . . . . . SARDTA..F.V . . K . . . . . . . ..A

100 110 120 130 140 150 160 170 180 * * * , . . * . * . * * . . . * * .

G S H T I Q I N Y G C D V G P D G R F L R G Y R Q D A Y D G K D Y I ~ E D L R S ~ ~ D ~ Q I T K ~ ~ E Q R R ~ L E G R C V D G L R R n ~ G K E T L Q R T . . . . L.R.V . . . L ..... L....E.Y.....................V...N.Q.....ADV..SM.A....Q..EW.P....K........ . . . . . . R . . . . . L.....L....H.S..... . . . . . . . . . . . . . . . . . N.Q . . . . . AGE . . . H.T . . . . E.L EW . . . . . . . . . . . . . . A . . . . Y.V . . . . . L . . . . . L....E.F.. . . . . . . . D . . . . . . . . L . . . N.Q . . . . . AGV . . . H.T . . . . E.L EW......... . . . . A . . . . F.R.V . . . L . . . . . L....E.Y....R........ . . . . . . . . . N.Q . . . . . AGE . . 1.A . . . . E.LEW . . . . . . . . . . . . . A . . . . . T . . . . . L.... L.. .D.S....R...... . . . . . . . . . . N.Q . . . . AGV .. W.A . . . . E.LES ... H . . . . . . . . . . A . . . . L.R . . . . . L . . . . . L... E.Y . . . . R N.Q AGE 1.A . . . . E.L EW A . . . . R . . . . . L..... L . . E.F . . . . R . . . . . . . . . . . . . . . . . . . N.Q . . . . . AG. . . . M.A . . . . E.LEW . . . H . . . . . . . . . . A . . . . . . R . . . . E...........L..........T..................Q.....ARE..RL.A.M..T..EW...H........... . . . . M.V . . . . . L . . . . . . . . . . E.H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ARR..........EQ.EW . . . . . . . . . . . . A . . . . L.N . . . . . . . . . . . L....H..... . . . . . . . . . S.... .T . . . . . Q... .ARV...L.A.. B . . EW . . . . . . . . . . . . . A

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W.R L D.F S......T.....Q.....ARE...F.A....L..ES........ A L.R L HD.S S......T.....Q.....ARE...L.A....L..EW.............. A

L.T.. .L... .L....H.Y..... F V.....Q.....ARE...V.A.. .T ..EW.............. A L.W L L....H.F........ N.Q AGE M.A T EW H.......... A

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . L.M.H . . . L . . . . . L....Y.R..............H......L...N.Q.....AG V. . . . A . . . . . . . EW . . . . . . K. . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...

. . . . . . . . . . . . . . . . .

. . . . Y.W . . . . . . . . . . . L....D.F . . . . . . . . . . Q.. . . . . V . . . N.Q . . . . . AGE . . . Q.A . . . . T..E W. . . . . . . . . . . A

. . . . L.R.S . . Y.E . . . . L... D Y . . . . R....... . . . . . . L...N.Q... .ARV...L.A... E.P EW.... M..Q....P.A

... V.W.H . . . L... .L....Y.F..... . . . . . . . . . . . . V . . . . . . N.Q . . . . . A.E .. R..A..Q.Q.LEW.............. A

. . L.W . . . . . L . . . . . L... E.F . . . . . . . . . . . . . . . . . . . . . . . RF.Q . . . . . - . . . . L.A . . . . K.LEW. . . . . . QN.S.L.A

. . . L.T . . . . . L . . . . . L....H.Y..... . F V Q.....ARE...V.A....T..EW.............. A . . . L.K.C . . . L . . . . . L....Y.S... R . . . S...........GE...N.Q.....AGE.....A.......EW......K....... A .V.T . . . . . . . . . . . L.. .E.F . . . DR . . . . . . . . . . . . . . . . . . . N.Q. . . .AR. . . KD.A . . . . P . . E.............. A

. . . . L.W.C . . . L . . . . . L....D.Y.....................T.....Q.....ARE.....A....T..EW.. . . . . . . . . . . . A

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A L.W.H L..........E.F.......LT........S.V.T....SE..SNDGSE..HQ.A...DT..BW.............. P

L.W.H EL R E.F LT V T SEQ.SNDASE HQ.A ...DT..EW. HK K. LHL . . . . . .

Isolation of eight MHC class I cDNAs from a rhesus monkey cDNA library

One of the problems with cloning MHC class I genes using PCR amplification from species for which there is no large database is that the genes or cDNAs cloned are dependent on the choice of PCR primers. Additionally, the use of primers designed for human MHC class I genes in phylogenetically divergent species may re- sult in an inability to isolate products or genes of certain MHC class I loci. All of the MHC class I cDNAs and gene cloning to date in nonhuman primates has been conducted using primers that were designed to amplify products of the human MHC class I loci. The exceptions to this are two cDNA libraries made in chimpan- zees (17, 18) and a cDNA library made in the cotton-top tamarin

(34). Therefore, to make any definitive conclusion about the MHC class I loci of any particular primate species, it is important to carry out a complete analysis of a cDNA library using nonspecific MHC class I probes.

Screening of a cDNA library from rhesus monkey 88090 re- sulted in the isolation of seven full-length MHC class I cDNAs and a cDNA that was missing exon 1 encoding the leader peptide (Ta- ble 11). Preliminary analysis of these cDNAs by inspection of their transmembrane and cytoplasmic domains reveals that these eight products were derived from one A locus, three B loci, and one E locus (Fig. 2). The two A locus products were characteristically longer than their B locus counterparts. The E locus product was clearly an E locus product, given its unique peptide binding region

4660 THE MHC CLASS I LOCI OF THE RHESUS MONKEY

Alpha 3

HLA-A'0101

Mamu-A'OZ Mamu A'Ol

Mamu-A*04 Mamu-A'O3 Mamu-A'OS Mamu-A'O6 Mamu-A'O7 Gogo-OK0 HLA-H HLA-8'2702 HLA-0'7901 Popy-6-01 Mamu-B*Ol Mamu-0*02

Mamu-0'04 Mamu-8'03

Mamu-0.05 Mamu-0'06 Mamu-0.07 Mamu-B'OB MamU-B'O9 Mamu-8.11 tL-C*0101 HLA-E"0101 Mamu-E'O5

FIGURE 2. (Continued)

HLA-A'0101

Maw-A'OZ Mamu-A'O1

M m - A * 0 4 Mm-A.03 Mamu-A'OS Mamu-A*06 Mamu-A'O7 Gogo-OK0 HLA-8'2702 HLA-B'7901 POpY-B*Ol Mamu-8'01 Mamu-8-02 Mm-8'03

Mamu-8.05 Mamu-8'04

Mamu-6-06 Mamu-6.07 Mamu-B.08

Mamu-B'11 HLA-C'O101 HLA-E'0101 Mamu-E'O5

M ~ ~ U - B * O ~

280 290 300 310

ELSSQPTIPIVGIIAGLULLGAUITGAWAAUMWRPXSS .PF . . .................................. .P . . . S..L............I.VI...I...I...... .P . . . .................................. .P. . S . . . .............................. .P...S . . . . . . . . . . . . . . T V . . . . . . . . . . . . . . . .P . . . .................................. .P . . ES . . . . . . . . . . . . . . . . V . . . . . . . . .W....

. * . . . . * *

.P..

.P..

.P.

. P. .

.P..

. P. .

.P..

.P..

.P..

.P

.P..

. P. .

. P. .

. P. .

. P. .

. PA.

.P

. . . . . . . . . . .

. s V . . . . . u.

. s . . . . . .V

. s . . . . . . . V .

. s . . . . . .u.

.s . . . . . . . V.

. s . . . . . .u.

.s V.

. s V-

. . . . . . . . V . . s . . . . . . . V. . s . . . . . . . V . .S....M..V. . s ......UT . . . . . . . . V .

. S .A,.....

. . . . . . .

. . . . . . .

. . . . . . . . . . .

(PBR) substitutions and the E-like nature of its cytoplasmic and transmembrane region.

HLA-A- and HLA-€3-specific nucleotide substitutions are present in the rhesus monkey MHC class I cDNAs

HLA-A-, HLA-B-, and HLA-C-specific nucleotide substitutions are present at several positions in human MHC class I cDNAs. Anal- ysis of these positions in the rhesus monkey MHC class I cDNAs revealed that many of these locus-specific substitutions were also present (Table 111). The presence of these human locus-specific substitutions was especially evident in exons 5, 6, and 7.

Gene tree analysis of the MHC class I loci of the rhesus monkey

Since the majority of the locus-specific features of the human A , B, and C loci are found in exons 4 to 8, we determined the relation- ship among the various human and rhesus MHC class I loci using gene tree analysis of this region. These trees confirmed our pre- liminary analyses of the predicted amino acid structure and HLA-A- and HLA-B-specific substitutions. The MHC class I loci of the rhesus monkey were shown to be derived from the A, B, and E loci when trees were made of exons 4 to 8 (Fig. 3). Mamu-A locus alleles clustered with HLA-A locus alleles and clustered differently from the Marnu-B locus alleles that clustered with their primate B locus counterparts.

We then determined whether any MHC class I allelic lineages present in humans were present in the rhesus monkeys. Previous

. . . . . . . . . .

. .AV.AV.'JI.

..AU.AV.VI.

. . AU . AV 'I

..AV.AV VI.

..AV.AU.V..

..AV.AV.V..

..AU.AV.V..

..AV.-..V.. AV . . . . VI.

..AV.AV.V..

. . AU . . . . FI. .AV.AV.V..

'. .AV.AU.V. . . AV . AULUL . . . . . . s.us. .: V....U..

. . . . . M...K...

. . . . . . . c... . T..C..... . . . .

I c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........:.. . . . T . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . u..c.... . . . . . . I..K... . . . . . . . . . . . . .

HLA-A.0101 Mm-A.01 Mmu-A'OZ Mamu-A.04 Mamu-A*03 Mamu-A*05 Mamu-A.06 Mamu-A*07 Gogo-OK0 HLA-8'2702 HLA-B*7901

Mamu-6.01 Mamu-B'OZ Mamu-B'O3 Mamu-B'O4 Mmu-B*05

Mamu-B'O7 Mamu-B'OB

Mamu-8'11 Mamu-B'OP

HLA-C'0101 HLA-E*0101 Mamu-E'05

POpy-6'01

~amu-n*06

3 2 0 330 340

DPXGGSYTQARSSDSAQGSDVSLTACKV' . . . . . . . s . . . . . . . . . . . . . . . . . . . .

. . . . * .

. . . . . . . . . . . . . . . . . . . . . . s s

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . s . . . . . . . . . . . . . . . . . . . .

. . . . . . . s . . . . . . . . . . . . . . . . . . . . .

. . . . . . . s . . . . . . . . . . . . . . . . . . . . G S................. f GG . . . . . S . . . C . . . . . . . . . . . . .' GG . . . . . S . . . . . . . . . . . . . . . . . * GG S * GG S C............ .'

GG . . . . . S . . . . . . . . . . . . . . . . . * GG. . . . . S . . . . N............ * GG S W............. * GG..... s.. . . . . . f

GG . . . . . S. . . . . . . . . . . . . . . . * GG . . . . . F... N . . . . . . . E.... * GG . . . . . S . . . . N........... . *

. . . . . . . . . . . . . . . . . . . . . . . . . . . . s . . . . . .

. . . . . ...

. . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

GG . . . . . S . . . . . N........... * GG.... S .... N........ ,.: GG . .CS . . . . . N......E..I... A. GG . . . . . SK.EW.... . . . ESHSL' G...W..S..VG...S....D,.,,Y,P.

analysis has shown that exon 2 encoding the Q 1 domain is well- conserved between chimpanzees and humans at both the A and B loci (21). This is not true for exon 3 encoding the a 2 domain, where although the A locus is conserved between these two spe- cies, the B locus has diverged considerably by putative segmental exchange events. We, therefore, made trees comparing exon 2 of the rhesus and human A and B locus alleles (Fig. 4A). All of the rhesus monkey A locus alleles clustered outside the six different families of human A locus alleles, suggesting that there was no sharing of allelic lineage over the evolutionary distances separat- ing humans and rhesus monkeys. Interestingly, some of the rhesus monkey A locus alleles (Mamu-A *03, -A*05, -A*06, and -A*07) clustered with exon 2 of other primate A and B locus alleles, in- dicating perhaps that these rhesus monkey A locus alleles were the product of an ancient segmental exchange event.

Like the rhesus monkey A locus alleles, the majority of the rhesus monkey B locus alleles clustered outside the other primate B locus alleles (Fig. 4B). The exception to this was Mamu-B*Ol, which clustered with human, chimpanzee, and gorilla B locus al- leles. Comparison of this rhesus monkey allele to its most similar human allele HLA-B*2702 revealed that it differed by 48 nucleo- tides and 25 amino acids in exons 2 and 3 and the a 1 and 2 domains, respectively. This number of nucleotide differences is as large as the number of nucleotide differences separating the most divergent HLA-B locus alleles (17). In contrast, Mamu- DRBl*O308 and HLA-DRB1*0301 differ by only six nucleotides and two amino acids in exon 2 and in the cy 1 domain, respectively

The Journal of Immunology 4661

Table 111. Conservation of locus-specific nucleotides" in the rhesus monkey MHC class i cDNAs

Human Rhesus monkey

Exon Position A B C A B

1 36 A G C 61 C G C

Gb

2 102 C A C C 125 C T T C/r 153 A A C G 155 A A C C

125 T C C L C 142 T C C T

4 2 C C A C 4 C C A C 8 C A A C

18 A G C - C 29 C C T C

125 C A A A 128 C T T T 155 G A G 5 158 T A A T 164 G A A & 168 G A G C

227 C A G C 236 G A G C 242 T T C T 248 T C G T 255 A A C &

5 15 C T C T 38 C T C C 39 A C C A 52 T C C T 54 C C G C 59 T A *' T 61 c c * C

c

3 2 T G G L C

21 2 C A A &

-

64 C T T c/r 65 T T G T 67 T T C T 68 C C T G

76 C G C C 77 A A T A 79 C C T C 80 T C C T 81 c c c C 95 c c c C

100 C C T C 110 C T T C

6 1 A G C A 3 A G C A

19 A A G A 32 A C C A 33 A T 7 A

7 5 T C C T 6 G A A C

28 T T A T 37 C C T C

- -

C

A T

C C - CfA TIc/A - ClC

A G

T C A A C A

-

-

- C/r

A/c C/r

C G Aic @ C

T T C - C/r C G A C T c

G A C T

38 A A C G N G 44 T A T T A

-

8 1 T d C T 2 G C C

' Locus-specific nucleotides for HLA alleles are defined by Parham et al. (52). Nucleotide substitutions that are not locus-specific in the rhesus monkey

MHC class I cDNAs are underlined. ' Positions at which gaps have been introduced to maximize homology. '' Exon 8 is not translated in HLA-5 alleles.

( 1 I ) . All of the other rhesus monkey B locus alleles clustered out- side both the A and B locus groups. Again, like the rhesus monkey A locus alleles, this pattern of clustering indicates that B locus

Mamu-8'09

- Mamu-B'O6

-I Mamu-8'07 Mamu-B"O5

Patr-6'01

I* HLA-6'0702

G o ~ o - C ' M

Mamu-A*04 Mamu-A'O5

* *

- Mamu-A'O2

HLA-E'O101 Mamo-E'05 Mafa-E'OI

Mamu-F

0 - d

.02

FIGURE 3. There is no evidence for a C locus in the rhesus monkey. Gene tree analysis of exons 4 to 8 of rhesus monkey cDNAs compared with other primate MHC class I cDNAs. Phylogenetic trees are based on nucleotide substitutions per site (d) in exons 4 to 8; *p c: 0.05, * *p < 0.01, and ***p < 0.001.

allelic lineages are not shared between humans and rhesus mon- keys. This is in sharp contrast to the DRBl and DQBl loci where trans-specific sharing of allelic lineages between humans and rhe- sus monkeys is seen ( 1 1).

A segmental exchange event between the A locus and other primate MHC class I loci has resulted in Mamu-A*03, -A*05, -A*06, and -A*07

Four of the Mumu-A locus alleles appeared to be recombinants between the A locus and other primate MHC class I loci. Although Marnu-A*03, -A*OS, -A *06, and -A*07 had transmembrane and cytoplasmic domains characteristic of the A locus (Fig. 2), con- tained A locus-specific nucleotide substitutions (Table HI), and clustered with A locus cDNAs in trees of exons 4 to 8 (Fig. 3), analysis of exon 2 revealed that they were more similar to other primate A, B, and H (35) locus alleles (Figs. 2 and 4A). These rhesus monkey A locus alleles showed some sequence similarity to Gogo-A*03, an allele that is a recombinant between the A and H loci (23,24). Inspection of the nucleotide sequence of Gogo-A*03 and HLA-H revealed a region from nucleotide 3 to 212 of exon 2 that differed by only two nucleotides. Construction of gene trees

4662 THE MHC CLASS I LOCI OF THE RHESUS MONKEY

Mamu-A*04

Patr-6.07

HLA-8'5701 Gogo-8'0101

Papa-B'OG

- HLA-B'7301

HLA-8'0801

-

Gogo-A'O3

Popy-A'O3 Popy-A'O2

B

r

04 O L - " i

d

Popy-8'02 Popy-8'07

HLA-8'0801

HLA-8'7501 HLA-9.4402

HLA-8'1801

HLA-8'1301 Papa-8'06

-

I Mamu-8'07

0 .04 I I

d

FIGURE 4. Alleles at the primate A and B loci do not evolve in a trans-specific fashion. Phylogenetic tree based on nucleotide substitutions per site (d) in exon 2 of primate MHC class I cDNAs, showing relationships among ( A ) A alleles, and ( B ) B alleles.

spanning this region revealed a clustering of HLA-H, Gogo-A*03, Popy-B*Ol, HLA-B*7901, and Mamu-A*03, -A*05, -A*O6, and -A*O7 (Fig. 5). Interestingly, these alleles also clustered more closely with HLA-B*3801, -B*3901, -B*1401, and -B*2701 than they did with other B locus alleles (Fig. 5 and data not shown). Since three of these rhesus monkey A locus alleles (Murnu-A*05, -A*06, and -A *07) were the only A locus cDNAs isolated from 84557, it is likely that they represent bona fide rhesus monkey A locus alleles.

Discussion Analysis of the MHC class I loci of the rhesus monkey by cDNA cloning and sequencing shows that a rhesus monkey MHC class I haplotype consists of at least one HLA-A homologue and at least two HLA-B homologues. No evidence for a HLA-C locus homo- logue was observed, suggesting that the C locus in gorillas, chim- panzees, and humans is of fairly recent origin. This confirms pre- vious data showing no evidence for the presence of HLA-C locus homologues in orangutans and gibbons. Interestingly, we could find no shared A or B locus allelic lineages between humans and rhesus monkeys. This observation contrasts with the finding that

many of the DRBI and DQBl allelic lineages are shared between rhesus monkeys and humans (1 I , 13, 14). It, therefore, appears that the MHC class I loci of primates are undergoing a continual pro- cess of segmental exchange and duplication and then expansion or deletion of their MHC class I loci.

One of the potential pitfalls of phylogenetic analysis using the PCR is that the amplification of similar genes in different species is dependent on the choice of PCR primers used. Previous analyses of the MHC class I genes of Old World primates have relied on PCR primers designed to amplify the MHC class I loci in humans. Although these studies produced useful results, it was difficult to know whether all of the MHC class I genes expressed in these various different primate species had been amplified. This makes it impossible to make definitive conclusions regarding the nature of the MHC class I loci in these species. Furthermore, this uncertainty makes the design of MHC class I specific primers that might be useful in an MHC class I typing scheme in these primates very difficult. For these reasons, we decided to synthesize a cDNA li- brary and screen it, hoping that this would give us all of the MHC class I loci expressed in the rhesus monkey. The isolation of A and B locus-derived cDNAs from a cDNA library from rhesus monkey

The Journal of Immunology

G o ~ o - B ' O ~ O I HLA-B14402

HLA-B'1.505 HLA-B'1301 Gogo-B'OIOl

Hyla-B'OI

Mamu-A'02 Mamu-A*01

Mamu-A'O4

I r

II 1

-r HLA-A9602 HLA-A'6802

Patr-A*02 Patr-A'04

Gogo-A '0201 HLA-A4402

HLA-A.2901 Patr-A'03

HLA-B'0702 HLA-B*4201

Papa-B'04 Patr-8'02

14 HLA-H'OZ HLA-H'04

Marnu-A '03 Mamu-A'O6

Mamu-A'05 Mamu-A'07 . I- GOgO-A*03

0 .06 - d

FIGURE 5. Unusual relationship among rhesus monkey A locus al- leles and alleles of the A, 5, and H loci. Gene trees were based on nucleotides 3 to 212 of exon 2. The references for the primate se- quences used in gene tree construction in this figure and Figures 2 to 4 are given in References 50 and 51.

88090 confirms our analysis of PCR products from the A and E loci of animal 84557. Additionally, screening of the cDNA library from rhesus monkey 88090 failed to provide any evidence for the existence of an HLA-C homologue in the rhesus monkey.

The isolation of three A locus alleles from rhesus monkey 84557 and five B locus alleles from rhesus monkey 88090 indicates that there are multiple A and B loci in the rhesus monkey. Analysis of cDNAs from the parents of 88090 shows that the father expresses Mamu-A*04 and -B*02 whereas the mother expresses Mamu- A*03, -B*03, and -B*04 (data not shown). Thus, it is likely that rhesus monkey MHC class I haplotypes can differ in their com- plement of MHC class I loci in a manner similar to that seen at the primate MHC class I1 loci. This is quite different from the con- stancy of the number of classical MHC class I genes seen in human haplotypes.

One of the unusual features of the rhesus monkey MHC class I loci is the presence of several HLA-B homologues that do not appear to be well expressed. Using a variety of monoclonal and polyclonal Abs we precipitated five different MHC class I mole- cules from 88090 (data not shown). In vitro translation of five of

4663

Table IV. Bw4 and Bw6 epitopes can be found in rhesus monkey MHC class I mo/ecu/es

Amino Acid Residue Alpha 1 Domain

Serological MHC Class I Epitope Locus 77 80 81 82 83

Mamu Allele

Bw4 HLA-A,B N I A L R HLA-B N T A L R H LA-A S I A L R HLA-B S T L L R HLA-B D T L L R Mamu-A,B N T L L R Af01,A*05,B*06 Mamu-A S N" L L R A*07 Mamu-B N I A L S B*Ol Mamu-B N N L L R B'11

Bw6 HLA-B S N L R G Mamu-A N N L R G A*02 Mamu-A A N L R G A*03,A*04 Mamu-A,B S N L R G A*06,B*05 Mamu-B C N L R G B*02,6*04 Mamu-B D N L R G B*03 Mamu-B N I A R G B*07,B*09 Mamu-B D I L R G B*08

Amino acid replacements not corresponding to known Bw4 or Bw6 motifs are underlined.

the cDNAs isolated from the cDNA library accounted for all five of these well-expressed molecules. There are, however, two B lo- cus cDNAs that were not well-expressed by the parental cell line. Similarly, in animal 84557 we were able to isolate cDNAs encod- ing all of the four well-expressed MHC class I molecules. Four other cDNAs were poorly expressed or we were unable to in vitro translate them. Whether the weakly expressed molecules are en- coded by cDNAs that are products of loci analogous to the HLA-C locus is a matter for speculation.

It has recently been shown that MHC class I molecules play a crucial role in the regulation of NK function (reviewed in Ref. 36). Despite evidence for low levels of surface expression (37), HLA-C products have been implicated in NK cell function (38, 39). The possibility that the C locus may be absent in the rhesus monkey raises the issue of which locus is now performing C locus-related NK functions in this species. Only one of the two sets of C locus substitutions (at positions 77 and 80 in the Q 1 domain) that appear to be important in NK cell recognition are found in the rhesus monkey. Although the combination of 77N, 80K is absent, 77S, 80N is present in a number of different rhesus monkey MHC class I molecules (Mamu-A*06, -A*07, -B*05). Similarly, the Bw4 and Bw6 motifs have been implicated in NK recognition (40,41). Both of these motifs can be found in the rhesus monkeys' MHC class I molecules (Table IV). Mutation of HLA-A*02 74 H + D provides protection against NK lysis in CIR-transfectants, suggesting that substitutions at this position may also be important in NK function (42). Both histidine and aspartic acid can be found at position 74 in rhesus monkey MHC class I molecules. It is possible, therefore, that at least some of the NK receptors are conserved between hu- mans and rhesus monkeys.

Although trans-specific sharing of allelic lineages is a hallmark of the DRBl and DQBl loci (8-15), there does not appear to be any such trans-specific sharing of allelic lineages at the A and B MHC class I loci of rhesus monkeys and humans. Chimpanzees and humans share an allelic lineage at the A locus (16-20) and examination of the Q 1 domain of the B locus products shows that some chimpanzee molecules can differ from their human counter- parts by only 2 amino acids (21). This suggests that exon 2 en- coding the Q I domain might be analogous to exon 2 of the DRBl

4664 THE MHC CLASS I LOCI OF THE RHESUS MONKEY

locus in its conservation among several primate species. Compar- ison of rhesus and other primate MHC class I alleles shows that there is little conservation of exon 2 at the A or 3 loci during the 35 million years since rhesus monkeys and humans last had a common ancestor. This indicates that the MHC class I loci are evolving differently from their MHC class I1 counterparts in primates.

Mamu-A *03, -A*O5, -A*06, and -A*07 are unusual in that they appear to be related to other primate A , E , and H genes in exon 2. Indeed, nucleotides 3 to 212 of exon 2 of this family of alleles are remarkably well-preserved in several different loci from several different primate species. It is difficult to explain how this might have arisen since one would have to propose several different re- combination events to explain the presence of this stretch of nu- cleic acids on three different primate MHC class I loci. It is also possible that these alleles are the result of convergent evolution, and selection for a particular function has maintained these alleles in primate populations.

One of the compelling reasons to analyze carefully the MHC class I loci of the rhesus monkey is the need for a definitive MHC class I typing scheme in these nonhuman primates. Rhesus mon- keys play an important role in the analysis of the immunogenetics of human diseases (43,44). They also provide models for several different areas of biomedical research. The utility of the rhesus monkey model in AIDS research (26-28,45-47) has increased the need for an accurate description of the various MHC alleles in this species. Homologues of HLA-A, -E, -E (48), -F (49), DRA, DR3, DQA, DQ3, and DP3 have now been found in the rhesus monkey. Unfortunately, however, without a suitably large database it will be difficult to design PCR-SSP primers for routine analysis of MHC alleles in this species. The first step to MHC analysis at the molecular level will, therefore, likely be based on the direct se- quencing of MHC alleles, given the ease with which sequence- based typing can now be performed.

Acknowledgments We thank Ronald Bontrop for access to unpublished rhesus monkey MHC class I sequences. We also thank Lettie Smith for preparation of this manu- script, and Jeff Rowell at the New Iberia Primate Center for providing us with the blood samples from rhesus monkey 84557.

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