the expressed class ii a-chain genes of the marsupial major

The Expressed Class II a-Chain Genes of the Marsupial Major Histocompatibility Complex Belong to Eutherian Mammal Gene Families

Robert W. Slade and Werner E. Mayer Max-Planck-Institut fur Biologie, Abteilung Immungenetik

The major histocompatibility complex (Mhc) is a multigene family found in vertebrates. Mhc genes code for heterodimeric cell-surface molecules involved in presentation of peptides to T-lymphocytes. There are two classes of Mhc) and in eutherian mammals four main families of class II genes have been recognized; DR, DQ, DP, and DN/DO. Each class II family contains genes that code for one or more a and p chains. Do the class II genes of marsupial mammals belong to any of these eutherian mammal class II families? The results to date are conflicting. The expressed class II P-chain genes could not be satisfactorily assigned to any eutherian class II gene family and were designated as new gene families, while, conversely, a partial sequence of an expressed a-chain gene was clearly very similar to the DNA gene of eutherian mammals. The aim of this study was to conduct a more thorough analysis of the cl-chain genes in a marsupial by obtaining full-length sequences of all the expressed a-chain genes in the red-necked wallaby, Macropus rufogriseus. Two class II u-chain genes were isolated from a spleen-derived cDNA library, and both have the potential to code for fully functional MHC molecules. Phylogenetic analysis indicated they belonged to previously identified eutherian class II families and are designated as Muru-DRA and MarcDNA. Northern blot data indicated processed transcript sizes of - 1.6 kb for Maru-DRA and -2.5 kb for ikfuru-DNA and that the latter was expressed at a lower level than the former. The phylogeny shows that the DR, DQ, DP, and DN/DO gene families diverged prior to the divergence of the marsupial and eutherian mammal lineages.

Introduction

Genes of the major histocompatibility complex (A&) code for glycoproteins that are central to the immune response of vertebrates because they provide the context for the recognition of foreign peptides by T-cells (Zinkernagel and Doherty 1974; see Klein 1986 for an overview). Short peptides, derived from both self and foreign proteins, are bound in the antigen-binding groove of MHC molecules for presentation to T-cells (Bjorkman et al. 1987; Falk et al. 1991; Rudensky et al. 199 1; Brown et al. 1993 ) . In general, class I molecules bind peptides derived from proteins in the cytosol, while class II molecules bind peptides derived from proteins captured within endosomes (reviewed in Germain 1994). The functional MHC molecule is a heterodimer, and the class I molecule is formed from the product of a class I gene

Key words: major histocompatibility complex, class II genes, molecular evolution, marsupial, red-necked wallaby.

Address for correspondence: Robert W. Slade, Department of Zoology, Univepity of Queensland, 4072 Australia. E-mail: ZL82 [email protected]

Address for reprints: Werner E. Mayer, Max-Planck-Institut ftir Biologie, Abteilung Immungenetik, D-72076 Tiibingen, Germany.

Mol. Biol. Evol. 12(3):441-450. 1995. 0 1995 by The University of Chicago. All rights reserved. 07374038195 / 1203~0009$02.00

( -330 residues) and the gene for Pz-microglobulin ( -98 residues). The class II heterodimer is formed from the roughly equally-sized products ( -220 residues) of an a- and a P-chain gene.

Evolution of the Mhc multigene family is characterized by rapid duplication and loss of the functional genes such that these genes are often not orthologous between taxa but instead are most closely related to each other within a taxa; this has been termed the accordion model of Mhc evolution (Klein et al. 1993). In eutherian mammals, this birth-and-death process of gene evolution is more rapid for class I genes than for class II genes (Nei and Hughes 1992). For example, the functional class I genes of primates are all more closely related to each other than to the functional class I genes of rodents (Hughes and Nei 1989). For class II genes, however, the same families are present among different orders of eutherian mammals, and so orthologous relationships between genes can be detected (Klein and Figueroa 1986; Hughes and Nei 1990). Thus, the a-chain gene of the DR class II gene family (i.e., the DRA gene) in humans is more similar to the D&f gene in the mouse, rat, cow, dog, and so forth, than to any other human class II cr- chain gene. However, there are no orthologous relation-

441

442 Slade and Mayer

ships among class II genes between mammals and birds, at least for P-chain genes (Hughes and Nei 1990). (A class II a-chain gene has not yet been obtained from a bird.) Duplications of class II genes do occur, and humans, for example, contain up to five DRB genes per haplotype, but similarity of these genes with the DRB genes of other eutherian mammal species is clear (Hughes and Nei 1990; Klein et al. 199 1).

Four main class II gene families, each with their respective a- and P-chain genes, have been described in eutherian mammals; DR, DQ, DP, and DN/DO (Klein and Figueroa 1986; Karlsson and Peterson 1992). A fifth family, DM, is very divergent from other class II genes (Cho et al. 199 1; Kelly et al. 199 1) and does not appear to present antigens to T-cells directly (Fling et al. 1994; Morris et al. 1994). The four main families diverged from each other at about the same time, some 100-200 million years ago (Mya) (Trowsdale et al. 1985; Klein and Figueroa 1986; Hughes and Nei 1990). The marsupial and monotreme mammals diverged from the eutherian mammal lineage approximately 1 OO- 150 Mya and 150-200 Mya, respectively (Hope et al. 1990)) and are therefore ideal groups in which to investigate the early evolution of these eutherian class II gene families. Studies of marsupial Mhc genes at the DNA-sequence level have demonstrated the existence of class I (Mayer et al. 1993) and class II genes (Schneider et al. 199 1; Slade et al. 1994). The class I genes evolve in a similar way to that described previously; that is, the three expressed genes isolated from the red-necked wallaby, Mm-opus rufogriseus, are more similar to each other than to class I genes in other taxa (Mayer et al. 1993).

The class II genes, however, present contrasting results. An investigation of the expressed class II P-chain genes in the red-necked wallaby showed that the three genes belong to two families but could not be assigned to any of the eutherian class II gene families and were therefore designated as new gene families, DA and DB (Schneider et al. 199 1). The 3’ untranslated region (3 ‘UTR) was used to assign orthology, but the suitability of this region to detect orthology over such a long divergence time was questioned (Slade et al. 1994). Nonetheless, phylogenetic reconstruction of the coding region sequences did not clearly demonstrate orthology of the wallaby DAB or DBB genes with any of the eutherian genes (Schneider et al. 199 1). The data on marsupial u-chain genes presented a different view as a gene with distinct similarity to the eutherian DNA gene was isolated from the tammar wallaby, A4. eugenii (Slade et al. 1994). However, the latter study obtained only the partial sequence of one expressed a-chain gene, therefore, the aim of this study was to conduct a more thorough analysis of the expressed a-chain genes in a marsupial, the red-necked wallaby.

Material and Methods Library Screening

Five hundred thousand plaque-forming units of a spleen-derived hgt 10 cDNA library from a red-necked wallaby were screened with full-length cDNA clones of human DRA (Korman et al. 1982), DQA (Auffray et al. 1982), and DPA (Trowsdale et al. 1985) genes (see Schneider et al. 199 1 for details of library construction). The probes were labeled by the random primer extension method (Feinberg and Vogelstein 1983) to a specific activity > 1 X 10 * cpm/ug. Hybridization conditions for the nitrocellulose filters were as previously described (Mayer et al. 1993). The filters were washed at room temperature for 5 min in 2 X SSC/O. 1% SDS (where 1 x ssc = 150 mM NaCl, 15 mM sodium citrate, pH 7.0), and then at 50°C for 30 min in 2 X SSC/O.l% SDS. Positive plaques were replated and screened to ob- tain clones for subsequent sequencing.

Polymerase Chain Reaction (PCR)

The 10X buffer was 500 mM KCl, 100 mM Tris- Cl ( pH 8.3 ) , 0.1% Nonidet P-40,0.1 % of filtered Tween 20. The PCR reaction also contained 1.5 mM MgC12, 100 p-M dNTP’s, and five units/ 100 ~1 of Taq DNA polymerase. The reaction was overlaid with par- affin oil. Primers dna 1 ( 5 ‘-TGTGTGGCGGCTTCC- AGAGTT-3 ‘) and dna2 ( 5 ‘-GTGAGGTAGTAGA- ACTTGCGGAA-3’) were designed from the tammar wallaby DNA-like sequence (Slade et al. 1994). These were used for anchored PCR on the cDNA library in combination with hgt 10 primers Tu 1244 ( 5’-ACA- AGCTTGTATTTCTTCCAGGGTAA-3 ‘) and Tu 1245 ( 5’-AGAGTCGACAAGTTCAGCCTGGTTA-3 ‘) . The cycle parameters were 94°C 1 min, 55°C 1 min, 72°C 3 min for 35 cycles. Primers dna3 ( 5’-GTGGGCAC- CATCCTTAT-3 ‘) and dna4 ( 5 ‘-AGTAAAGCCCA- TAGTGC-3 ‘) were designed from red-necked wallaby sequence to amplify a part of the 3’UTR, and the cycle parameters were 94°C 1 min, 50°C 1 min, 72°C 2 min for 30 cycles. Negative controls were always included to check for contamination.

Sequencing

The insert from the hgt 10 library was subcloned into the EcoRI site of M 13mpl9 and sequenced using standard universal and reverse M 13 primers containing a fluorescent label which were provided in the Pharmacia AutoRead sequencing kit. The sequence was completed with internal labeling of Fluorescein- 15- *dATP (Voss et al. 1992) using the same sequencing kit and with site-specific primers 1247 ( 5 ‘-TGGGTCTGGTGGG- CATC-3 ‘) , 1249 ( 5 ‘-TCCGCAAATTCCACTAT-3 ‘) , 125 1 ( 5 ‘-ATAGCTGTGGACAAAGC-3 ‘) , dra 1 ( 5 ‘-

Mhc Genes in a Marsupial 443

TTATTGGATCGTTTCAT-3 ‘) , dra2 ( 5 ‘-ACAGGT- TGTTCCAGTCC-3 ‘) , dra5 ( 5 ‘CATGCCCTTGATA- ATAA-3’)) and dra6 ( 5’-TCCATTCCACTCTCTAC-3 ‘) . The products from the anchored PCR were kinased and end filled and then ligated to the SmaI site of M 13mp 19 using standard protocols (see, e.g., Sambrook et al. 1989) and then sequenced with Ml3 primers as described above. Multiple clones of the PCR products were sequenced to check for misincorporation errors that occur during amplification.

Northern Blot

Poly (A) RNA from red-necked wallaby spleen was isolated as previously described ( Schneider et al. 199 1) , and 3 pg was electrophoresed overnight through a 1.2% agarose-formaldehyde gel. Two samples of total RNA isolated from tammar wallaby small intestine were included as positive controls, and Lambda phage DNA digested with Hind111 was included as a molecular weight marker. The RNA was vacuum-blot transferred at a pressure of 45 cm HZ0 for 2 h to a Hybond N + filter. Hybridization was at 52°C overnight in 50% formamide, 5 X SSPE, 0.5% SDS, 5 X Denhardt’s, 100 p,g/ml salmon sperm DNA. The first hybridization was with Maru- DNA as probe (exon 2 to the end of the 3 ‘UTR) and then with Maru-DRA (full-length cDNA sequence). The probes were labeled as described above to a specific activity > 1 X lo9 cpm/ pg. The filter was washed in 2 X SSPE/0.2% SDS at room temperature for 20 min and then exposed to X-ray film for 3 d. Equivalent conditions were used for each hybridization.

Genetic Analysis

Overlapping sequences were aligned with either the AssemblyLIGN program ( IBI-Kodak) , which also created the contiguous sequence, or with the MacVector program ( IBI-Kodak), in which case the contiguous sequence was created manually. Multiple alignments were created with the CLUSTAL V program (Higgins and Sharp 1988) and adjusted manually to increase similarity. Phylogenetic reconstruction used the neighbor-joining method (Saitou and Nei 1987). The genetic distances were calculated from the inferred amino acid sequence (after removal of all gaps and missing data) and included a Poisson correction for multiple substitutions. The re- liability of clustering patterns was assessed by boot- strapping the data 500 times (Felsenstein 1985). Phy- logenetic analyses were conducted with the MEGA suite of programs (Kumar et al. 1993).

Results

From the initial screening of the cDNA library with a cocktail of human D&t, D&t, and DPA probes, 35

positive clones were selected. The rescreening revealed only one positive clone, which has been designated as MarcDRA (see later), and this contained a - 1.5-kb insert after digestion with EcoRI. The complete sequence of Mat-u-DRA is shown in figure 1. The 1,527 base pairs (bp) contain a 77 I-bp open-reading frame coding for 257 residues. The sequence also includes 3 1 bp of 5 ‘UTR and 725 bp of 3 ‘UTR, and a classical polyadenylation signal (AATAAA) starts 27 bp before the end of the sequence. A polyA tail was not present, probably due to the method of library construction in which first- strand cDNA synthesis was primed with random hex- amers.

All mammals so far investigated have at least two functional class II gene families, which, combined with the presence of two P-chain gene families from the red- necked wallaby, led us to assume that two cl-chain gene families should be present in the cDNA library. As a DNA-like gene had previously been shown to be expressed in the closely related tammar wallaby, this was a likely candidate for the gene of a second a-chain gene family. Primers were designed from the tammar wallaby sequence and used in anchored PCR with an aliquot of the cDNA library as template. The amplified products were cloned and sequenced, and the overlapping sequences were made contiguous to give what is designated here as the Maru-DNA gene (see later). The sequence is shown in figure 1. The 2,099 bp of sequence contain a 762-bp open-reading frame coding for 254 residues. The sequence is truncated at the 5’ end, and so no start codon is present; however, by analogy with the human and mouse DNA sequences, this is expected to be 9 bp (i.e., three residues) upstream. The 3’UTR is 1,337 bp, and again no polyA tail is present. There is a classical polyadenylation signal (AATAAA) 430 bp before the end of the sequence and a nonclassical polyadenylation signal ( AGTAAA ) 63 bp before the end of the sequence. However, it is likely that the 3’UTR is truncated by -400 bp (see below) and that the actual polyadenylation signal is beyond the end of the clone.

Poly( A) RNA from red-necked wallaby spleen was probed with both the Maru-DNA and Maru-DRA genes to determine the length of the processed transcripts and to gauge the relative levels of expression. This northern blot showed a -2.5-kb processed transcript for the Maru-DNA gene (fig. 2, lane 1) which is 400 bp longer than the cDNA sequence, and so we assume that the 3’UTR is truncated and that the actual polyadenylation signal has not been sequenced. An alternative possibility, albeit unlikely, is that an unusually long 5 ‘UTR has been truncated. The Maru-DRA gene shows the expected - 1.6-kb transcript (fig. 2, lane 2). The -2.5-kb band in lane 2 is most likely the result of incomplete probe

444 Slade and Mayer

Maru-DRA Maru-DNA

Manx-DRA Warn-DNA

Xatu-DRA Maru-DNA

Maru - DRA Maru-DNA

Maru-DRA Maru-DNA

Manx-DRA Marts-DNA

Maru-DRA Manx-DNA

Maru -DRA Maru-DNA

Manx-DRA Maru-DNA

Max-u -DJU Maru -DNA

Maru-DRA Manz-DNA

Maru -DIu Maru-DNA

Maru -DRA Maru-DIU

Iyaru-DRA Max-u-DNA

Max-u -DRA Maru -DNA

Maru -DRA Maru -DNA

Maru -DNA

Maru-DNA

Maru -DNA

Manx-DNA

Maru -DNA

5’ UTR 31/men 1 100 CTQAQATTGCAQTQTQTACCCCAAGAAQAAG/ATG ACT TCC MC AAA TCC TTG ATC CTA GGA QCC TTC ATT CTQ TCA GTG CTG CTG QGT CCC TQQ GQA QCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AQC ADA GTG CTG ATC CTA AQG ACC CTG TCC CTQ GTT GTG CTQ CTQ AGT CCC CAA QQA ACT TCT

113/ltxon 2 193 AQG QCC ATT AAA G/AD MC CAT GTG l +* ATC ATC CM QCA GAG TTC TAC CA0 ACC CAC OAA CCC TCT GGA GAG TTT ATG TTC QAC TTT (UT QQQ CA0 TCC ATT QAA G/CT GAC CAT GTG QGG ATC TAC GGC ACA QGT GTA TAC CAG TCC TAT GAG TCC TCA QQC CM; TAC AC0 CAQ QAA TTT GAT GAG

286 OAT GAG ATT TTC CAT GTG QAT TTG MC AAG MA QAG ACA OTC TGG AGQ CTT CCT QAC TTC AGC MA TTT GCC AQC TTT GAG QCT CAG GGT QCC GAC GAG CTG TTT TAC GTA GAC CTG CAG MO MG GAG ACT GTG TGG CGG CTT CCA GAG TTT AGC CAT TTT AQC AGC ‘MY QAC CCT CAG GQA GGG

362/6x011 3 379 TTG QCC MT CTT QCT GTG QAC AAA QCC MC CTG QM ATC ATG AT0 AAA CGA TCC AAT MC ACC CCT GAC ACC AAT G/M GGC CCT GAA GTG ACA CTG CGT U AM GCC ACA TQC MG TAC AAC CTG GAC ATC CM ATC MG CQC TCC MC AQA AGC AQQ GCC ATC AGT G/n; CCC CCT GAA GTQ ACT

472 GTG TTT CCC AAA QQT CCA GTG GAG CTQ QGC CAQ CCC MC ATC CTT OTC TQC TTC ATT GAC MQ TTC TCT CCT CCQ QTA CTT ACT GTQ ACC TGQ GTG TTC TCA GAG AGT CCT GTG GAG TTG GGC CA0 CCA MC QTA CTC ATC TGC TTQ QTQ GAC MC ATC TTC CCT CCA GTG QTC MC ATC AILA TQG

565 CTT CAT MT GGQ OTT CCC ATC ACT OAT GQT QTG TTT GAA ACT OTC TTC CTC CCT CQC TCT QAC CAT QCC TTC AQA AAA TTC CAC TAT CTC ACC ClT CQT MT GQC CAG GTQ ATC ACC ACT GGT GTG TCT GAQ ACA GAT TTC TAC TCT COO CCT GAC CAC MA TTC CGC AAQ TTC TAC TAC CTC ACT

Cu/E%on8 4 to rent TTC ATC CCC TCT QCC ACC OAT TAC TAT GAC TQT MO QTT QAQ CAC TOO QQA CTG QAA CAA CCT OTT QTC m CAC TGG Q/M CCA QAA QTA COD TTT CTC CCC MC ACA GAG QAC TTT TAT GAC TGC AAA GTG QAG CAC TQQ QGC TTQ QAG CAQ CCA CTC CTC AAQ CAC! TG6 Q/M CCC CA0 ATT CCA

751 ACC CCA CTG CCA GAO ACA ACA QAQ ACT QTG OTC TGC GCC CTA QQC CTG QCC ATA QGC CTT QTG QGC ATC ATC QTA QGC ACC ATT CTT ATT ATC TCC CCA GTQ CCA GAQ ACA ACA GAA ACT QTQ OTC TGT QCC CTT QQT CM QCA GTQ QQC TTG QTQ QQC ATC ATT QTQ QGC ACC ATC C’FI’ ATA ATC

005 MO QQC AT0 AQA TCA MC MC ACT TCC COT GQT GGC TCC CQT QQA CCC CTQ TGA/ AQA QQC ATQ COT TCC AGC AGT AQQ TTC CAA CAT CAA QGG CCT CTQ TM/

1045 TCAQACCAQTTACTCTTCTMCTTGTTTTCCTTTTOAAAAI CATAATTCCA_ TCACCCCCCCCCATACATMTCCCMCTACTACACATQCTCCCACACTCQCCCAAACCCA TQCCTTC~~CCnTCC~TQTCTCCTCTaTCTATCT

1525

1765

CTCCTAAATCACMGTGTGACCATATCCTTGAACTCTTCAAQAAATTCTAGTMCTGCCMCCCT~ TACMQCTCCTCTGGATTTAAAAQCCCTTCACAATTQGCACTATQQ

1885

DCTTTACTTTOCAOCC~~CCTG~C~ATACM~CA~TMTCTCT~T~CQTTT~TQ~~~~CATG~ TTTQCATAGATTQTTCCCTQTQCCTAQAAT

2005 OCCTTCCCCCTCACCTTTOTC~ATAOCTTCCTA~T~C~CATMCTCA~TCATQTCCTC~CTAC~CCTTATCCT~T~~AG~~TAGT~AC~C~GTA~~

2125 ACGTTOTATATCCTTTATA~A~CCTATDTAM TTTTTTAQTACAGAGACTGTTTTQAQGCTTTTQTTTTTG

2142 TTATCTTTGTATCTTCA

FIG. 1 .-Nucleotide cDNA sequence of Guru-DRA and Muru-DNA genes, where an asterisk (*) indicates a gap to increase similarity, and a period (.) indicates that a sequence was not obtained. The exon borders (/) were inferred from comparison with eutherian class II u-chain genes. There are 1,527 bp in the Muru-DRA sequence and 2,099 bp in the Muru-DNA sequence. A 16-bp perfect palindrome in the 3’UTR of the Muru-DNA sequence is underlined, and potential polyadenylation signals are underlined and italicized. The codons which code for the potential glycosylation sites in the al (exon 2)‘and a2 (exon 3) domains of eutherian mammals are shown by bars over the sequence. Note that both Mum-DRA and -DNA contain only the al domain glycosylation site. The GenBank accession numbers for Muru-DRA and Muru-DNA are U I8 110 and U 18 109, respectively.

removal after hybridization with the Mat-u-DNA probe. intensity from the northern blot also shows that Maru- For technical reasons, we were not able to repeat the DRA is expressed at a higher level than Mar-u-DNA. hybridization so that alternative explanations for the The deduced amino acid sequences show the length presence of the -2.Skb band in lane 2 of differential and conserved residues expected of a class II a-chain splicing, alternative polyadenylation sites, and cross-hy- molecule ( fig. 3 ) . The leader peptide of MawDRA con- bridization with other a-chain genes must also be contains 25 residues, while the leader peptide of Maw- sidered possible. A qualitative appraisal of relative band DNA contains at least 23 residues. The al domain of


Man-DNA (- 2.5 kb) III)c

Maru-DRA (- 1.6 kb) e

FIG. 2.-Northern blot of red-necked wallaby poly(A) RNA probed with Mum-DNA (lane I) and -DRA (lane 2) clones. Exposure time was 3 d. The molecular weight was determined by reference to Lambda phage DNA cut with HindIII. The 2.5kb band evident in lane 2 is probably the result of incomplete removal of the Mm-u-DNA probe before hybridization with the Maw-DRA probe (see text).

MarsDRA and -DNA contains 84 and 85 residues, respectively; the a2 domain of both genes contains 94 residues; the connecting peptide contains 13 residues; the transmembrane region contains 23 residues; and the cytoplasmic tail of Maru-DRA and -DNA contains 17 and 15 residues, respectively. The cysteine residues that form a disulphide bridge in the a2 domain are present, but the a2 domain potential glycosylation site is absent in both Maru sequences. For Maw-DRA, the corresponding sequence is T-V-T, which is one substitution away from being an N-V-T glycosylation site (i.e., a codon change from AAT to ACT; fig. 1). For Maru-DNA, the corresponding sequence is N-I-K, and this also is only one substitution away from being an N-I-T glycosylation site (i.e., a codon change from ACA to AAA; fig. 1). In common with all eutherian mammal class II a-chain molecules (with the exception of Bota-DYA and HLA-DPA2), there is a potential glycosylation site in the a 1 domain (N-N-T for DRA and N-R-S for DNA). For examples of other residues conserved among class II a-chain molecules, the reader is referred to Stiltmann et al. (1993).

A phylogenetic tree was constructed to ascertain the relationship between the Maru class II a-chain genes and those of eutherian mammals (fig. 4). The residues used for creating the pairwise distances are 1 to 22 1 in figure 3-that is, the al, cr2, connecting-peptide, and transmembrane domains. The Maru-DRA sequence

forms a distinct clade with the eutherian DRA sequences, and this pattern is observed in 100% of the resamplings. The position of Maru-D&4 within that clade also reflects the expected phylogenetic position of a marsupial sequence as being an outgroup to eutherian sequences. A similar clustering pattern is observed for the Maru-DNA sequence which forms a clade with the human and mouse DNA sequences, and that pattern is observed in 67% of the resamplings. The phylogeny also shows that the divergence of the marsupial and eutherian mammal lineages, as represented by the divergence of the orthologous DRA and DNA genes, occurred after the divergence of the four class II gene families of DR, DQ, DP, and DN/DO.

Discussion

Two expressed class II a-chain genes were isolated from a spleen-derived cDNA library of the red-necked wallaby, Macropus rufogriseus. Both a-chain sequences have the potential to code for fully functional Mhc molecules as neither possesses any obvious de- fects, and it is likely that these two a-chain genes code for molecules that pair with the two P-chain molecules isolated from the same library (Schneider et al. 199 1) to form two mature class II heterodimers. However, we cannot exclude the possibility that other expressed a- and P-chain genes are present, particularly ones that are divergent from the eutherian mammal genes

446 Slade and Mayer

CONSENSUS ==> Maru -DRA Maru -DNA H-2Oa HLA-DNA Susc-DRA Bota-DRA HLA-DRA W-2Ead Orcu -DPA HLA-DPA H-2Aak Susc-DQA IiLA-DQA Bota-DQA Bota-DYA Brre-DAA

.a -31 -21 -11 1 11 21 31 41 . &DtPEDMMAMI GLVVLGLOAG ALLTPLSASG A ***IRADHV *SYGAEFYQS YGPSGEFTFE FDGDEIFYVD LEKKETVWRL l *****_,..N KSLI__uI- m_LGpw- -***--EN-- *,“Q-_-m-T ~--_--,,+D ~~~_---~~~.~~~-------

. . . . . . ..SR V-ILRT-SLV ,,__,+QG’&Q Se**-Em--- GIn.-T~--- -ES--QY-Q- --E--L---- -Q--------

l ****_TJ’LW E__PV’L_- SF-S-R-* _***_____M ____pA____ -D&-Q__& ___EQ--S-m _~-V----

*****vu= ---LQWTm T--S-Q&G* -***T-_--M _---PA-__- --A--Q__& __EEQL-S-m -K-S-A----

l *******AT I_G_pV__F” ITIm_QK_” _***-mN__ *IIQ____L_ pDK____M6-,, _____--H-m M--R------

.,........ . . . . . . . . . . . . . . . . . . . . . . . . . ..N-- l IIQ----Lx PEE-A--M-D _____--H-m MG--------

******_-Is -VP---FFII -V-MSAQE-W -***--EE-- l IIQ____LN PDQ---_~-D _____--H-m ~_--_----

**t***_d& -AL--WFP’1 -V-MSSQK-W -***--BE-T *IIQ----LL PDm___+D -------~-- ~--S--~---

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-m *--I-A-T W----CM-- --~--V-~-- -D--QAI-Y-

_----R-FH_ m-I--S- _F_LS-Ra_ _***------ *-m-A-V-T ~-T-__+- --~--M---- -D------H-

l r******_p nS~_r__v_ --T_M--Lc- GEnn-E-__- _---r~--- P-DI_QY--- -----L---- -~------&

*******e-V pGR--~--- _-T-~--C_ G__*-A_--- A---L~--- -__R_Y__H- ---_-Q---m ________--

*+t+t**e__ L--M____ --T-~-PC- G--*-,,-_-_ A------- ---__QYSH- -----,&--- _-R-----Q-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . __I _,+-ISI_BT -----W-H_ _____E-mm- ---~------

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..--- -T---_-T --T--Q_--- _____L-___ -0-m---_--

**et****** ****m-FOP L-IFmIV-N R l **AQ-E-R 'DFDFTGCSD TE**KDDLTG ---E-EYHT- FIR--G-WI'-

51 61 PEFGDFA*S* FEAQGYMANI -D-SK__*_' _______--L ___SH_s*_* -DP--G-REK _______*R* SDF_SG_~_ ,-_----*R* -DP--G--G-

R-_-R__*_* __________

____H__*_* _______-_M

E---R__*_* _______---

E--m__*_* _______ ____

____-•_* V-_--G_---

E___QAp_* _____G____

---AQLR*R* _-p--G-Q--

-L-S&T*-* _,,p____R__

-L-RR-R*R* _Dp_F__T--

-V-SR--*T* -Dp____R--

S--SNfT*R* _-V-S--R--

-D-A-PYSYP GGYE?&V- -M

CONSENSUS = Maru - DRA Maru-DNA H-2Oa HLA-DNA SUSC-DRA Bota-DRA HLA-DRA H-2Ead Orcu-DPA HLA-DPA H-2Aak SUSC-DQA HliA-DQA Bota-DQA Bota-DYA Brre-DAA

=> 71 81 _ 91 101 111 121 _ 131 141 151 161 AVDKANLDIL IKRSNNTPAT NVPPEVTVFP KSPVELGQPN TLICFVDKIF PPVINVTWLR NGQPVTEGVS ETVFLPRSDH S FRKFHYLTF LPSAEDVYDC _------R-M M-___-m-D_ --G__--mm- -G-------- I-V_-I--FS ---LT----R --V-I-D--F __________ A--------- I---T-Y--- -~-Y----- -----RSR-I S--------S E------mm- V---L--N-- ---V-IR--- ---VI-T--- __D-YS_P__ R----Y---- --NT--F--_ SMI__H____ Vg+__R_R-V S-_-R-_-L_ _TR____R__ V-_-I--D__ -_________ -S--I-R--A Q-S-YSQPN- R------_-- V-_-------

-AI--H____ ~___RsR_I ____R_-_L_ __R_______ ~___~__N__ _____I____ ---T-----A Q-S-ySQp__ L-------p- ,,---------

_-----_E__ --_____-N- ----_---LS ,,K_-m--E__ I--_-I--FS ---V___--_ --S---R--- -_---m-E__ L-------~- M--T-__---

_-M------M ----m-_-N- --___--LL_ m__---Em_ _----I_-FS ----S-me-- --K-_-D--_ Q_---e-N-- L-------p- --~___---

_------&&f T----Y--I- --------LT N__---m-- V---_I--FT ---V------ --K---T--- -------,+- L-------p- ---T----mm

__L_____~ m____ --DA --A---_-LS ~_~-~-~~~~,~-~~~~~~~~ ---V-m-e-- --R------- __---e-D__ L---_----- ---~-F---

-~-S--- -Q&-H-Q-P -E----~--- -,+----m-s ----H~--F- ---L--mm-- ________-_ __--_~~F R-------A- V---A__---

-ILNN--NT- -Q-_-&Q-- -D-------- _E_------- ----HI--F- ---L-----C --EL-----A -SL----T-Y --~---_--- V-----F---

-~-H--E-- T----S---- -~-QA---_ ___-L----m _------N-e _____ I____ -SK&-D--Y --S---y --H-L,+--- I--D&I---

_TL-H--N-V T--_-_-A-V -~------S ___-I-____ ---_~--S-- ----_I---K --HS-*e-F- --,‘J--Sm-- --L-IS_--- ---D&F---

_-L-H--&V -----S-A-- -~------S ----T----v ----L--N-- -_-V-I_--S --~,+----- --S--SK--- --F-IS---- ----DEI---

_T,CJ-~-~_ -Q---S-A-- -~------S :__-&____ ----~--N-- -----~---- --US------ --S--IK-_y --L-IN---- ---DD-m-m_

~S-R----- --N-SF---- SEI---A--- --S-V--~-- _---Q--N-- -----~--pY --m_A--IA --T-Y-K--- --L--S---- --TS--F---

EIC-Q--ATD --AY-SPEEQ LD--VTSIYS EDE-V-DEK- ----H-TGF- --PV--S-TK -NDI-M-DI- FSQYRDN--G T-NM-SA-K- T-AEG-I-S-

171 181 191 201 211 221 231 CONSENSUS ==> KVERWGLD*E PLLKXWEPEI PTPLPETTET WCALGLAVG LVGIWGTVL IIKGLRSGNA SRRRGPL**

Maru -DRA _------E*Q -Wee-_-m,, R---_--s-w -__-----I_ _---I--_I- ----M--N-T --(3(3SRGpL

Manx-DNA _------E*Q -----w--Q- -S-V------ __________ ----~---~- --R-M--SSR FQHQ-mm**

H-2Oa __------*T ---Q----QV L--&D---- LI-G---m- -M-t&L---- &T-T-~SI R-e******

HLA-DNA Q_______*A ---R-__LQV -I-p---- L__-----I_ _--FL-___- --M--SW pm**+*+**

Susc-DRA Q_------*K -------F-A Q--------N T------I-A ----~----- ----V-K--- TE--mm-**

Bota-DRA HLA-DRA H-2Ead

----L--N'- -------Y-A -A-_-----N A-_----I-A _---IA--IF ----V-KS-- ~___--** K_------*- --_-___F~ -S____---N ___--m-T__ _---II--IF ---_--KS-- M-_--m** E-D----E*_ _-R-T--F-E K-L----K-N -~-----F-- -------~~- -M--I--V m--QoAL*

Orcu-DPA HLA-DPA

Km_-----*A ---T---AQE -~Q~------ -_-----V-s -A-,,---I-- -T-A---SPD P-A-R--**

R______-‘Q -------~QE -IQ&----- -L_----~- _---I-____ ---S--_-~ PmAQ_T-•*

H-2Aak SUSC-DQA HLA-DQA Bota-DQA Bota-DYA Brre-DAA

__---m-E*_ -V-------- -A-=5-L--- -------S-D --------IF --Q-----(3T --HP---**

__----__*K __________ -A-MS-L--- --__---~-- _------m-F --Q-_---Gp --BQ-SD**

__------*- ________-- --_~S-L--- -------S-m __________ --R----VG- --HQ-mm’*

__----m-t- ----____D- -A-MS-L--- ___-m--T-_ _---&__I_ --Q____-Gp _-wQ_--**

K_---D-K*- --V------- --_TS-L-m- ____----I_ -~-------- -LK~~L-A- -----*' T_K-RSIQOQ -m-T--- **E--$j.$,GPA -F_~--~- _L-Vu--FF L---ma** l **e*****

FIG. 3.-Alignment of deduced amino acid sequences of MHC class II a-chain genes where a dash (-) indicates an equivalent residue; an asterisk (*), a gap to increase similarity; and a dot (.), that the sequence was not obtained. The al domain is from residues l-90, a2 domain from 9 1- 185, the connecting peptide from 186- 198, the transmembrane region from 199-22 1, and the cytoplasmic tail from 222-end. The potential glycosylation sites in the al and a2 domains of eutherian mammals are shown by bars over the sequence. Note that both Maru-DRA and -DNA contain only the al domain glycosylation site. The species shown are red-necked wallaby, Macropus rufogriseus (Maru); human, Homo sapiens (HLA); pig, Sus scrofa (Susc); rabbit, Oryctylagus cuniculus (Orcu); cow, Bos taurus (Bota); mouse, Mus musculus (HZ); and zebrafish, Brachydanio rerio (Brre). The references for the sequences are H-2Oa, Karlsson and Peterson (1992); HI&DNA, Trowsdale and Kelly (1985); Susc-DRA, Hirsch et al. (1992); Bota-DRA, van der Poe1 et al. (1990); HLA-DRA, Koppelman and Cresswell (1990); H-2Ea“, Hyldig-Nielsen et al. (1983); Orcu-DPA, Sittisombut et al. (1988); HLA-DPA, Lawrance et al. (1985); H-2Aak, Bishop et al. (1988); Susc-DQA, Hirsch et al. ( 1990); HLA-DQA, Auffray et al. (1987); Bota-DQA, van der Poe1 et al. (1990); Bota-DYA, van der Poe1 et al. (1990); and Brre- DAA, Stiltmann et al. (1993).

used as probes (and see later). The lack of a glycosylation site in the a2 domain may be unusual for an a-chain gene, but there is a potential site in the al domain, and the presence of at least one site appears common to most, if not all, expressed a-chain genes. The corresponding sequence in the a2 domain is one substitution away from being a potential glycosylation site, and characterization of the a-chain genes of monotreme mammals may help in determining the ancestral mammalian condition regarding this particular character.

It is intriguing to note the similarity between wallaby, human (Trowsdale and Kelly 1985), and mouse (Karlsson et al. 199 1) with respect to the reduced level of expression of the DNA gene compared with the DRA gene. For the human DNA gene, this has been attributed to a mutation in the polyA signal from AATAAA to ACTAAA (Trowsdale and Kelly 1985), a mutation shared by the mouse DNA sequence (Karlsson and Pe- terson 1992). However, it is not possible, at this point, to also ascribe the reduced level of expression in the wallaby DNA gene to a similar nonclassical polyA signal


87 DQA family 95

Beta-DQA

90 H-2Aak I

Both-DYA

Man-DRA

50

100

H-2Ea d

HLA-DRA

Brre-DAA

DRA family

0.0 0.2 0.4 0.6

Genetic distance

FIG. 4.-Phylogenetic tree of the class II u-chain amino acid sequences shown in fig. 3. The sequence used is from residues 1-221 and contains the al, (r2, connecting-peptide, and transmembrane domains. After removal of gaps, a total of 195 residues were used to create the pairwise genetic distances.

as the polyA signal in MarcDNA has, most likely, not been sequenced. The lack of polymorphism within the eutherian DNA genes (Jonsson and Rask 1989; Karlsson and Peterson 1992) is probably also the case for marsupials as there is only one variant site between the tammar wallaby and the red-necked wallaby in 39 1 bp of exons 2 and 3, and this is a synonymous substitution in exon 3.

The designation of orthology of genes within the A4hc can be difficult. This is because it is a multigene family which is characterized by a relatively rapid rate of gene duplication. In mammals, for instance, a class II gene family in a species will often contain two or more gene copies so that while it is generally simple to designate which family a particular gene belongs to, it is not necessarily so easy to designate which of the multiple genes within a family is orthologous (i.e., related by spe- ciation events only) with which of the multiple genes in the same family of another species. Nonetheless, the placement of the Maru class II a-chain genes within previously designated eutherian gene families is clear. The inclusion of Mar-u-DRA with the DRA family in eutherian mammals is well supported by the bootstrap value ( 100%). The Maru-DNA gene forms a single clade

with the eutherian DNA family, although it is not as well supported by the bootstrap value (67%). When the phylogeny is reconstructed using a nonsynonymous distance measure (with Jukes and Cantor [ 19691 correction ) , instead of the amino acid distance measure, the bootstrap value for the DNA clade increases to 88% (data not shown). Consistent with previous suggestions (Slade et al. 1994)) the 3 ZJTR is not suitable to detect orthology over long divergence times as, for this region, neither of the wallaby a-chain genes could be satisfactorily aligned with their eutherian counterparts (data not shown).

The phylogenetic tree (fig. 4) also shows that the DNA and DQA gene families arose by duplication of a single ancestral gene, and this model is well supported by the bootstrap value of 90%. This had been previously suggested by other studies but without strong support (Figueroa et al. 1990; Rask et al. 1990). The increased support in the phylogeny presented here appears to be due to the inclusion of the Alarm-DNA gene which shares more amino acid residues with DQA sequences than do the mouse and human DNA genes. The duplication of a different ancestral gene into the DPA and DRA genes is also indicated in the phylogenetic tree, and this had also been previously suggested (Hughes and Nei 1990).

448 Slade and Mayer

Table 1 Genetic Distance (%) of Nonsynonymous Substitutions (with Jukes-Cantor correction) between Exon 3 Sequences of Various Mhc Class II a- and B-Chain Loci

Class II Family Species Compared a-Chain Locus P-Chain Locus

DR .

DQ . . .

DP . . . . DO . . .

. Human/cow 9.60 + 2.21 Human/mouse* 11.76 + 2.48 Cow/mouse* 14.51 + 2.79

. . Human/cow* 6.41 f 1.76 Human/mouse 15.77 + 2.91 Cow/mouse 13.41 + 2.65

. . Human/rabbit 10.03 + 2.26 . Human/mouse 8.05 + 2.01

9.60 + 2.21 28.97 + 4.29 32.59 + 4.63

3.32 + 1.26 11.30 + 2.41 12.34 _+ 2.54 7.21 + 1.88

12.14 f 2.53

NOTE.-An asterisk (*) indicates a significant difference between a-chain and p-chain gene at the 5% level. The anom- alously high rate of evolution of DRB compared with DRA is found only in comparisons involving the mouse sequence, and this remains true after inclusion of pig and sheep DR sequences (data not shown).

The position of the wallaby sequences shows that the DR, DQ, DP, and DN / DO gene families diverged prior to the divergence of the marsupial and eutherian mammal lineages.

The a-chain genes clearly belong to previously identified eutherian class II gene families, but this is not so for the P-chain genes (Schneider et al. 199 1). Ac- cording to Schneider et al. ( 199 1)) the MawDBB gene does form a clade with the eutherian DOB gene family in a neighbor-joining tree of nonsynonymous distances when the peptide-binding codons in exon 2 are removed. This then would make it the partner to the Maw-DNA gene. However, the grouping is not very stable, and inclusion of Maru-DBB in the clade is dependent on which sequences are included and on whether amino acids or nonsynonymous sites are used to construct distances (data not shown). The same instability of position ap- plies to the Mat-u-DAB gene (data not shown). This difference between the a- and P-chain class II genes of the red-necked wallaby in their similarity to eutherian mammal gene families is surprising because other data indicate that origin of the eutherian class II gene families occurred at the same time for both the a- and P-chain genes within each family. That is, the distance between the eutherian mammal class II gene families is roughly the same for both a-chain and P-chain genes (see, e.g., the phylogenetic trees with distance scale in Hughes and Nei 1990 and in Klein et al. 1993). Also, within a family, the cz- and P-chain genes tend to be closely associated physically ( see, e.g., Campbell and Trowsdale 1993).

There are at least two explanations for the contrasting results. First, as is the case for the a-chain genes, the expressed P-chain genes of the red-necked wallaby do belong to the DR and DN/DO gene families, but similarity with eutherian DRB and DOB genes has been obscured by a more rapid rate of P-chain gene evolution compared with a-chain gene evolution. However, a

comparison of conserved exon 3 sequences among eutherian mammals shows that this explanation is not supported by the available data (table 1). In general, a- chain and P-chain genes within a family evolve at similar rates. Second, as suggested by Schneider et al. ( 199 1 ), the Maru-DAB and -DBB genes represent two new mammalian gene families. If so, this would mean either that the a- and P-chains of the mature MHC heterodimers in marsupials have coevolved differently from those in eutherian mammals or that there are other expressed class II genes in the marsupial yet to be found.

Acknowledgments

We thank Petra Schwarzmaier and Beate Piimmerl for technical assistance, Colm O’hUigin for data presentation programs, and Jan Klein for comments. We thank three anonymous referees for their helpful comments.

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NAOYUKI TAKAHATA, reviewing editor

Received July 18, 1994

Accepted December 2 1, 1994

the expressed class ii a-chain genes of the marsupial major

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