Introduction

Eutherians (‘placentals’) and metatherians (marsupials) arethe two most successful groups of mammals, with the onlyother extant group being the egg-laying prototherians(monotremes). The evolutionary pathways of eutherians andmetatherians are thought to have diverged approximately130 million years ago1–3 and it appears that their anatomicaland physiological characteristics have developed in parallel,even though numerous differences are known to exist.4 Todate, mammalian immunological studies have focused on theeutherian systems, with little comprehensive work havingbeen carried out on marsupials. This is despite the fact thatsuch information would be particularly illuminating for anunderstanding of the evolution of mammalian-specificimmune mechanisms.

A number of functional studies have suggested that sub-stantial differences may exist at the cellular and/or molecularlevel between the immune system of marsupials and that ofeutherians. In total, these studies indicate a lowered level ofT cell responses, as measured by mixed lymphocyteresponses; delays in second-set graft rejection; and differ-ences in B cell regulation as demonstrated by delay in classswitching and poorly elicited memory response (reviewed byStone et al.5). Such observations have prompted a recentrevisitation of fundamental aspects of immune function inmarsupials, including complement function,6 humoralresponses to vaccination,7 response to allografts and mixed

lymphocyte responses.8,9 In addition, a number of importantimmunological genes have been isolated and sequenced,including the immunoglobins,10–12 the genes controlling theirjunctional diversity13,14 and some cytokines, includingTNF,15,16 lymphotoxin (LT),17,18 IL-1019 and IL-1β.20

The present paper is concerned with the isolation andcharacterization of the genes encoding two key moleculesinvolved in T cell function, the T cell receptor α and β chains.The TCR genes have been studied in a variety of eutherianspecies, including humans, rodents, pigs and sheep. Ineutherian mammals, the T cell receptor plays a central role inantigen recognition and subsequent activation of T cell pop-ulations.21 The TCR is a heterodimer, which occurs in twoforms, alpha-beta (αβTCR) and gamma-delta (γδTCR).22

Together, they are expressed at a frequency of between20 000 and 40 000 per T cell.23 In humans and rodents, the αβreceptor is the more prevalent, being present on more than95% of T lymphocytes, and binds both MHC I and MHC IImolecules.24 However, in some species, such as sheep, rumi-nants and chickens, a higher portion of γδT cells are foundin the T cell pool.25 The components of the αβ heterodimerare both approximately 40–50 kDa in molecular weight andare linked to one another by a disulfide bond.26 The αβ chainscontain two extracellular domains, an amino-terminal vari-able (V) domain and a carboxyl-terminal constant (C)domain.23 The genes encoding the TCR have a ‘translocon’genetic configuration analogous to that of immunoglobulingenes and undergo similar selection processes duringontogeny.27 The β and δ chains contain variable (V), joining(J), and diversity (D) regions, as well as a constant (C) generegion, which rearrange to increase the ability to recognizeforeign peptides.28 Unlike the β and δ chains, the α and γchains do not have any D gene segments.28 As with

Immunology and Cell Biology (2000) 78, 103–109

Research Article

Cloning of marsupial T cell receptor α and β constant regioncDNAs

PETER D ZUCCOLOTTO, GAVAN A HARRISON and ELIZABETH M DEANE

School of Science, University of Western Sydney, Nepean, Kingswood, New South Wales, Australia

Summary Partial cDNAs encoding the tammar wallaby (Macropus eugenii) T cell receptor alpha constant region(TCRαC) and T cell receptor beta constant region (TCRβC) were obtained using reverse transcriptase-coupledpolymerase chain reaction (RT-PCR). These PCR products were used to screen a brushtail possum (Trichosurusvulpecula) lymph node cDNA library, resulting in the isolation of clones containing the complete coding regionsfor TCRαC and TCRβC. These constitute the first marsupial T cell receptor sequences to have been elucidated.Sequence analysis of the T. vulpecula constant region revealed a considerable level of sequence identity with TCRof other species, particularly eutherian mammals, at both the nucleic acid and amino acid levels. At the nucleotidelevel, 65.8% sequence identity was calculated for the T. vulpecula and human TCRαC sequences, with 55.9% identity at the amino acid level. For TCRβC, the T. vulpecula and human β1 sequence identity at the nucleotidelevel was 75.1% and at the amino acid level, 67.0%. Phylogenetic analyses based on the T. vulpecula sequencesindicated that these sequences are basal to, but also most closely related with, TCRαC and TCRβC homologuesfrom eutherian mammals, consistent with the current views of both mammalian and TCR evolution.

Key words: evolution, Macropus eugenii, marsupial, T cell receptor constant genes, Trichosurus vulpecula.

Correspondence: Elizabeth M Deane, School of Science, Univer-sity of Western Sydney, Nepean, PO Box 10, Kingswood, NSW2747, Australia. Email: e.deane@uws.edu.au

Received 13 August 1999; accepted 23 September 1999.

immunoglobulins, diversity of the TCR is further enhancedby deletion and insertion of nucleotides by terminal deoxy-nucleotidyltransferase (TdT) enzymes and this gene, as wellas the Recombination Activating Gene-1 (RAG-1), hasrecently been isolated from a South American marsupial,Monodelphis domestica.13,14 During the foetal stage ofdevelopment, the γδ thymocytes are abundant in most vertebrates and they are subsequently superseded by the αβthymocytes as the animal develops. This is particularly thecase in humans and rodents,25 but has not yet been investi-gated in marsupials due to a fundamental lack of reagents.

To date there have been no reports on the structure of marsupial TCR and this has severely limited our ability toinvestigate T cell ontogeny and function in this mammaliansubclass. Such information is crucial for generating an under-standing of the apparent differences in T cell functionobserved in marsupials. Moreover, characterization of themarsupial TCR will permit further elucidation of the evolu-tionary relationship between marsupials and other mam-malian groups.

Materials and Methods

Production of tammar wallaby probes by polymerasechain reaction

Blood was collected from the caudal vein of an adult female tammarwallaby into an EDTA-coated tube. Total RNA was isolated by theguanidinium thiocyanate method29 using Total RNA IsolationReagent (Advanced Biotechnologies, London, UK). Approximately1 µg of total RNA was used for synthesis of cDNA using avianmyeloblastosis virus (AMV) reverse transcriptase and a 15nucleotide oligo-dT primer (Promega, Madison, WI, USA). Poly-merase chain reaction (PCR) was performed in reactions containing3.5 mmol/L MgCl

2, 0.2 mmol/L of each dNTP, 50 mmol/L KCl,

10 mmol/L Tris-HCl (pH 8.0), 10 pmol of each primer (see later) and2 U Taq Polymerase (Promega), in a total volume of 50 µL. For boththe α and β constant genes, denaturation was carried out at 95°C for1 min and extension at 72°C for 2 min. Annealing was carried out for2 min at 40°C for the α constant gene and 55°C for the β constantgene. After the completion of 35 cycles, a final extension cycle of72°C was performed for 10 min. The oligonucleotide primers usedwere designed from known TCR sequences obtained from theGenBank database.30 The sequences of the consensus primers wereas follows: Alpha Forward, 5′-TCTGCCTNTTCACCGANTTTGA-3′; Alpha Reverse, 5′-CAGCGTCATGAGCAGNTTAAANCC-3′;Beta Forward, 5′-CGNAACCACTTCCGNTGNCAAGT-3′; and BetaReverse: 5′-TTTNTTGACCATNGCCATNA-3′. The PCR productswere then separated by 2% agarose gel electrophoresis, purifiedusing a Bresaclean DNA purification kit (Bresatec, Adelaide, SA,Australia) and cloned into the pGEM-T Easy Vector (Promega).

Screening of a brushtail possum lymph node cDNAlibrary

A brushtail possum lymph node cDNA library10 was screened withthe cloned tammar wallaby TCRαC and TCRβC fragments. ThiscDNA library was constructed using the λ ZAP Express vector(Stratagene, La Jolla, CA, USA). The cloned fragments were used asDNA probes by labelling them with α-[32P]-dATP using a RandomPrimed Labelling System (Boehringer, Mannheim, Germany). Afterprehybridizing for 3 h, probe hybridization was carried out for 24 hat 60°C with constant rotation. After incubation, the filters were

washed in 2 × SSC (1 × SSC is 0.15 mol/L NaCl, 0.015 mol/Lsodium citrate) and 0.1% (w/v) SDS. Two higher stringency washesusing 0.15 × SSC and 0.1% (w/v) SDS were then carried out. Allwashes (for both TCRαC and TCRβC) were conducted at 60°C. Thewashed filters were then exposed to X-ray film (Amersham, LittleChalfont, Buckinghamshire, UK) for 48 h at – 70°C with intensify-ing screens. Positive plaques were selected and further libraryscreens carried out until single plaques could be selected. A total of40 plaques were selected from each of the secondary library screensfor TCRαC and TCRβC. Once selected, the pBK-CMV phagemidvectors were excised from the ZAP Express vectors according to themanufacturer’s instructions (Stratagene). Primers corresponding tothe pBK-CMV vector, T3 and T7, were used to sequence the isolatedclones by the dideoxy chain termination method.31 Internal primers,derived from the sequences obtained with T3 and T7, were alsodesigned to completely sequence the isolated clones in both directions (sense and antisense).

Phylogenetic analysis of brushtail possum TCRαC andTCRβC

Phylogenetic reconstruction, based on the maximum parsimonymethod, was performed on the deduced protein sequences for thebrushtail possum TCRαC and TCRβC regions using the P H Y L I P

computer package (J Felsenstein, University of Washington) throughthe Australian National Genomic Information Service. Prior to treeconstruction, protein sequences were first aligned using the P I L E U P

program (Genetics Computer Group, Madison, WI, USA) and 100replicates produced for analysis by the boostrap resampling proce-dure with the S E Q B O OT program. Parsimony trees were generatedusing P ROT PA R S on the replicate data sets with gaps having beenrecoded as missing data. Consensus trees were displayed using theT R E E V I E W program.32

Results

Brushtail possum TCRαC cDNA sequence

A 340 b.p. fragment was generated from cDNA synthesizedfrom tammar wallaby PBMC RNA. This fragment had52.4% average sequence identity with eutherian TCRαCsequences (data not shown) and we therefore conclude that itrepresents a partial coding region of the tammar TCRαCcDNA. The use of this fragment as a probe for screening abrushtail possum lymph node cDNA library led to the isola-tion of the complete brushtail possum TCRαC region clones.

The longest brushtail possum TCRα clone identified was1340 b.p. in length, including a 3′ poly-A repeat of 16 b.p.This clone consisted of a partial coding sequence for the vari-able region (212 b.p.), the complete joining region (63 b.p.)and the complete constant region (387 b.p.). Figure 1 showsthe nucleotide and amino acid sequences of the possumTCRαC cDNA. The deduced amino acid sequence for thepossum TCRαC region is 129 residues in length and wasfound to have three cysteine residues and three sites forpotential N-linked glycosylation. An alignment of the aminoacid sequence of the possum TCRαC with that of otherspecies is shown in Fig. 2 and the percentage of sequenceidentity, at both the nucleotide and amino acid levels, isshown in Table 1. At the amino acid level, the highest degreeof sequence identity was found to be with eutheriansequences, including human (55.9%), followed by pig

PD Zuccolotto et al.104

Marsupial TCR C regions 105

Figure 1 Nucleotide and aminoacid sequences of brushtailpossum (Trichosurus vulpecula)TCRαC (GenBank accessionnumber AF133097). Numberingto the left of sequence representsnucleotide position; numbering toright, in bold, represents the aminoacid position. Cysteines arecircled and potential N-linked glycosylation sites are boxed. Thepolyadenylation signal is under-lined and shown in bold.

Figure 2 Alignment of the deduced brushtail possum TCRαC amino acid sequence with TCRαC sequences from other vertebrates (seeTable 1 for GenBank accession numbers). Gaps that have been introduced to optimize the alignment are indicated by dots and positionsof agreement with the possum sequence are shaded. The different TCRαC domains are also indicated: EX, extracellular domain; TM,transmembrane domain; CY, cytoplasmic domain.

PD Zuccolotto et al.106

Table 1 Percentage sequence identity of brushtail possum TCRαC and TCRβC with homologues from other vertebrate species

TCRαC TCRβCDNA Protein DNA Protein

Human 65.8 55.9 β1, 75.1; β2, 74.8 β1, 67.0; β2, 68.6Mouse 62.2 50.4 73.6 63.0Rat 63.0 50.0 74.5 63.2Rabbit 61.3 50.4 73.6 62.7Pig 62.0 54.5 73.3 64.4Sheep 60.7 53.5 71.0 62.1Chicken 40.1 31.2 50.9 41.7Salamander 45.3 33.1 53.0 47.4Catfish 39.1 20.0 45.8 29.6

Percentage identities (ignoring gaps) were calculated using the H O M O L O G I E S computer program (Genetics Computer Group, Madison, WI,USA). Accession numbers for GenBank entries used in the analyses for TCRαC are: human, M12423; mouse, U46581; rat, M18853; rabbit,M12885; pig, L21158; sheep, M55622; chicken, U04611; salamander, U50992; catfish, U39194. Those used for TCRβC are: human β1, L07294;human β2, M12888; mouse, U46841; rat, X14319; rabbit, M13895; pig, L21159; sheep, M94182; chicken, M37806; salamander, L08498;catfish, U39193.

Figure 3 Nucleotide and aminoacid sequence of brushtail possum(Trichosurus vulpecula) TCRβC(GenBank accession numberAF133098). Numbering to the leftof sequence represents nucleotideposition; numbering to right, inbold, represents the amino acidposition. Cysteines are circled andpotential N-linked glycosylationsites are boxed. The polyadenyla-tion signal is underlined andshown in bold.

(54.5%), sheep (53.5%), rabbit (50.4%), rat (50.4%) andmouse (50.0%). Lower levels of identity were observed forthe salamander (33.1%), chicken (31.2%) and catfish(20.0%) sequences.

Brushtail possum TCRβC cDNA sequence

Likewise, the use of consensus TCRβC primers led to the iso-lation of a 260 b.p. fragment from tammar wallaby PBMCRNA. This fragment had 67.8% average sequence identitywith eutherian TCRβC and was used as a probe in screeningthe possum lymph node library.

The longest TCRβ clone isolated was 868 b.p. and con-sisted of the partial coding region for the joining region (93b.p.) and the complete constant region (537 b.p.). Figure 3shows the nucleotide and deduced amino acid sequences ofthe possum TCRβC. The possum TCRβC region is 179 aminoacids long and contains three cysteine residues and three sitesfor potential N-linked glycosylation. A comparative align-ment of the amino acid sequence of the possum TCRβC withother species is shown in Fig. 4. Similar to the resultsobtained for the TCRαC comparison, levels of amino acidsequence identity for possum TCRβC were highest amongeutherian species. As shown in Table 1, the human β2sequence showed the highest level of sequence identity(68.6%), followed by human β1 (67.0%), pig (64.4%), rat(63.2%), mouse (63.0%), rabbit (62.7%) and sheep (62.1%).Lower levels of sequence identity were obtained for the sala-mander (47.4%), chicken (41.7%) and catfish (29.6%).

Phylogenetic relationships of brushtail possum TCRαCand TCRβC

Phylogenetic trees based on maximum parsimony analyses ofthe deduced possum TCRαC and TCRβC amino acid

sequences are shown in Figs 5 and 6, respectively. In bothcases, the possum sequence formed part of a mammalianclade but was sister to all eutherian species. However, therewere some differences between the tree based on TCRαCsequences and that based on TCRβC. Most notably, the position of human and ungulates (pig and sheep) are inter-changed between the two trees and the salamander sequenceoccupies a spurious position in the TCRβC tree, which wouldindicate that it is more closely related to the mammaliansequences than is the chicken.

Discussion

As is evident from Table 1, a comparison of the possumTCRαC sequence with orthologous sequences from otherspecies revealed that the highest degree of sequence identitywas with eutherian mammals. Lower levels of sequence iden-tity were also calculated for non-mammalian vertebrates,including the salamander and chicken, and this is to beexpected considering their early divergence from mammals.33

More variation was evident in the extracellular portion of theTCRαC compared with that located around the transmem-brane and cytoplasmic regions. For example, the level ofamino acid sequence identity of the possum TCRαC with themouse was 47% for the extracellular (EX) region, 60% forthe transmembrane (TM) region and 83% for the cytoplasmic(CY) region (data not shown). The fact that the TCRαC CYand TM domains and part of the EX domain in the immedi-ate vicinity of the TM domain are conserved is of interestbecause it is thought that these regions have a role in deter-mining a non-covalent interaction with CD3 chains and informing a disulfide bond with the TCRβ chain.30,34

A comparison of the possum TCRβC sequence with othervertebrate TCRβC sequences also revealed the highest levelsof sequence identity with eutherian sequences (see Table 1).

Marsupial TCR C regions 107

Figure 4 Alignment of the deduced brushtail possum TCRβC amino acid sequence with TCRβC sequences from other vertebrates (seeTable 1 for GenBank accession numbers). Gaps that have been introduced to optimize the alignment are indicated by dots and positionsof agreement with the possum sequence are shaded. The different TCRβC domains are also indicated: EX, extracellular domain; TM,transmembrane domain; CY, cytoplasmic domain. Arrowheads indicate residues referred to in the text.

The possum TCRβC contains three cysteine residues, two ofwhich are conserved in all of the species compared and theremaining cysteine is absent only in the catfish (see Fig. 4).Two of the conserved cysteines (positions 31 and 132, inFig. 4) are thought to be involved in the formation of a disul-fide bond within the TCRβ chain, whereas the cysteine atposition 96 may have a role in forming the linkage betweenTCRα and TCRβ in the heterodimer.30 In contrast to TCRαC,the different domains of TCRβC displayed similar levels ofsequence conservation. For example, a comparison of thepossum TCRβC EX domain with the same domain of thehuman TCRβC region revealed an amino acid sequence iden-tity of 70%, while the amino acid sequence identity for thepossum and human TM domain was 72%. It is thought thatboth of these regions may play a crucial role in the differen-tiation of thymocytes.35 Specific amino acids within thetransmembrane domain have also been shown to be crucialfor TCR expression. The lysine at position 156 in Fig. 4 isconserved in all the species compared and it is thought that itmay have a role in the association of the αβTCR with theCD3 complex.36 Within the extracellular domain, two aminoacids are thought to be important in the interaction of theTCRα and TCRβ chains. Located at positions 96 and 100 ofthe TCRβ chain, respectively, this cysteine and phenylalanineare conserved in all of the species studied (Fig. 4). Substitu-tion of these amino acids has been shown to prevent TCR cellsurface expression.28

Due to the relatively close homologous relationshipbetween TCRαC and TCRδC that has been noted by others,37

phylogenetic reconstruction using TCRαC and TCRδCsequences and including the possum TCRαC was performed(data not shown). This indicated that the possum sequence

indeed forms part of a TCRαC monophyletic group and thus confirmed its identity as a bona fide TCRαC rather than a TCRδC homologue. Having demonstrated this, phylo-genetic analyses of the deduced possum TCRαC and TCRβCsequences revealed a close, but nevertheless basal, relation-ship between these and orthologous sequences from euther-ian mammals. This is consistent with the current view ofmammalian evolution, which holds that the stem marsupialdiverged some 130 million years ago, well before the mainradiation of the eutherian orders 65–115 million years ago.3

Consequently, the possum sequences reported here representthe most phylogenetically distinct mammalian TCRsequences currently available and should prove to be usefuloutgroups in assessing the evolution of mammalian TCR.

The present report describes the cloning of the cDNAsencoding tammar wallaby TCRα and TCRβ constant regions.Sequencing of these regions revealed considerable levels ofsequence identity with eutherian species and highly con-served regions important in the expression of the αβTCR.These sequences, as well as previously characterized genes,will allow us to investigate the ontogeny of adaptive immu-nity and the specific role of the αβTCR in T cell function inmarsupials and to begin to address novel aspects of marsupialimmune function. Future work will also concentrate oncloning of the brushtail possum γ and δTCR constant regionsand the sequencing of the complete possum TCR α and βchain (V, D and J segments) for a better understanding of theevolution of the marsupial immune system and physiologicaland structural adaptations not shared by other groups.

PD Zuccolotto et al.108

Figure 5 Unrooted protein parsimony tree based on TCRαCsequences from various vertebrate species. Phylogenetic recon-struction was based on the alignment shown in Fig. 2, in whichgaps were treated an uninformative. The numbering refers to thebootstrap values (out of 100) giving rise to each branchingarrangement.

Figure 6 Unrooted protein parsimony tree based on TCRβCsequences from various vertebrate species. Phylogenetic recon-struction was based on the alignment shown in Fig. 4, in whichgaps were treated as uninformative. The numbering refers to thebootstrap values (out of 100) giving rise to each branchingarrangement.

Acknowledgements

We thank Ron Claassens and Dr Steve McLoud for assistancein animal handling procedures. For financial support, we aregrateful to the University of Western Sydney, Nepean, and the Cooperative Research Centre for the Conservation and Management of Marsupials.

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Marsupial TCR C regions 109