Characterizing T-cell receptor g -variablegene in Aotus nancymaae owl monkey

peripheral blood

J.E. Guerrero

D.P. Pacheco

C.F. Suarez

P. Martınez

F. Aristizabal

C.A. Moncada

M.E. Patarroyo

M.A. Patarroyo

Authors’ affiliations:

J.E. Guerrero1,2*,

D.P. Pacheco1*,

C.F. Suarez1*,

P. Martınez1,2,

F. Aristizabal2,

C.A. Moncada1,

M.E. Patarroyo1,2,

M.A. Patarroyo1,2*

1Molecular Biology

Department, Fundacion

Instituto de Inmunologıa de

Colombia, Santa Fe de

Bogota, Colombia

2Universidad Nacional de

Colombia, Bogota, Colombia

Correspondence to:

M. A. Patarroyo

Fundacion Instituto de

Inmunologıa de Colombia

Calle 26 No 50-00

Santa Fe de Bogota,

Colombia

Tel: þ57 1 3244672x141

Fax: þ57 1 4815269

e-mail: mapatarr@fidic.org.co

Abstract: gd T lymphocytes have a heterodimeric complex formed by the

association of g and d chains as receptor. Proliferation of this lymphocyte

population has been observed, when infection by several pathogens such as

Mycobacterium tuberculosis and Plasmodium spp. occurs. The New World

Monkey Aotus nancymaae has become a very good experimental model for

the immunological and physiopathological study of these infectious agents.

The A. nancymaae g-variable region was characterized from peripheral blood

samples by using cDNA and genomic DNA polymerase chain reaction

amplification, DNA sequencing, and dot-blot hybridization techniques.

Seventeen different T-cell receptor g-variable (TCRGV) sequences wereobtained. These sequences were distributed among TCRGV subsets 1, 2, or 3,

according to human subset classification. Although no subset 4 amplification

was obtained, this subset was detected by dot-blot hybridization. The

presence of these 4 subsets resembles the behavior displayed by ‘gd-lowspecies’ (humans and mice), where high diversity among these lymphocytes

can be observed. Homologies greater than 70% were found with respect to

humans. Sequence convergence between human and A. nancymaae subsets

1 and 3 highlights Aotus as a promising model for studying these lymphocyte

functions.

gd T lymphocytes use a heterodimer complex formed by g- and

d-chain association (1). The gd population is variable in different

species; the ‘gd-low’ species include humans and mice, whose gd

T cells constitute a very limited proportion, about 5% of total

T lymphocytes in peripheral blood. However, within these species,

gd T lymphocytes in tissues, especially in gut and reproductive

epithelia, may account for almost half of the T cells (2). The ‘gd

high’ group includes sheep, cattle, rabbits, and chicken, where gd

T cells comprise up to 60% of the peripheral T-cell population (3).

In humans, gd T lymphocytes have been classified into groups

under different properties, for example, classified by particular

Key words:

Aotus; evolution; gd T lymphocytes; Platyrrhini;

T-cell receptor

Acknowledgments:

We are greatly indebted to P. Cardenas, Y.P. de

Coana, F. Carrillo, M. Mancera, and F. Guzman

for their valuable help in preparing this

manuscript, to R. Rodrıguez for his help in

obtaining the monkey blood samples, and to

J. Garry for patiently revising the manuscript. This

work was financed by the Colombian Ministry of

Public Health.

Received 10 May 2003, revised 19 June 2003,

accepted for publication 15 July 2003

Copyright � Blackwell Munksgaard 2003

Tissue Antigens.

Tissue Antigens 2003 62: 472–482

Printed in Denmark. All rights reserved*The authors made equal contributions to this work.

472

expressed g-variable gene segments as well as their specific

anatomical location. Subset 1 (S1) is formed by the group displaying

any of the variable g segments (Vg2, Vg3, Vg4, Vg5, and Vg8), these

lymphocytes being mainly located in bowel and colon mucosa (4–6).

Subset 2 (S2) comprises Vg9 segment-expressing lymphocytes,

preferentially located in peripheral blood (7). Finally, subsets 3 (S3)

and 4 (S4) are conformed by Vg10 and Vg11 segments, respectively;

these last subsets do not have a specific location. Those lymphocytes

prevailing both in peripheral blood and secondary lymphoid tissues

carry out an immune-response regulatory function, whilst gd

T lymphocytes located in mucosa act as cytotoxic lymphocytes (8, 9).

T-cell receptor diversity is mainly generated by germline recombin-

ation of variable (V), diversity (D), and joining (J) gene segments to

form the variable domain (V-J in g and V-D-J in d chains, respect-

ively). This variable domain is responsible for interacting with the

antigen. Each variable segment (V) has been divided into five regions

according to its function: two complementarity-determining regions

(CDRs) and three framework regions (FRs). CDRs form loops and are

in direct contact with antigen, whilst FRs adopt b-sheet conformation

providing structural support (10). Previous studies have shown that

positive Darwinian selection is displayed in immunoglobulin (Ig)

CDRs and T-cell receptor b (TCRb) but not in their respective FRs.

This selection is associated with high diversity at CDR level.

gd T lymphocytes have been proposed as ancestral to ab T and B

cells, because they posses characteristics of both cellular types, such

as direct antigenic recognition (like B cells) and a membrane receptor

(like abT cells) (2, 3). Gamma and delta variable genes are grouped in a

restricted chromosome region and constitute a multigene family, as

occurs in other Ig superfamily members. The evolution of these genes

may be driven by some processes concerning the genomic organization

pattern, such as the concerted evolution process or the birth-and-death

evolution model. These phenomena have been demonstrated for major

histocompatibility complex (MHC) gene families, light and heavy Ig

chains, and variable b TCR gene families (11).

gd T-lymphocyte proliferation has been observed in pathogenic

processes caused by Mycobacterium tuberculosis, Plasmodium sp.,

Toxoplasma gondii, Yersinia enterocolitica, Epstein–Barr virus and

early human immunodeficiency viral infections (12–17), responding

primarily to non-peptide antigens, phosphoantigens, alkylamines,

heat-shock proteins and non-classical MHC class I molecules, such

as MIC A and B (8, 18). Their recognition is carried out directly,

contrary to what happens with ab T lymphocytes in which such

recognition is indirectly mediated via MHC-dependent antigen

presentation (19).

Aotus monkeys can be infected by Plasmodium parasites causing

human malaria, therefore becoming a valuable animal model for

malaria vaccine development and antimalarial drug screening

(20–22). The Aotus immune system [ab T-lymphocyte receptors

(23), class I (24) and class II MHC molecules (25, 26), Igs (27), and

cytokines (28)] has been molecularly characterized at our institute for

better understanding of the parasite–host interaction in this experi-

mental model. High molecular similarity has generally been shown to

date between human and Aotus sequences.

Because gd T lymphocytes are involved in immune response

modulation against malaria, genetic rearrangement studies of those

gene segments encoding splenic owl monkey gd T-cell receptor

molecules have been done at molecular and functional level (29),

demonstrating that this primate constitutes an excellent animal

model for investigating the role of T cells during immune responses

against this infectious disease.

This study focuses on characterizing the gd T-cell peripheral blood

population, emphasizing molecular diversity, subset identification,

comparative analysis with previously reported primate sequences,

and synonymous and non-synonymous substitutions within the dif-

ferent regions constituting the molecule. An explanation of the

evolutionary relationship phenomena occurring in this type of multi-

gene family is also discussed.

Materials and methods

Animals

About 3ml of blood was obtained from each A. nancymaae monkey

(14 animals) by venipuncture. Monkeys were kept at our institute’s

experimental primate station in Leticia (Amazon), in accordance with

CCAC (Committee on Care and Use of Laboratory Animals, US)

guidelines and the Colombian National Institute of Health guidelines

for the use of laboratory animals, supervised by the Colombian

Wildlife Corporation (CORPOAMAZONIA). No antigenic stimulus

was applied, in order to study gd T-lymphocyte diversity in healthy

animals.

RNA and genomic DNA extraction, cDNA synthesis,

cloning, and sequencing

Total A. nancymaae heparin-anticoagulated peripheral blood samples

were obtained for mononuclear cell isolation. Lymphocytes were

separated by gradient centrifugation on Ficoll–Hypaque.

Total RNA was extracted from peripheral blood lymphocytes with

Trizol reagent (Gibco BRL, Rockville, MD) and used for cDNA

synthesis. Reverse transcription mixture contained 200U Moloney

murine leukemia virus reverse transcriptase (MMLV-RT), 5� RT

buffer, 10mM dNTPs, and 5mM oligo dT (Promega, Madison, WI).

The reaction was incubated for 1 h at 42 and 95�C for 5min for

Guerrero et al : T-cell receptor g-variable gene

Tissue Antigens 2003: 62: 472–482 473

first-strand cDNA synthesis. Genomic DNA was extracted with

Wizard DNA genomic Preps (Promega).

Primers used in the study were designed based on human sequences

and classified into three groups: the first group (1P) was designed to

amplify complete gamma (V-J-C) rearrangements in A. nancymaae

cDNA. The forward primers for the T-cell receptor g-variable (TCRGV)

were as follows: PS1F (50-TGCTTCTAGCTTTCCTG-30, variable region

signal sequence) for the variable segments belonging to subset 1 (Vg2,

Vg3, Vg4, Vg5, and Vg8); Vg9 (50-ATCAACGCTGGCAGTCC-30,

variable region signal sequence) for subset 2; for subset 3, PS3F

(50-TCCTCCTGGGCTCCTGA-30, beginning of variable region) and

PS4F (50-TTCTTCTCCCTCTGGGT-30, variable region beginning) for

subset 4. Reverse universal primer Cg (50-AAGGAAGAAAAA-

TAGTGGGC-30, constant region beginning) was used for all subsets

(Fig. 1). The Vg9 and Cg primers have been previously reported (29).

The second primer group (2P) was designed for amplifying a smaller

TCRGV gene segment, specific for each subset: subset 1, SB1-F

50-TTGGAAGGGAGAACGAAGT-30, SB1-R 50-TCATAGTACAGAA-

GACGCTGTGG-30; subset 2, SB2-F 50-TCACCTAGAGCAACCTCAAA-

30, SB2-R 50-TTCTCTACATTGTGAATGGTG-30; subset 3, SB3-F

50-GCAGTTCCAGCTATCCATTTC-30, SB3-R 50-TTCTTGCCTCCACT-

TTGTTG-30; and subset 4, SB4-F 50-CAACCTGAAATATCTATTTCC-

AGACC-30, SB4-R 50-AGTGGAAGTGGAAGTGTGAGCATT-30 (Fig. 1).

A third primer group (3P) was designed for specifically amplifying

subset 1 variable segments from genomic DNA; forward primer PS1F

and reverse variable segment-specific primers (located in different

sectors within the variable region): Vg2 (50-TACTTCCCTGGACT-

GAC-30); Vg3 (50-AGATTTTGCAGTCTCAATA-30); Vg4 (50-GTT-

CTTCCTTGTGCTTC-30); Vg5 (50-TTCAATTAGATTTCGTAGTA-30)

and Vg8 (50-GATACTTTTCTCGACTGAT-30) (Fig. 1).

cDNA and genomic DNA polymerase chain reactions (PCR) were

carried out with an initial 5-min denaturing cycle at 95�C, followed by

35 cycles consisting of 1min annealing at primer pair-specific

temperature, 1min extension at 72�C, and 1min denaturing at 94�C.

A final 5-min extension cycle was run at 72�C.

PCR products were purified by Wizard PCR preps Kit and cloned in

pGEM-T vector (Promega). Plasmid DNA from recombinant bacteria

was purified according to phenol chloroform DNA extraction protocol.

Three independent recombinant clones per amplified product were ana-

lyzed in both directions using an automatic sequencer (ABI PRISM 310

Genetic Analyzer, PE Applied Biosystems, Foster City, CA).

Dot blot and hybridization

One cDNA aliquot and one gDNA aliquot from both Aotus and

human origin were spotted on prewet HybondTM-Nþ membranes

(Amersham Pharmacia Biotech, Piscataway, NJ). Simultaneously,

one human and one Aotus PCR product of each subset were also

spotted on the membranes (excepting Aotus subset 4 that did not

amplify) to be tested for cross-reactivity with each one of the PCR-

labeled subsets used as probes. Labeling and detection were carried

out with the Gene ImagesTM and CDP-StarTM systems, using an

antifluorescein alkaline phosphatase (AP) conjugate, according to

manufacturer’s instructions. Final detection was made with CDP-

StarTM chemiluminescent detection reagent, followed by exposing

the blot for 2 h on autoradiography film.

Sequence analysis

In addition to our A. nancymaae sequences, other functional gamma

variable segment sequences (excluding joining segment) fromBos taurus

(Bota), Mus musculus (Mumu), Oryctolagus cuniculus (Orcu), Ovis aries

(Ovar), Gallus gallus (Gaga), Homo sapiens (Hosa), Hylobates lar (Hyla),

Pan paniscus (Panpa), Pan troglodytes (Pantro), Pongo pygmaeus (Popy),

and Gorilla gorilla (Gogo), as reported in GenBank were used. The

alignment was made by using the CLUSTALX software (http://www-

igbmc.u-strasbg.fr/biolnfo/clustalx) (30). Those sequences displaying an

PS1F

SignalPeptide

FR1 FR2 FR3CDR1 CDR2Joining Segment

Constant Segment

SB1D

SB1R SBR3 SBR2 SBR4SB2DSB3DSB4D

Cg

3P

1P

2P

PS1F

PS3FPS4F

Vg9

Vgamma8Vgamma2

Vgamma5Vgamma3

Vgamma4

Fig. 1. Designed primer positions along the T-cell receptor g-variable mRNA molecule.

Guerrero et al : T-cell receptor g-variable gene

474 Tissue Antigens 2003: 62: 472–482

intraspecific identity value higher than 98%, detected by GENEDOC

software (http://www.psc.edu/biomed/genedoc/) (31), were eliminated.

Homology analysis was carried out with respect to the shortest sequence

in all cases. The alignment reported in the EMBLALIGN alignment

database (http://srs.ebi.ac.uk/) (32), accession number ALIGN_000537,

contains the GenBank accession numbers for each sequence. Partial

sequences were excluded.

Phylogenetic trees were constructed with MEGA-2.1 software (http://

www.megasoftware.net/) (33) using parsimony (34) and distance

methods such as minimum evolution (ME) (35) and neighbor-joining

(NJ). Genetic distances were estimated by using the P, Jukes-Cantor

(36), and Kimura two-parameter (37) distances for nucleotide and P,

gamma and Poisson-corrected distances for amino acid sequences.

Bootstrap analysis and internal branch test (38) (1000 replicates each)

were used to assign confidence levels to branch nodes (values greater

than 70% for bootstrap and 95% for internal branch test were

considered significant). Rates of synonymous and non-synonymous

substitutions and associated variance (assessed by the bootstrap

method with 1000 replicates) were calculated by Nei and Gojobori’s

method (39) using MEGA-2.1 for FRs and CDRs. Maximum likelihood

(ML) analyses were performed with PHYLIP-3.6 software (40).

Nomenclature and variable segment classification of sequences

employed in this analysis agree with that previously reported in

1995 (41), using the IMMUNOGENETICS database (IMGT, http://imgt.

cines.fr/home.html).

Results and discussion

TCR gamma segments were specifically amplified by PCR, either

from DNA or from cDNA extracted from A. nancymaae peripheral

blood (findings are summarized in Table 1).

Eleven TCRGV sequences from cDNA and 12 other sequences

from genomic DNA were obtained. All primer combinations success-

fully amplified human controls.

Only subsets 1 and 2 from A. nancymaae cDNA were successfully

amplified by 1P primers, while no amplification was obtained when

using subset 3 and 4 primers. Subset 1, subset 2, and subset 3 were

amplified from both cDNA and genomic DNA when 2P primers were

used. We performed genomic DNA specific amplification of this

subset in A. nancymaae using 3P primers, because subset 1 possesses

higher allelic groups than other subsets in humans. Only the

segment-specific variable primers for Vg5 and Vg8 showed a positive

amplification.

Sequences obtained were included in a group when the identity

among them at nucleotide level was higher than 98%. According to

this, those subset 1 sequences obtained were classified into four

Sequences obtained in the present work

Specimens

Subset Source Primers Groups GenBank ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14

S1 cDNA PS1F-Cg GVa AY245680 X

AY245681 X X X

AY245682 X

AY245683 X

AY245684 X

AY245685 X

AY245686 X

Genomic DNA PS1F-Vg5 GVb AY245676 X

AY245675 X

GVc AY245677 X

PS1F-Vg8 AY245678 X

GVd AY245679 X

S2 cDNA Vg9-Cg GVe AY245688 X

AY245687 X

S3 Genomic DNA SB3-D SB3-R Gvf AY289743 X

Gvg AY289744 X X X X X

Gvh AY289745 X

The sequences chosen for subsequent analysis are shown in bold.

Table 1

Guerrero et al : T-cell receptor g-variable gene

Tissue Antigens 2003: 62: 472–482 475

groups: GVa, GVb, GVc, and GVd. Only one sequence group could be

distinguished in subset 2 (GVe). Subset 3 showed three groups: GVf,

GVg, and GVh. GVf and GVg had high similarity to previously

reported human subset 3. Some of the GVa subgroup (subset 1) and

GVe (Vg9, subset 2) sequences were identical or had a high homology

to previously reported Aotus sequences (29). Only two sequences

were observed in more than one individual (Table 1).

The presence of subset 3 sequences, as well as the evolutionary

relatedness of A. nancymaae to humans, suggested that A. nancy-

maae diversity would likely behave as ‘gd-low’ species do, having a

high subset number. Dot blot hybridization, using a human PCR-

labeled subset 4 sequence as probe, was performed for screening

subset 4 presence in A. nancymaae, because no PCR amplification

was obtained with the primer sets used. Positive results were

obtained from both A. nancymaae gDNA and cDNA, ratifying this

subset presence in owl monkeys, suggesting primer bias as being

responsible for the negative PCR result. Subset 1, 2, 3, and 4 probes

were tested against each subset-specific amplification product in dot

blot assays to discard intersubset cross-reactivity. No cross-reactivity

was observed (Fig. 2). These results suggest that owl monkeys

belong to the ‘gd-low’ species (3).

The tree corresponding to TCR gamma variable segment from

mammals (Fig. 3) shows four well-defined clusters corresponding to

subsets 1 (S1), 2 (S2), 3 (S3), and 4 (S4). This grouping agrees with the

existing human TCRGV subset classification (41). The tree shows

A. nancymaae sequences clustered into S1, S2, and S3 groups. It can

be seen that primates cluster separately within these groups, espe-

cially in S1 and S3, where they form statistically well-supported

groups. This result agrees with previous reports (42) showing gd

T-lymphocyte similarity among primates.

An independent analysis was performed because the sequences

obtained for subset 3 differed in length respecting S1 and S2 (starting

from FR1 and ending at the beginning of FR3 in Fig. 4) but yielded

equivalent results. The different subsets clustered clearly in unique,

1 2 3 4 5 6

a

b

Subset 1probe

a

b

Subset 2probe

a

b

Subset 3probe

a

b

Subset 4probe

C -

C -

C -

C -

Fig. 2. Dot blot hybridization for subsets 1–4.

Rows: Human template (a); Aotus nancymaae

template (b); Columns: cDNA (1); genomic DNA (2);

subset 1 polymerase chain reaction (PCR) product (3);

subset 2 PCR product (4); subset 3 PCR product (5);

subset 4 PCR product (6); negative control: unrelated

plasmid DNA (Row b, column 6).

Guerrero et al : T-cell receptor g-variable gene

476 Tissue Antigens 2003: 62: 472–482

well-supported groups. S3 sequences showed a close relationship to

primate S3 sequences. Aona-GVh was the most divergent sequence in

the primate S3 cluster; however, Aona-GVg was identical to the

human TCRGV10 sequence and only differed in one residue respect-

ing Aona-GVf (Fig. 3B).

Topologies obtained for both nucleotide and amino acid sequences by

several methods (ME, NJ, ML, and Parsimony) displayed the same groups,

these being supported by significant CP and bootstrap values (Fig. 3

denotes eachnode significantly supported in all calculationswithin a circle).

Global distribution throughout subsets was also maintained when

only FR regions were considered (both at nucleotide and at amino

acid level). Although the relationship between sequences inside each

FR might has varied, A. nancymaae and human FR sequences were

always related (data not shown).

CDRs maintained this global cluster pattern only at nucleotide

level, but the S4 cluster was not maintained at amino acid level;

however, S4 sequences remained related to each other and conserved

their clustering to S3. Such subset grouping was maintained in

primates. Considering CDR2 only, A. nancymaae and human

sequences were not found in a unique cluster (A. nancymaae

sequences are related to TCRGV from Bovidae, followed by human

sequences in S1), but when CDR1 or CDRs as a whole were consid-

ered, A. nancymaae and human sequences formed an exclusive group

(data not shown).

These findings agree with IMGT database analysis (Fig. 4) show-

ing A. nancymaae FR and CDR region delimitation with their human

counterparts. All A. nancymaae sequences analyzed conserved T-cell

receptor characteristic positions in the variable segments (shown by

squares in Fig. 4).

Sequences belonging to the three subsets found in owl monkeys

had a high level of homology respecting human segments. Subset 1

displayed Aotus vs human homology ranging between 76 and 83% at

nucleotide level and 72 and 84% at amino acid level; this is a little

lower than that observed in TCRBV genes among Aotus and humans

[77–90% at amino acid level (23)]. Subset 2 showed homology higher

than 90% at both nucleotide and amino acid levels.

A. nancymaae subset 3 sequences had high identity with humans

in the analyzed sector. Two sequences (Aona-GVf and Aona-GVg)

Aona-GVaAona-GVb

Aona-GVdAona-GVc

Aona-S

1Hosa-TRGV8Hosa-TRGV2Hosa-TRGV4Hosa-TRGV3

Hosa-TRGV5

Hosa-S

1

Prim

ate-S1

Bota-TRGV5S1Bota-TRGV5S6

Ovar-TRGV5S2Ovar-TRGV1S1

Bota-TRGV1S3Ovar-TRGV2S4

Bota-TRGV2S1

Bovidae-S

1

Orcu-TRGV1.4Orcu-TRGV1.3

Orcu-TRGV1.2Orcu-TRGV1.5

Orcu-S

1

Mumu-TRGV7Mumu-TRGV6

S1

Hosa-TRGV9Patr-TRGV9

Aona-GVeS2

Gogo-TRGV10Papa-TRGV10Hosa-TRGV10

Popy-TRGV10Hyla-TRGV10

Orcu-TRGV1.6Bota-TRGV3S2

S3

Mumu-TRGV3Mumu-TRGV1

Mumu-TRGV2Bota-TRGV4S1

Mumu-TRGV4Hosa-TRGV11

S4

100

100

100

7992

82100

99

7378

97

100

6599

99

85 100

96

98

6696

99

99

65

99

66

99

0.1

Hosa-TRGV10Aona-GVg

Aona-GVfPapa-TRGV10Gogo-TRGV10

Popy-TRGV10Hyla-TRGV10

Aona-GVhBota-TRGV3S2

Orcu-TRGV1.6

70

7193

99

99

A

B

Fig. 3. Minimum evolution (ME) tree for mammal T-cell receptor g -variable (TCRGV)-deduced protein sequences. Aotus nancymaae sequences

are indicated by bold lines. The bold circles denote each subset root.

Guerrero et al : T-cell receptor g-variable gene

Tissue Antigens 2003: 62: 472–482 477

showed homology between 99 and 100% at both nucleotide and

amino acid levels; Aona-GVh showed higher variation with 91%

homology at both levels. This might indicate a greater diversity in

this subset regarding humans, and the convergence shown by Aona-

GVg and Aona-GVf with human sequences suggests narrow func-

tional similarity.

Most of the differences between humans and A. nancymaae were

condensed at the CDRs. Remarkably, the changes between them were

generally conserved within each species, precluding polymorphism

generated by somatic variation process within each segment.

Mean homology between Aotus subgroups in subset 1 varied

between 89 and 97% at nucleotide level and 83 and 95% at amino

acid level. The extent of divergence between S1 subgroups was

slightly smaller than that observed in the human S1 group.

Mean homology between human TCRGV S1 segments varied

between 86 and 96% at nucleotide level and 79 and 94% at amino

<---------------------- F R 1 - I M G T ---------------------------------------------->

1 5 10 15 20 25

S S N L E G R T K S V I R Q T G S S A E I T C D L AHoSa TRGV2 TCT TCC AAC TTG GAA GGG AGA ACG AAG TCA GTC ATC AGG CAG ACT GGG TCA TCT GCT GAA ATC ACT TGT GAT CTT GCT - - K - - - - - - - - T G R - - - - - A - - - - - PAotus GVa --- --- --G --- --- --- --- --- --- --- --- -CT G-- CGT --- --- --- --- --- -C- --- --C --- --- --- C-- S S N L E G R T K S V I R Q T G S S A E I T C D L AHosa TRGV4 TCT TCC AAC TTG GAA GGG AGA ACG AAG TCA GTC ATC AGG CAG ACT GGG TCA TCT GCT GAA ATC ACT TGT GAT CTT GCT - - K - - - - - - - - T G R - - - - - A - - - - - -Aotus GVb --- --- --G --- --- --- --- --- --- --- --- -CT G-- -GT --- --- --- --- --- -C- --- --C --- --- --- --- S S N L E G R T K S V T R P T G S S A V I T C D L PHoSa TRGV8 TCT TCC AAC TTG GAA GGG AGA ACA AAG TCA GTC ACC AGG CCA ACT GGG TCA TCA GCT GTA ATC ACT TGT GAT CTT CCT - - M - - - - - - L - - G R - E Y S - - - - - - C -Aotus GVc --- --- -TG --- --- --- --- --G --- -T- --- --T G-- -GT --- -A- -AT --T --- --- --- --C --- --- TG- --- - - K - - - - - - - - - G R - - - - - A - - - - - -Aotus GVd --- --- --G --- --- --- --- --G --- --- --- --T G-- -GT --- --- --- --T --- -C- --- --C --- --- --- --- A G H L E Q P Q I S S T K T L S K T A R L E C V V SHosa TRGV9 GCA GGT CAC CTA GAG CAA CCT CAA ATT TCC AGT ACT AAA ACG CTG TCA AAA ACA GCC CGC CTG GAA TGT GTG GTG TCT - A - - - - - - - - - - - - - - - - - - - - - - - YAotus GVe --- G-T --- --- --- --- --- --- --- --- --- --- --- --T --- --- --- --- --- --- --- --- --- --- --- -A- L S K V E Q F Q L S I S T E V K K S I D I P C K I SHosaTRGV10 TTA TCA AAA GTG GAG CAG TTC CAG CTA TCC ATT TCC ACG GAA GTC AAG AAA AGT ATT GAC ATA CCT TGC AAG ATA TCG - - - - - - - - - - - - - - - - - - - - -Aotus GVf G --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- - - - - - - - - - - - - - - - - - - - - -Aotus GVg G --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- - - - - - - - - - I - - - - - - - - - - -Aotus GVh G --- --- --- --- --- --- --- --- --- A-A --- --- --- --- --- --- --- --- --- --- ---

<------------------- F R 2 - I M G T --------------> ___________________ CDR1 - IMGT ________________ 30 35 40 45 50 55

E G S N G Y I H W Y L H Q E G K A P Q R L Q YHoSa TRGV2 GAA GGA AGT AAC GGC TAC ... ... ... ... ... ... ATC CAC TGG TAC CTA CAC CAG GAG GGG AAG GCC CCA CAG CGT CTT CAG TAC V E N T R - - - - - - - - - R - - - - - - L -Aotus GVa -T- -A- -A- -C- A-A --- ... ... ... ... ... ... --- --- --- --- --C --- --- --A C-- --- --- --- --- --- --- -T- --- E G S T G Y I H W Y L H Q E G K A P Q R L L YHosa TRGV4 GAA GGA AGT ACC GGC TAC ... ... ... ... ... ... ATC CAC TGG TAC CTA CAC CAG GAG GGG AAG GCC CCA CAG CGT CTT CTG TAC - G - T R - I - - - - - - - R - - - - - - - -Aotus GVb --- -G- --- A-- AGA --- ... ... ... ... ... ... –T- --- --- --- --T --- --- --A C-- --- --- --- --- --- --- --- --- V E N A V Y T H W Y L H Q E G K A P Q R L L YHoSa TRGV8 GTA GAA AAT GCC GTC TAC ... ... ... ... ... ... ACC CAC TGG TAC CTA CAC CAG GAG GGG AAG GCC CCA CAG CGT CTT CTG TAC - - - T R - I - - - - -Aotus GVc --- --- --- A-- AGA --- ... ... ... ... ... ... AT- --- --- --- --C --- - V - T R - I - - - - - - - R - - - - - - - -Aotus GVd --- -T- --- A-- AGA --- ... ... ... ... ... ... AT- --- --- --- --C --- --- --A C-- --- --- --- --- --- --- --- --- G I K I S A T S V Y W Y R E R P G E V I Q F L V SHosa TRGV9 GGA ATA AAA ATT TCT GCA ACA TCT ... ... ... ... GTA TAT TGG TAT CGA GAG AGA CCT GGT GAA GTC ATA CAG TTC CTG GTG TCC - A - - - E - - I - - - - - - - - - A - - L - - YAotus GVe --- GC- --- --- --- -AG --- --- ... ... ... ... A-- --- --- --- --- --A --- --- --- --- -C- --- --- C-- --- --- -A- S T R F E T D V I H W Y R Q K P N Q A L E H L I YHosaTRGV10 AGC ACA AGG TTT GAA ACA GAT GTC ... ... ... ... ATT CAC TGG TAC CGG CAG AAA CCA AAT CAG GCT TTG GAG CAC CTG ATC TAT - - - - - - - - - - - - Q - - - - - - - - - - - -Aotus GVf --- --- --- --- --- --- --- --- ... ... ... ... --- --- --- --- -A- --- --- --- --- --- --- --- --- --- --- --- --- - - - - - - - - - - - - - - - - - - - - - - - - -Aotus GVg --- --- --- --- --- --- --- --- ... ... ... ... --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- - - N - - - - - - - - - - - - - - - - - - - - - -Aotus GVh --- --- -AC --- --- --- --- --- ... ... ... ... --- --- --- --- --- --- --- --G --- --- --- --- --- --- --- --- ---

<---------------------------------------------------- F R 3 ---- ____________ CDR2 - IMGT _______________ 60 65 70 75 80 Y D S Y N S K V V L E S G V S P G K Y Y T Y A S THoSa TRGV2 TAT GAC TCC TAC AAC TCC AAG GTT ... ... GTG TTG GAA TCA GGA GTC AGT CCA GGG AAG TAT TAT ACT TAC GCA AGC ... ACA - - T - Q I - - - - D - - I - - - - - D I - - I -Aotus GVa --- --- A-- --- C-G AT- --- --- ... ... --- --- --T --- --- A-- --- --- --A --- --- G-- -T- --T --- -T- ... --- Y D S Y T S S V V L E S G I S P G K Y D T Y G S THosa TRGV4 TAT GAC TCC TAC ACC TCC AGC GTT ... ... GTG TTG GAA TCA GGA ATC AGC CCA GGG AAG TAT GAT ACT TAT GGA AGC ... ACA - - T - H I K - - - D - - - - S R - D - I - A I -Aotus GVb --- --- A-- --- CA- AT- -AA --- ... ... --- --- --T --- --- --- --- T-C C-- --A G-- --- -T- --- -C- -T- ... --C Y D S Y N S R V V L E S G I S R E K Y H T Y A S THoSa004 TAT GAC TCC TAC AAC TCC AGG GTT ... ... GTG TTG GAA TCA GGA ATC AGT CGA GAA AAG TAT CAT ACT TAT GCA AGC ... ACA - - T - Q - Q - - - D - - - - P G - - N I - - I -HoSa TRGV8 --- --- A-- --- C-G --- CA- --- ... ... --- --- --T --- --- --- --- -C- -G- --- --- A-- -T- --- --- -T- ... --- - - T - Q I K - - - D - -Aotus GVd --- --- A-- --- C-G AT- -A- --- ... ... --- --- --T --- --- I S Y D G T V R K E S G I P S G K F E V D R I P EHosa TRGV9 ATT TCA TAT GAC GGC ACT GTC ... ... ... AGA AAG GAA TCT GGC ATT CCG TCA GGC AAA TTT GAG GTG GAT AGG ATA CCT GAA - - S - - A - R M - - - - T - - - - - - - - V - -Aotus GVe --- --- -C- --T --- G-- --- ... ... ... --- -T- --- --A --- --- A-- --- --- --- --- --- --- --- --- G-- --- --- I V S T K S A A R R S M G K T S N K V E A R K N S QHosaTRGV10 ATT GTC TCA ACA AAA TCC GCA GCT ... ... CGA CGC AGC ATG GGT AAG ACA AGC AAC AAA GTG GAG GCA AGA AAG AAT TCT CAA - - - - - - - - - - - - - - - - - - - - - -Aotus GVf --- --- --- --- --- --- --- --- ... ... --- --- --- --- --- --- --- --- --- --- --- --- --- --- - - - - - - - - - - - - - - - - - - - - - -Aotus GVg --- --- --- --- --- --- --- --- ... ... --- --- --- --- --- --- --- --- --- --- --- --- --- --- V A L - - - S - G G - - - E R - - - - - - -Aotus GVh G-- -C- -T- --G --- --- T-- --- ... ... G-- G-- --- --- --- G-- -GG --- --- --- --- --- --- ---

------------------------- F R 3 - I M G T ---------------------->

85 90 95 100 R N N L R L I L R N L I E N D S G V Y Y CHoSa TRGV2 AGG AAC AAC TTG AGA TTG ATA CTG CGA AAT CTA ATT GAA AAT GAC TCT GGG GTC TAT TAC TGT - R S W K - V - Q - - - - D - - - - - F VAotus GVa --- -GG –G- -G- -A- --- G-- --- -A- --- --- --- --- G-- --T --- --- --- --- -TT GTG R K N L R M I L R N L I E N D S G V Y Y CHosa TRGV4 AGG AAG AAC TTG AGA ATG ATA CTG CGA AAT CTT ATT GAA AAT GAC TCT GGA GTC TAT TAC TGT - - S W K L V - - - - - -Aotus GVb --- --- -G- -G- -A- T-- G-- --A --- --- --A --- --- G K S L K F I L E N L I E R D S G V Y Y CHoSa004 GGG AAG AGC CTT AAA TTT ATA CTG GAA AAT CTA ATT GAA CGT GAC TCT GGG GTC TAT TAC TGT R R - W - L V - R - - - -HoSa TRGV8 A-- -G- --- TGG --- --G G-- --A CG- --- --- --- --- T S T S T L T I H N V E K Q D I A T Y Y CHosa TRGV9 ACG TCT ACA TCC ACT CTC ACC ATT CAC AAT GTA GAG AAA CAG GAC ATA GCT ACC TAC TAC TGT - - - - - - - - - - - - - K - - - - - - -Aotus GVe --- --- --- --T –-C --- --- --- --- --- --- --- --- A-A --- --- --- --- --T --T ---

Fig. 4. Amino acid sequence alignment

corresponding to T-cell receptor g -variable(TCRGV) region (FR1, CDR1, FR2, CDR2,

FR3, and partial CDR3) from Aotus nan-

cymaae and its human homolog, as analyzed

by IMMUNOGENETICS. FR, framework region.

Guerrero et al : T-cell receptor g-variable gene

478 Tissue Antigens 2003: 62: 472–482

acid level, indicating that A. nancymaae have a similar degree of

intraspecies divergence to that exhibited by human subset 1 and the

same degree of sequence conservation as subset 2.

Although both A. nancymaae and humans clustered together, owl

monkey sequences were grouped in a well-defined cluster in subset 1

(Fig. 3A), whereas human sequences showed more instability within

the S1 primate group (there was low statistical support for its

branches and variant clustering pattern, depending on the phylo-

genetic inference method used). The precise relationship between

humans and A. nancymaae remains ambiguous, independently of

the phylogenetic method employed. The extent of divergence

between A. nancymaae and human sequences is equivalent to the

divergence observed among just human sequences. This divergence

is greater than that observed among A. nancymaae sequences only.

This type of TCRGV primate segment clustering was consistent with

a species-specific evolution pattern.

Accordingly, specific primers for each TCRGV segment subset 1

deduced from consensus human FR3 regions were used to perform

the PCRs from A. nancymaae genomic DNA. A. nancymaae

sequences obtained did not cluster with the human segments from

which the primers had been deduced. This finding reveals that

species-specific changes at segment level inside each species is

occurring, indicating the conservation of some motifs at FR3 level

between humans and owl monkeys.

This species-specific pattern was shared with rabbits, but not with

Bovidae (sheep and cows), where each segment clustered together,

maintaining a trans-specific pattern. Other analyses based on the

available sequences, including non-mammal sequences (chicken,

skate, and clawed frogs, data not shown) have shown that a spe-

cies-specific cluster pattern is the most common in subset 1. The

species-specific behavior of S1 contrasts with the trans-specific rela-

tionship observed in a, b, and d TCR genes (3, 11). The remaining

TCRGV subsets showed noticeable low polymorphism, also contrast-

ing with a and b TCR genes. These phenomena (low diversity and

species-specific patterns) might indicate a different way of TCRGV

gene evolution agreeing with a homogenization process as shown in

the tree describing the origin of several groups (Bovidae S1, Orcu S1,

and Aona S1) (Fig. 3). Other groups (S2 and S3) showed a pattern

0

5

10

15

20

25

30

All Molecule FRs FR1 FR2 FR3 CDRs CDR1 CDR2

Num

ber

of d

iffer

ence

s (a

a) w

ithin

spe

cies

Homo Aotus

Fig. 5. Average number of intraspecies amino acid pair-wise

differences in the subset 1 primate species studied. The average

was obtained from all possible pair-wise sequence comparisons for each

sequence group. The error bars indicate the maximum and the minimum

values observed.

Synonymous vs non-synonymous changes in T-cell receptor g -variable (TCRGV) (FR-CDR)

FR CDR

Number dN dS dN� dS S dN dS dN� dS S

1 AonaS1 0.051 0.078 �0.027 NS 0.098 0.05 0.048 NS

2 HosaS1 0.093 0.121 �0.028 NS 0.315 0.068 0.247 þ

3 PrimateS1 0.105 0.161 �0.055 NS 0.369 0.103 0.265 þ

4 OvarS1 0.301 0.407 �0.106 NS 0.372 0.451 �0.078 NS

5 BosS1 0.287 0.381 �0.094 NS 0.253 0.487 �0.234 NS

6 BovidaeS1 0.27 0.359 �0.089 NS 0.29 0.393 �0.103 NS

7 OrcuS1 0.099 0.147 �0.049 NS 0.18 0.125 0.054 NS

8 S1 0.325 0.487 �0.161 – 0.606 0.563 0.042 NS

9 S2 0.042 0.123 �0.082 – 0.138 0.117 0.02 NS

10 S3 0.13 0.217 �0.087 – 0.207 0.269 �0.061 NS

11 S4 0.256 0.643 �0.387 – 1.006 1.134 �0.128 NS

dN, non-synonymous substitution; dS, synonymous substitution; NS, non-significant; S, selection.

Table 2

Guerrero et al : T-cell receptor g-variable gene

Tissue Antigens 2003: 62: 472–482 479

similar to species phylogeny, while S4 displayed a trans-specific

pattern. The overall TCRGV evolutive forms are more heterogeneous

than that observed in other TCRV genes (a, b, and d).

A. nancymaae is the least variable species within subset 1. Primate

S1 showed variability equivalent to that observed in rabbits and

lower than that observed in Bovidae. These tendencies were

maintained in each of the gene sectors. Figure 5 shows that the

average difference in owl monkeys was at least half of that observed

in humans at amino acid level; the same was true at nucleotide level

(data not shown). This was the general tendency in all of the gene

sectors, with the exception of FR1, where the variability observed

between these species was similar.

Previous studies have shown the presence of positive selection in

CDRs, both in Ig and in TCRBV (11, 43). A selection pressure can be

detected if the number of non-synonymous substitutions is signifi-

cantly different to synonymous substitutions (dN> dS, positive pres-

sure selection; dN< dS, negative pressure selection). Table 2 and

Fig. 5 show the results of the Nei–Gojobori test in FRs and CDRs,

with a pair-wise deletion option for each group shown in the tree.

Each subset (as a group) showed a significant negative pressure

for the whole molecule (data not shown); this tendency was also

shared when FRs were considered altogether or separately (Table 2,

Fig. 6A), more synonymous than non-synonymous substitutions

always being shown. Nevertheless, these disequilibrium results are

non-significant for some groups (data not shown). As can be seen in

Table 2 and Fig. 6B, CDRs showed more heterogeneous behavior, but

significant results can only be observed for positive selection in

primate subset 1, specifically for human CDR1 (data not shown).

0

0.2

0.4

0.6

0.8

1

0

A

B

0.2 0.4 0.6 0.8 1

Synonymous substitutions dS

Synonymous substitutions dS

1 92 7

3

65 4 8

11

10Non

-syn

onym

ous

subs

titut

ions

dN

Non

-syn

onym

ous

subs

titut

ions

dN

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

19

7

23

10

65

4

8

FR dN/dS

CDR dN/dS

Fig. 6. Relationships between synonymous (dS) vs non-synonymous (dN) substitutions in TCRGV FR(A) and CDR(B). The highest marks

represent significant selection. Species associations are numbered from 1 to 11, according to Table 2. The straight line represents the expected relationship in the

case of strict neutrality.

Guerrero et al : T-cell receptor g-variable gene

480 Tissue Antigens 2003: 62: 472–482

A. nancymaae also showed little tendency to accumulate non-

synonymous substitution in CDRs, but such behavior was negligible.

This pattern contrasts with that observed in TCRBV, where a clear

purifying selection in the FRs and a sharp positive selection in CDRs

exist (11).

Although many groups were close to neutral behavior, these

results suggest a conserving phenomenon for TCRGV (including

A. nancymaae), generally comprising all sequence components.

The presence of four subsets in A. nancymaae (S4 could not

be amplified, but was identified in cDNA and gDNA), equivalent

to those present in humans, suggests that A. nancymaae

monkeys belong within the ‘gd-low’ group (including humans

and mice, as they have a high degree of diversity in the TCR-V

and IGV genes) (3, 44). On the contrary, ‘gd-high’ species display

a lower diversity, compensated maybe by a higher gd cell

proportion.

Although the function of gd lymphocytes is not fully understood,

their presence correlates with an immunosurveillance role, as demon-

strated by studies where a high level of expression for subsets 1 and 2

was found in humans infected with P. falciparum malaria (45). The

presence and homology of the same subtypes among A. nancymaae

and humans at sequence level lead to the hypothesis of a similar role

for this kind of cell in A. nancymaae. Human and owl monkey subset 1

convergence at CDR level provides evidence of such homology,

suggesting similar recognition properties. Subset 3 also exhibits con-

vergence; functional similarity among owl monkey human subset 2 has

previously been shown (Daubenberger). These findings highlight

A. nancymaae as a promising model for studying TCRGV function.

References

1. Hayday AC. Gamma delta cells: a right time

and a right place for a conserved third way of

protection. Annu Rev Immunol 2000: 18:

975–1026.

2. Richards MH, Nelson JL. The evolution of

vertebrate antigen receptors: a phylogenetic

approach. Mol Biol Evol 2000: 17: 146–55.

3. Su C, Jakobsen I, Gu X, Nei M. Diversity and

evolution of T-cell receptor variable region

genes in mammals and birds. Immunogenetics

1999: 50: 301–8.

4. Kabelitz D, Glatzel A, Wesch D. Antigen

recognition by human gammadelta T

lymphocytes. Int Arch Allergy Immunol 2000:

122: 1–7.

5. Lahn M. The role of gammadelta T cells in the

airways. J Mol Med 2000: 78: 409–25.

6. Hanninen A, Harrison LC. Gamma delta

T cells as mediators of mucosal tolerance: the

autoimmune diabetes model. Immunol Rev

2000: 173: 109–19.

7. Haas W, Pereira P, Tonegawa S. Gamma/delta

cells. Annu Rev Immunol 1993: 11: 637–85.

8. Kaufmann SH. Gamma/delta and other

unconventional T lymphocytes: what do they

see and what do they do? Proc Natl Acad Sci

USA 1996: 93: 2272–9.

9. Steinle A, Groh V, Spies T. Diversification,

expression, and gamma delta T cell

recognition of evolutionarily distant members

of the MIC family of major histocompatibility

complex class I-related molecules. Proc Natl

Acad Sci USA 1998: 95: 12510–5.

10. Allison TJ, Winter CC, Fournie JJ, Bonneville M,

Garboczi DN. Structure of a human

gammadelta T-cell antigen receptor. Nature

2001: 411: 820–4.

11. Su C, Nei M. Evolutionary dynamics of the

T-cell receptor VB gene family as inferred

from the human and mouse genomic

sequences. Mol Biol Evol 2001: 18: 503–13.

12. O’Brien RL, Happ MP, Dallas A, Palmer E,

Kubo R, Born WK. Stimulation of a major

subset of lymphocytes expressing T cell

receptor gamma delta by an antigen derived

from Mycobacterium tuberculosis. Cell 1989:

57: 667–74.

13. Havran WL, Boismenu R. Activation and

function of gamma delta T cells. Curr Opin

Immunol 1994: 6: 442–6.

14. Perera MK, Carter R, Goonewardene R,

Mendis KN. Transient increase in circulating

gamma/delta T cells during Plasmodium vivax

malarial paroxysms. J Exp Med 1994: 179:

311–5.

15. Tanaka Y, Morita CT, Nieves E, Brenner MB,

Bloom BR. Natural and synthetic non-peptide

antigens recognized by human gamma delta

T cells. Nature 1995: 375: 155–8.

16. Poquet Y, Halary F, Champagne E et al.

Human gamma delta T cells in tuberculosis.

Res Immunol 1996: 147: 542–9.

17. Bukowski JF, Morita CT, Brenner MB. Human

gamma delta T cells recognize alkylamines

derived from microbes, edible plants, and tea:

implications for innate immunity. Immunity

1999: 11: 57–65.

18. Groh V, Steinle A, Bauer S, Spies T.

Recognition of stress-induced MHC molecules

by intestinal epithelial gammadelta T cells.

Science 1998: 279: 1737–40.

19. Davis MM, Chien Y. Issues concerning the

nature of antigen recognition by alpha beta

and gamma delta T-cell receptors. Immunol

Today 1995: 16: 316–8.

20. Lujan R, ChapmanWL, HansonWL, Dennis VA.

Leishmania braziliensis: development of

primary and satellite lesions in the

experimentally infected owl monkey, Aotus

trivirgatus. Exp Parasitol 1986: 61: 348–58.

21. Collins WE. Nonhuman primate models. II.

Infection of Saimiri and Aotus monkeys with

Plasmodium vivax. Methods Mol Med 2002:

72: 85–92.

22. Noya O, Gonzalez-Rico S, Rodriguez R,

Arrechedera H, Patarroyo ME, Alarcon de

Noya B. Schistosoma mansoni infection in owl

monkeys (Aontus nancymai): evidence for

early elimination adult worms. Acta Trop

1998: 70: 257–67.

23. Vecino W, Daubenberger C, Rodriguez R,

Moreno A, Patarroyo M, Pluschke G. Sequence

and diversity of T-cell receptor beta-chain

V and J genes of the owl monkey Aotus

nancymaae. Immunogenetics 1999: 49: 792–9.

24. Suarez CF, Cardenas PP, Llanos-Ballestas EJ

et al. Alpha 1 and Alpha 2 Domains of Aotus

MHC Class I and Catarrhini MHC Class Ia

share similar characteristics. Tissue Antigens

2003: 61: 362–73.

Guerrero et al : T-cell receptor g-variable gene

Tissue Antigens 2003: 62: 472–482 481

25. Diaz D, Naegeli M, Rodriguez R et al.

Sequence and diversity of MHC DQA and

DQB genes of the owl monkey Aotus

nancymaae. Immunogenetics 2000: 51:

528–37.

26. Nino-Vasquez JJ, Vogel D, Rodriguez R et al.

Sequence and diversity of DRB genes of Aotus

nancymaae, a primate model for human

malaria parasites. Immunogenetics 2000: 51:

219–30.

27. Diaz OL, Daubenberger CA, Rodriguez R et al.

Immunoglobulin kappa light-chain V, J, and C

gene sequences of the owl monkey Aotus

nancymaae. Immunogenetics 2000: 51: 212–8.

28. Hernandez EC, Suarez CF, Mendez JA,

Echeverry SJ, Murillo LA, Patarroyo ME.

Identification, cloning, and sequencing of

different cytokine genes in four species of owl

monkey. Immunogenetics 2002: 54: 645–53.

29. Daubenberger CA, Salomon M, Vecino W et al.

Functional and structural similarity of

V gamma 9V delta 2 T cells in humans and

Aotus monkeys, a primate infection model for

Plasmodium falciparum malaria. J Immunol

2001: 167: 6421–30.

30. Thompson JD, Gibson TJ, Plewniak F,

Jeanmougin F, Higgins DG. The CLUSTAL_X

windows interface: flexible strategies for

multiple sequence alignment aided by quality

analysis tools. Nucleic Acids Res 1997: 25:

4876–82.

31. Nicholas KB, Noicholos HBJ, Deerfield DWI.

Genedoc. Analysis and visualization of genetic

variation. EMBNEW NEWS 1997: 4: 14.

32. Lombard V, Camon EB, Parkinson HE,

Hingamp P, Stoesser G, Redaschi N. EMBL-

Align: a new public nucleotide and amino acid

multiple sequence alignment database.

Bioinformatics 2002: 18: 763–4.

33. Kumar S, Tamura K, Jakobsen IB, Nei M.

MEGA2: molecular evolutionary genetics

analysis software. Bioinformatics 2001: 17:

1244–5.

34. Fitch W. Toward defining the course of

evolution: minimum change for a specified

tree topology. Syst Zool 1971: 20: 406–16.

35. Rzhetsky A, Nei M. Theoretical foundation of

the minimum-evolution method of

phylogenetic inference.Mol Biol Evol 1993: 10:

1073–95.

36. Jukes T. Evolution of protein molecules. In:

Munro HN, ed. In: Mammalian Protein

Metabolism. New York: New York Academic,

1969: 21–132.

37. Kimura M. A simple method for estimating

evolutionary rates of base substitutions

through comparative studies of nucleotide

sequences. J Mol Evol 1980: 16: 111–20.

38. Sitnikova T. Bootstrap method of interior-

branch test for phylogenetic trees. Mol Biol

Evol 1996: 13: 605–11.

39. Nei M, Gojobori T. Simple methods for

estimating the numbers of synonymous and

nonsynonymous nucleotide substitutions. Mol

Biol Evol 1986: 3: 418–26.

40. Felsenstein J. PHYLIP – Phylogeny Inference

Package (Version 3.6). Cladistics 1989: 5:

164–6.

41. Arden B, Clark SP, Kabelitz D, Mak TW.

Human T-cell receptor variable gene segment

families. Immunogenetics 1995: 42: 455–500.

42. Sturm E, Bontrop RE, Vreugdenhil RJ, Otting N,

Bolhuis RL. T-cell receptor gamma/delta:

comparison of gene configurations and

function between humans and chimpanzees.

Immunogenetics 1992: 36: 294–301.

43. Tanaka T, Nei M. Positive Darwinian

selection observed at the variable-region genes

of immunoglobulins. Mol Biol Evol 1989: 6:

447–59.

44. Sitnikova T, Nei M. Evolution of

immunoglobulin kappa chain variable region

genes in vertebrates. Mol Biol Evol 1998: 15:

50–60.

45. Ho M, Tongtawe P, Kriangkum J et al.

Polyclonal expansion of peripheral gamma

delta T cells in human Plasmodium

falciparum malaria. Infect Immun 1994: 62:

855–62.

Guerrero et al : T-cell receptor g-variable gene

482 Tissue Antigens 2003: 62: 472–482