Immunogenetics 38: 210-222, 1993 / m / / / / / / / o - geneO'cs

© Springer-Verlag 1993

Mhc-DRB genes of platyrrhine primates Katka Trtkov~ 1, Heike Kupfermannl, Bla~enka Grahovac 1, Werner E. Mayer1, Colm O'hUiginl, Herbert Tichy 1, Ronald Bontrop2, Jan Kleinl, a

Max-Planck-Institut far Biologie, Abteiltmg Immungenetik, Corrensstr. 42, W-7400 Ttibingen, Germany 2 TNO Institute of Applied Radiobiology and Immunology, Lange Kleiweg 151, R O. Box 5815, NL-2280 Rijswijk, The Netherlands " 3 Department of Microbiology and Immunology, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 3310l, USA

Received November 30, 1992/Revised version received January 27, 1993

Abstract. The two infraorders of anthropoid primates, Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys and the hominoids) are estimated to have diverged from a common ancestor 37 million years ago. The major histocompatibility complex class II DRB gene and haplotype polymorphism of the Catarrhini has been characterized in several recent stud- ies. The present study was undertaken to obtain infor- mation on the DRB polymorphism of the Platyrrhini. Fifty-five complete exon 2 DRB sequences were ob- tained from six species of Platyrrhini representing both the Callitrichidae and the Cebidae families. Combined with the results of a parallel contig mapping study, our data indicate that at least three loci (DRB1 *03, DRB3, and DRB5) are shared by the Catarrhini and the Platyr- rhini. However, the three loci are occupied by functional genes in the former infraorder and mostly by pseudogenes in the latter. Instead of the pseudogenes, the Platyrrhini have evolved a new set of apparently functional genes - DRBll and DRB*W12 through DRB*W19, which have thus far not been found in the Catarrhini. The DRB*W13, *W14, *W15, *W17, *W18, and *W19 genes seem to be restricted to the Cebidae family, whereas the DRB*W16 locus has so far been documented in the Callitrichidae family only. The DRB alleles of the cotton-top tamarin, and perhaps also those of the common marmoset (both members of the family Callitrichidae), are characterized by low nucleotide di- versity, possibly indicating that they diverged from a common ancestral gene relatively recently.

Correspondence to: J. Klein.

Introduction

The primate order is subdivided into two suborders, Prosimii and Anthropoidea (Martin 1990). The Pro- simii are represented by lemurs, lorises, and tarsiers; the Anthropoidea can be subdivided further into two infraorders, Platyrrhini and Catarrhini. The latter in- clude Old World monkeys (represented, for example, by the rhesus macaque), apes (represented by the gib- bon, the orangutan, the gorilla, and the chimpanzee), and humans. The Platyrrhini consist of two families, Callitrichidae (marmosets and tamarins) and Cebidae (true New World monkeys; this designation is, how- ever, often applied to all Platyrrhini which are confined in their distribution to South and Central America). The earliest fossil record of Anthropoidea dates back to Early and Middle Eocene of North Africa 55-45 mil- lion years (my) ago (Godinot and Mahboudi 1992). The earliest platyrrhine fossil from South America (Bolivia) is from the Late Oligocene, which corresponds to ap- proximately 25 my ago (Hoffstetter 1969; Fleagle et al. 1987). Molecular evidence, on the other hand, suggests that Platyrrhini and Catarrhini separated from each other at least 37 my ago (Sarich and Wilson 1967; Hasegawa et al. 1987) and, since there is no record of Platyrrhini outside the neotropical regions, some 12 my of their evolution on the South American continent remains undocumented. The currently prevailing view maintains that the Catarrhini-Platyrrhini split occurred in Africa after South America had separated from Africa and that the ancestral Platyrrhini reached South America by island hopping (the drifting continents may have remained loosely linked by a chain of islands and the primates may have rafted from one island to the next until they reached South America; see Hershkovitz 1977; Ciochon and Chiarelli 1980). In South America the ancestors radiated into the various genera and spe- cies of the Cebidae; the Callitrichidae are considered to

K. Trtkovfi et al.: Platyrrhine Mhc-DRB genes 211

be dwarf forms derived from the larger-bodied ances- tors of the Cebidae (Ford 1980), although some authors (Hershkovitz 1970) regard the marmosets and tamarins as primitive forms of the Platyrrhini.

If there is any truth to the notion of Platyrrhini origin by island hopping, the founding population of these neotropical primates must have been rather small. As random genetic drift is an important evolutionary force in small populations (Nei 1987), it is of interest to compare the major histocompatibility complex (Mhc) of the Platyrrhini and Catarrhini and thus explore the effect of drift on the Mhc. Of the different Mhc regions, the DRB region is particularly suitable for this type of study because it displays haplotype polymorphism in addition to allelic polymorphism. Several DRB haplo- types have been identified in humans (Klein et al. 1991), chimpanzee (BrSndle et al. 1992; Mayer et al. 1992; Kenter et al. 1992a), gorilla (Kasahara et al. 1992; Kupfermann et al. 1992; Kenter et al. 1992b), and orangutan (Sch6nbach et al. 1992), each haplotype occurring at an appreciable frequency in the population. The haplotypes differ in their length as well as in the number and disposition of the DRB genes they contain. Nine DRB loci (DRB1 through DRB9) have thus far been identified in the Catarrhini (Klein et al. 1992); of these, a single haplotype can contain five genes at the most. In each haplotype, however, only one or two genes are functional, while the rest are either bona fide pseudogenes or genes for which functionality has not been demonstrated. Most, if not all, of the nine DRB genes originated before the divergence of Cercopithe- coidea (Old World monkeys) and Hominoidea (apes and humans; see Klein et al. 1992), an event dated to more than 23 my ago (Martin 1990). An unresolved question is whether any of these genes occurred in the ancestral primate stock before the Platyrrhini-Catar- rhini split. To gather information on the DRB of the Platyrrhini has been the main aim of the present study.

derived from the [~1 domain-encoding part of the chimpanzee DRB cDNA clone C4-2 (Fan et al. 1989).

Polymerase chain reaction (PCR). Ten to twenty nanograms of genomic DNA in 50 B1 of 1 xPCR buffer (50 mM KC1, 1.5 mM MgCI2, 10 mM Tris-HC1, pH 8.3, 0.001% gelatin), 0.2 mM of each of the four deoxyribonucleotide triphosphates, 0.5 ~tM of each of the two primers, and 2.5 units of Taq polymerase (Beckman and Mul- timed, Kirchheim, Germany) were used for amplification by PCR (Saiki et al. 1988). The primers corresponded to the intron sequences flanking exon 2 of human or cotton-top tamarin DRB genes; they contained Barn HI and Sal I sites to ease subcloning. The sequences of the upstream oligonucleotide primers Tu139 and Tu215 were: 5'-CCGTCGACCGGATCGTTCGTGTCCCC-3' and 5'-CCGTCGACCGGATCGTTTGTGTCCCC-3', respectively; the sequences of the downstream primers Tu68 and Tu216 were: 5'-AAGGATCCCGCCC(G/T)CC(G/A)CC(G/A)TGCTCA-3' and 5'-AAGGATCCCCGCCGC(G/A)CTCA-3', respectively. The DNA was amplified by 30 cycles consisting of 1 min denaturation at 94 ° C, 30 sec primer annealing at 48 ° C to 55 ° C, and 2 rain primer extension at 72 ° C in the Hybaid Thermo Reactor TR1 (MWG-BI- OTECH, Ebersberg, Germany). The reaction was completed by an extension step of 10 min at 72 ° C. Five microliters of the mixture were analyzed on a 2% NuSieve 3 : 1 agarose gel (FMC - Biozym Diagnostik, Hameln, Germany), the remainder was extracted with phenol and chloroform, the DNA was precipitated with ethanol, resuspended in TE buffer (10 mM Tris-HC1, pH 7.4, 1 mM ethylene- diaminetetraacetic acid), and digested with the restriction enzymes Barn HI and Sal I. The DNA fragments were purified by electro- phoresis in a 1% low melting point agarose gel (GIBCO BRL, Eggenstein, Germany) and ligated to M13mp18 or M13mp19 vector DNA. Escherichia coli JM103 bacteria were transfected and positive phage clones were identified by plaque-lifting and hybridization to the chimpanzee DRB ~1 domain-encoding probe C4-2~1.

DNA sequencing and analysis. Single-stranded DNA was isolated from single clones and sequenced by the dideoxy chain-termination method (Sanger et al. 1977) using the Sequenase kit (US Biochemi- cals, Bad Homburg, Germany). At least three clones derived from one or two independent PCR were sequenced from each gene. The sequences were read and analyzed using the gel reader and the MacVector software from International Biotechnologies, Inc. (Tecnomara, Feruwald, Germany). Genetic distances were calcu- lated by the Kimura (1980) two-parameter method and dendrograms were constructed using the Saitou and Nei (1987) neighbor-joining method.

Materials and methods

Source ofDNA. Genomic DNA was isolated from peripheral blood leukocytes of six cotton-top tamarins (Saguinus oedipus), two com- mon marmosets (Callithrix jacchus), three common squirrel monkeys (Saimiri sciureus), one northern night monkey (Aotus trivirgatus), two black-capped capuchins (Cebus apeUa), and two dusky titis (Callicebus moloch). The first two species belong to the family Callitrichidae, the remaining species to the family Cebidae. The monkeys were obtained from colonies maintained at the Univer- sit~t Kassel, Kassel, Germany (A. trivirgatus and C. apella, C. moloch), Universit~t Bielefeld, Bielefeld, Germany (S. oedipus), and the TNO Institute of Applied Radiobiology and Immunology, Rij- swijk, The Netherlands ( C jacchus and S. sciureus). The S. oedipus individuals 28 and 32 are siblings; the other tamarins are not related to one another. C. jacchus XR and XS are also unrelated; the degree to which all other monkeys are related is not known. DNA was extracted following the protocol of Blin and Stafford (1976). The DRB probe C4-2~ 1 was a 200 base pair (bp) Dde I/Pst I fragment

Construction ofa minilibrary. Genomic DNA from the cotton-top tamarin cell line SC2 was digested with Hin dIII and electrophoresed in 0.8% low melting point agarose gel. Fragments in the size range of 3 - 4 kilobase (kb), which contained a gene hybridizing to the 180 bp-long Taq I fragment of the tamarin DRB cDNA clone 9-2 (Grahovac et al. 1992) were recovered using ~]-agarase (New Eng- land Biolabs, Schwalbach, Germany). The Hin dIII ends were par- tially filled in with deoxyadenosine triphosphate and deoxygua- nosine triphosphate using the Klenow enzyme (Gibco BRL). Likewise, the ends of )~ZAPII vector DNA (Stratagene, Heidelberg, Germany), which was digested with Xba I, were partially filled in with deoxycytidine triphosphate and deoxythymidine triphosphate to yield complementary overhanging ends. The genomic DNA frag- ments were ligated to the vector DNA, packaged into phage particles using the Gigapack II Gold packaging extracts (Stratagene) and transfected into E. coli CL1-Blue. The minilibrary had a complexity of 3 × 105 plaque-forming units. Plaque-lifts of the library were screened by hybridization using the 180 bp-long Taq I fragment of cDNA clone 9 - 2 as a probe.

Consensus

Saoe-DRB3*0501

Saoe-DRB3*0502

Saoe-DRB3*0503

Camo-DRB3*0501

Calao-DRB3*0502

Aotr-DRB3*0601

Camo-DRB3*0601

Camo-DRB3*0701

Saoe-DRB5*0701

Saoe-DRBII*OIOI

Saoe-DRBII*OI02

Saoe-DRBII*0103

Saoe-DRBII*0104

Saoe-DRBII*0105

CaIno-DRBII*OIOI

Caja-DRBII*OIOI

Caja-DRB*WI601

Caja-DRB*WI602

Caja-DRB*WI603

Camo-DRB*WI701

Saoe-DRBI*0301

Saoe-DRBI*0302

Saoe-DRBI*0303

Saoe-DRBI*0304

Saoe-DRBI*0305

Calno-DRBI*0301

Camo-DRBI*0302

Camo-DRBl*0303

Camo-DRBI*0304

Camo-DRBI*0305

Caja-DRBI*0301

Aotr-DRBI*0301

Aotr-DRBI*0302

Saoe-DRB*WI201

Saoe-DRB*WI202

Caja-DRB*WI201

Sasc-DRB*WI2 01

Sasc-DRB*Wi202

Sasc-DRB*WI203

Sasc-DRB*WI204

Sasc-DRB*WI901

Sasc-DRB*WI902

Sasc-DRB*WI903

Sasc-DRB*WI401

Sasc-DRB*WI402

Sasc-DRB*WI403

Sasc-DRB*WI404

Camo-DRB*WI401

Camo-DRB*WI402

Ceap-DRB*WI501

Ceap-DRB*WI502

Aotr-DRB*WI801

Aotr-DRB*WI301

Aotr-DRB*WI302

Ceap-DRB*WI301

Ceap-DRB*WI302

Ceap-DRB*WI303

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---

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A .

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Fig

. la

-e.

Ali

gnm

ent

of n

ucle

otid

e se

quen

ces

of d

iffe

rent

DR

B a

llel

es f

ound

in

Sagu

inus

oed

ipus

(Sao

e-D

RB

), C

alli

thri

x jac

chus

(C

aja-

DR

B),

Sai

mir

i sci

ureu

s (S

asc-

DR

B),

Aot

us t

rivi

rgat

us

(Aot

r-D

RB

), C

alli

cebu

s mol

och

(Cam

o-D

RB

), a

nd C

ebus

ape

lla

(Cea

p-D

RB

). A

das

h in

dica

tes

iden

tity

wit

h th

e pr

imat

e co

nsen

sus

sequ

ence

, an

ast

eris

k in

dica

tes

a de

leti

on.

The

num

beri

ng o

f co

dons

is a

ccor

ding

to

the

amin

o ac

id p

osit

ion

in t

he 1

31 d

omai

n.

to

to

o

,<

%

g

35

45

55

Consensus

GAG

GAG

TAC

GTG

CGC

TTC

GAC

AGC

GAC

GTG

~

GAG

TAC

CGG

GCG

GTG

ACG

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CTG

GGG

CGG

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GCC

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C-C

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C .

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C-C

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TC---

TGC

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Saoe-DRB3*0503

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C-C

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..

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C .

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T

---

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..

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CaIno-DRB3*0501

..

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T-

C-C

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C-C

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Aotr-DRB3*0601

......

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Camo-DRB3*0601

......

CTT

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T .

..

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C .

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G

Camo-DRB3*0701

......

C-G

..

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C .

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Saoe-DRB5*0701

A .

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Saoe-DRBll*OlOl

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TG

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AG

Saoe-DRBII*OI02

..

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TG

..

..

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..

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AG

---

GAG

TAC

TGG

AAC

AGC

CAG

.... C .

..

..

..

CA-

-G-

....

C .

..

..

..

CA-

-G-

.... C .

..

..

..

CA-

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.... C .

..

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C--

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..

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C--

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.... C .

..

..

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G

-T

--

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..

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G-

Saoe-DRBII*0103

..

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T .

..

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AG

Saoe-DRBII*OI04

..

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TG

..

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A

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..

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Saoe-DRBII*0105

..

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TT

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G

CGG

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G-

Caja-DRB11*0101

..

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TG

..

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Caja-DRB*WI601

..

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Caja-DRB*WI602

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G

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Caja-DRB*WI603

......

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T .

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T .

..

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..

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Camo-DRB*WI701

......

CTT

..

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G

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Saoe-DRBI*0301

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G .

..

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GG

AG

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---

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..

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G-

Saoe-DRBI*0302

..

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GG

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..

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Saoe-DRBI*0303

..

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G .

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Saoe-DRBI*0304

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G .

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G-

Saoe-DRBI*0305

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AG

---

A .

..

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.

Camo-DRBI*0301

......

CTT

..

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T .

..

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G

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G-T

---

Camo-DRBI*0302

..

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G

..

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G-T

---

Camo-DRBI*0303

......

C-T

..

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T .

..

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Camo-DRBI*0304

..

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..

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G

..

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..

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G-T

---

Camo-DRBI*0305

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Saoe-DRB*WI20Z

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Ceap-DRB*WI301

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T-

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CT .

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.

Fig.

lb.

o 2,

?

0Q ~o

65

75

85

Consensus

AAG

GAC

ATC

CTG

GAG

CAG

AAG

CGG

GCC

GAG

GTG

GAC

ACC

TAC

TGC

AGA

CAC

AAC

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~

GTT

GTT

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Aotr-DRB3*0601

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Camo-DRB3*0701

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A ....

Saoe-DRB5*0701

......

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Saoe-DRBII*OIOI

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CA

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---

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Saoe-DRBII*0102

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Saoe-DRBII*0103

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CA

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C ....

i

Ca

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16

02

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---

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Saoe-DRBI*0301

---

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TA-

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GC .

..

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C .

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GT

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---

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Saoe-DRBI*0302

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A .....

G-T

C-C .

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GT

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---

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Saoe-DRBI*0303

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GC .

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GT

***

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T ....

A ....

Saoe-DRBI*0304

.....

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GT

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---

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Saoe-DRBI*0305

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---

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A ....

C~mo-DRBI*0301

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Sasc-DRB*WI902

.- ....

L-G-

--H--R

.....

L-A-LI

.....

F .........

F .........

I--NH--R

.-Y .......................

IH .

..

. F .

..

..

..

..

..

..

..

..

..

S--KL---

.- ....

L-G-

--H--R

.....

L-A-LI

.....

F .........

F .........

I--NL--R

.- ..........

HL---Q--

-L .

..

..

..

..

..

..

..

..

..

.

F .........

S--H--R-

.- ....

R-G

....

L---Q--

-L---C

......

I .........

F .........

T--HY---

.- ....

F-D-

--HL---QQM

---A-LI

.....

FA ........

F .........

T--QL---

.C---HN-F-

--HL---Q--

-Y-H-FI

.....

IA .........................

R

.----HL-F

..

..

..

..

..

..

Y-V-EI

.....

I .........

F .........

T---F---

-EV--YIG-A

---F

.....

K

--L---R

......

V .....

E

-EV--YM--A

---F

.....

K

E-FM-R

.......

V .....

Q

--F--RM--Q

---V

.....

Q

--V--LA

.....

RL .....

E

--Y-

--R .....

E* .....

Q

E-F

......

A

---V ..... Q

Sasc-DRB*WI903

Sasc-DRB*WI401

Sasc-DRB*WI402

Sasc-DRB*WI403

Sasc-DRB*WI404

Camo-DRB*WI401

Camo-DRB*WI402

Ceap-DRB*WI501

Ceap-DRB*WIS02

Aotr-DRB*WIS01

Aotr-DRB*WI301

Aotr-DRB*WI302

Ceap-DRB*WI301

.C---HN-F-

--HL---Q--

-Y-H-FI

.....

IA .........................

R

--Y---R--A

.........

Q

---K-V-H-

--H-K-M--*

*--F-HI

..

..

..

..

..

..

..

.

F ............

S ....

E .....

M .....

K*--F--E

---K-V-H-

--H-K-M--*

*--F-HI

..

..

..

..

..

..

..

.

F ..........

E-S--R-

E .....

M .....

K*--F---

---KHV-H-

--H-K

........

F-DI

..

..

. F .........

F ..........

E-S--R-

--L---M

.....

RF .....

E

---K-V-H-

--H-K-M--*

*--F-HI

..

..

..

..

..

..

..

.

F ..........

E-S

....

E .....

M .....

K*--F---

.....

L-F-

--H

........

L-V-F

......

F .........

F-P

..........

S---R

E ....

R .......

M .....

E

.....

L-G

..

..

..

..

..

..

L-V-NIH

..

..

F .........

F-P

.......

V--S

.....

EL--R

.......

V .....

E

---R-F-P

....

L .......

SSE-HI

.....

N .........

F .

..

..

..

..

..

..

..

..

..

YM--R-

-A

.........

E

---R-F-P

....

L .......

L-E-HI

.....

F .........

F .

..

..

..

..

..

..

..

..

..

YM--R--A

.........

E

IS-R-L-P--

..........

Y ........

Q--E

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

K---G-

Q ....

L--GQ

--N

........

F ........

.....

F-P

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.

E---F--L

--FM-ET--A

.....

K .....

F ........

---F-TT--

-Y .

..

..

..

..

..

..

..

..

..

..

..

..

..

..

.

F .

..

..

..

..

..

..

..

..

..

F--DR--A

......

Y .

..

. G ........

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

L .

..

..

..

..

..

..

..

..

..

..

..

..

..

.

F .........

S ............

DA-GQ

--N

........

G ........

IF-R-L

....

GF-M-L-P--

IF-T-L-P--

GF-M-L-P--

IL-R-L-P--

IL-R-L-P--

IL-R-L-P--

IF-R-L---~

IS-R-L-P--

IF-R-L

....

-L-R-L-PQ-

-L-R-L-PQ-

IS-R-L-P--

-L-R-L-PQ-

-S-R-L-P--

FS-RI

.....

IS-R-L-P--

Ceap-DRB*WI302

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

L .

..

..

..

..

..

..

..

..

..

..

..

..

..

.

F .........

S ............

DA-GQ

--N

........

G ........

Ceap-DRB*WI303

.....

F-P

....

L .......

Y-Q-LI

......

A .

..

..

..

..

..

..

..

..

.

V---L

.....

Y---R--A

--N

........

G ........

Fig

. 2.

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216

Table 1. Platyrrhini DRB genes found in the animals tested.

K. Trtkovfi et al.: Platyrrhine Mhc-DRB genes

Species Individual Alleles sequenced

Saguinus oedipus SC2 Saoe-DRB 1 "0301 * 3"0503" 5"0701 *+ 08 1 *0302 * 1 *0305 3 "0501 * 28 1"0302" 1"0303" 3"0501" 32 1"0303" 3*0502* 11"0102 36 1"0302" 3"0501" 11"0102 37 1"0304" 3*0502* *W1202

Callithrixjacchus XR Caja-DRB 1 *030] 1 1 " 0 1 0 1 *W1201 XS 1 "0301 * W 1 6 0 2 *W1603

Aotus trivirgatus O01 Aotr-DRB 1 "0301 1 *0302 3"0601

Callicebus moloch 002 Camo-DRB 1 "0301 1"0302 3"0701 005 1 *0303 1 *0304 l *0305

Cebus apella O01 Ceap-DRB * W 1 3 0 1 * W 1 5 0 1 *W1502 002 *W1302 *W1303

Saimiri sciureus 291 Sasc-DRB * W 1 2 0 1 * W 1 2 0 2 *W1401 306 *W1203 * W 1 2 0 4 *W1402 307 *W1202 * W 1 4 0 4 *W1903

11"0105" 11"0103" *W1201 + 11 "0104 11 "0102 11 *0101 11"0101 11 "0101

*W1601

*W1301 * W 1 3 0 2 *W1801

*W1401 * W 1 4 0 2 *W1701 3"0501 3"0502 3"0601

*W1901 *W1403 *W1902

11"0101

* Pseudogene. + From minilibrary. * From Grahovac and co-workers (1991, 1992).

R e s u l t s

We amplified the polymorphic D R B exon 2 from a panel of six New World monkey species. We obtained a total of 333 sequences which identified 55 different genes. The new exon 2 D R B nucleotide sequences are given in Figure 1 and are stored at the GenBank under the accession numbers L12472-L12528 . The amino acid translation of these sequences appears in Figure 2. The phylogenetic relationship among the Platyrrhini sequences are depicted in Figure 3 and the relationships of the Platyrrhini sequences to selected Catarrhini sequences are shown in Figure 4.

Saguinus oedipus. In the cotton-top tamarin, twelve new sequences were obtained (Table 1), in addition to the four sequences reported by Grahovac and co-work- ers (1991, 1992). Eight of the 16 sequences were derived from pseudogenes characterized by several deletions, insertions, and premature stop codons. Two closely related sequences, Saoe-DRB3*0501 and *0502, which differ from each other by a synonymous transversion at position 12, have an insertion of one nucleotide at codon 47, a deletion of one nucleotide at codon 86, and two stop codons, one at position 57 in the frameshifted region and another at position 94 outside the frameshifted region. [In accordance with the pro- posed nomenclature for the M h c genes of the different species (Klein et al. 1990), we designate the Platyrrhini genes by a four-letter abbreviation of the scientific genus (first two letters) and species (third and fourth letters) names, followed by the locus designation based

on the homology relationship with human genes, and then, after an asterisk, by a serial number of the partic- ular allele]. The two sequences are identical with Saoe- DRB3*0503 (formerly "stretch 1 of Saoe-DRB2") iden- tified by Grahovac and co-workers (1992) except that the latter sequence lacks the one nucleotide insertion at codon position 47. However, because it shares with the two sequences the one-nucleotide deletion at codon 86, the resulting frameshift leads to a stop codon at position 90; without the frameshift, the sequence would have a stop codon at position 94. The scattered pattern of sub- stitutions, seen in comparison with the primate D R B consensus sequence, also distinguishes the three genes from functional genes, in which most nonsynonymous substitutions are concentrated at sites specifying the peptide binding region (PBR).

Two other cotton-top tamarin pseudogenes, Saoe- DRB1 *0303 and *0304, are also closely related to each other. They are characterized by a deletion of two nu- cleotides in codon 31 which shifts the reading frame and leads to a termination codon immediately downstream. A second frameshift is introduced by an insertion of one nucleotide in codon 66. Here again, even the correct reading frame contains a stop codon, namely at position 47. The two sequences share a codon deletion at position 78 with several other New World monkey D R B sequences and they are closely related to another pseudogene, Saoe-DRB1 *0302, and to a gene without detectable defects in the exon 2, Saoe- DRB1 "0301. The Saoe-DRB1 *0302 sequence does not contain the deletion of codon 31 and the stop codon 47 but shares with Saoe-DRB1 *0303 and *0304 the inser-

K. Trtkovfi et al.: Platyrrhine Mhc-DRB genes 217

Camo-DRB3*0501 ~ _ _ _ _ ~ C a m o - D R B 3 * 0 5 0 2

Saoe-DRB3*0501 i ~ ~ L Saoe-DRB3*0502

t--.--- Camo-DRB3*0701 Aotr-DRB*WI801

Camo-DRBI*0301 Camo-DRB3*O601

Aoir-DRB3*06Ol Aoir-DRB* WI301

Ceap-DRB*WI303 ~ C e a p - D R B * W I 3 0 1

I I ~Ceap-DRB*WI302 I ~q--Aotr-DRBI*0301 ~ - - - ~o t r - DRBI*0302

Aotr-DRB*WI302 _ ~ Camo-DRB* W1402

Camo-DRB*W1401

I - Sasc-DRB*WI401 Sasc-DRB*W1402

[ ~ - ~ - ~ Sasc-DRB*WI404 I Sasc-DRB*W1403

- - Saoe-DRB*WI201 [--- Saoe-DRB*WI2O2

r-4 L SasC-DRB*WI201 I I Caja-DRB*W1201

~ 1 ~'S Sasc-DRtR*WI9ol asc-DRB*W1903

Sasc-DRB*WI202 Sasc-DRB*WI2O3

Camo-DRBIl*Ol01 ~[- Saoe-DRBll*OIOI

[ I ~ LSa°e'DRB11*0102 ~ - ~ i ! aOe-DRBII*0103

Saoe-DRBII*OI04 oe-DRBI 1 "0105

. ~ _ c C a j a'DRB*WI601 Caja-DRB*WI602 aja-DRB* W I 603

Saoe-DRBI*0301 Saoe-DRBl*0302 Saoe-DRBI*0303 Saoe-DRB I*0304

Coja-DRBI*0301 - - Saoe-DRBI *0305

~ e Ceap-DRB*WI501 ap-DRB*WI502

Camo-DRBI *0303 Camo-DRBI*0304 amo-DRBl*0302

'amo-DRB l*0305 -DRB*WI701

-Saoe-DRB5*0701

i ~ I ~ I

0.0 0.04 0,08 0,12 0,16 G e n e t i c d i s t a n c e

Fig. 3. Nearest-neighbor tree of Platyrrhini DRB alleles.

tion at codon 66. Unlike the first group of pseudogenes, the substitutions in this group are focused on the PBR- encoding sites.

The Saoe-DRB1 *03 group of sequences shares sev- eral features. Codons 10-12 specify the YST amino acid motif found in the primate DRB1 *03 allelic lin- eage (O'hUigin et al. 1993) and in some artiodactyl DRB genes (Andersson et al. 1991). Codons for F, L, P, and R are present at positions 37, 38, 48, and 53, re- spectively, and codon 78 is deleted. Two clusters of replacements in comparison to the consensus sequence are located at positions 56-77 and 84-92. The first cluster is characteristic for this group of alleles. The second cluster appears to be restricted to the Platyrrhini infraorder and occurs in several other sequences. More distantly related to this group is the sequence Saoe- DRB1 *0305, in which the YST motif has been replaced by YRMC specified by codons 10-13. Exon 2 of the

Saoe-DRBI 1 *0101 { . ~ Caja-DRB*W160I

Saoe-DRB*WI201 S'asc-DRB*W1403

- - Ceap-DRB*WI501 Camo-DRB*WI701

~ B HLA'DRB3*0201 Patr-DRB3*020I HLA -DRB3*OIO1

Aotr-DRB3*0601 Patr-DRB1 *0302

I Mane.DRB1 "0301 Paha-DRBI*0301

Camo-DRB I*0301 -DRBI*1601 atr-DRBl*0201 HLA -DRB1 "0701

1"0101 RBI*IO01

I I ~ HLA~DRBI*0401 ~ Saoe-DRB5*0701

t------------------~_ Patr.DP~IPY-oDRIB5*060I

Aotr-DRB*W1301

0.0 0.02 0.04 0.06 0.08 0.1 i i t I i t

G e n e t i c d i s t a n c e

Fig. 4. Nearest-neighbor tree of Platyrrhini DRB alleles and selected Catarrhini DRB alleles. The sequences were taken from Marsh and Bodmer (1992), Mayer and co-workers (1992), Kupfermann and co-workers (1992), Sch6nbach and co-workers (1993), Zhu and co- workers (1991), Riess and co-workers (1990), and this communica- tion.

Saoe-DRB5*0701 gene, which was isolated from a cot- ton-top tamarin genomic minilibrary, is characterized by codons specifying amino acids K9, D11, Y13, H28, I31, D37, and several others across the exon, which it shares with most Catarrhini DRB5 genes. A deletion of two nucleotides at codon 72 causes a shift in the reading frame that renders it a pseudogene.

Five tamarin sequences, Saoe-DRBl l *0101," "0102, "0103, "0104, and "0105 share codons specifying the amino acid motif EQAKA at positions 9-13 and also the New World monkey-restricted motif at positions 84-92.

Finally, the tamarin sequences Saoe-DRB*W1201 and *W1202 are distinguished by the codon 10-13 specifying motifs QFKY and QLKG, respectively; they also display the New World monkey-restricted motif at the 3' end of exon 2.

Callithrixjacchus. From the second species of the Cal- litrichidae, the marmoset (C. jacchus), we obtained six sequences from two individuals. All possess variants of the motif characteristic for the Platyrrhini at codons 84-92. One of the sequences, Caja-DRBll*OIO1, is identical to Saoe-DRBll*OIO1. Another sequence, Caja-DRB1 "0301, which is present in both individuals studied, specifies the YST motif at positions 10-12 just as Saoe-DRB1 "0301 and *0302 do, but is distinct from these two sequences at most other informative posi- tions, in particular by the absence of substitutions in the segment at positions 56-77 and of the deletion at posi- tion 78. Another four marmoset sequences have the tyrosine-specifying codon 7. Three of them, Caja- DRB*W1601, *W1602, and *W1603 are closely related

218 K. Trtkov~ et al.: Platyrrhine Mhc-DRB genes

to each other; they share the codons for H17, Y26, E28, A74, R77, E84, L88, and P90, as well as the deletion of codon 78. The fourth sequence, Caja-DRB*W1201, displays a unique pattern of substitutions that distin- guishes it from other known genes; its closest relatives in our sequence collection appear to be Sasc- DRB*W1201 and Saoe-DRB*W1202.

Saimiri sciureus. In the three individuals of S. sciureus we identified 11 genes. The products of the genes Sasc-DRB*W1401, *W1402, *W1403, and *W1404 are related to one another in that they have the amino acids K9, V11, H13, H17, K19, F28, I31, F47, $60, and M71 in common. In addition, the sequences Sasc- DRB*W1401, *W1402, and *W1404 contain a two- codon deletion at positions 24 and 25, which is also found in the DRB7 (Andersson et al. 1987) and DPB (Marsh and Bodmer 1992) genes, and a deletion of codon 78. The Sasc-DRB*W1403 sequence is similar to these three sequences, but it lacks the deletions. The remaining squirrel monkey genes belong to two groups. The first group consists of Sasc-DRB*W1201 through *W1204. The product of one gene, Sasc-DRB*W1201, is similar to that of Saoe-DRB*W1202, with which it shares L l l , G13, H17, R20, L26, A28, L30, I31, F37, F47, and several other amino acid residues specified by codons at the 3' end of exon 2. Several other positions relate the products of these *WI2 genes to one another: G13 and M71 are shared by the products of Sasc- DRB*W1201 and *W1203; L18 and Q22 are common to the products o f Sasc-DRB*W1202, *W1203, and *W1204; and A28, L30, I31, F37, L61, V67 are found in the products of the Sasc-DRB*W1201 and *W1204 genes. The genes Sasc-DRB*W1901 through *W1903 form the second group characterized by codons speci- fying H10, F13, Y26, I31, I37, and Q84.

Aotus trivirgatus. The six sequences we obtained from A. trivirgatus are characterized by the absence of the New World monkey-restricted sequence motif in the 3' end of exon 2. In this region, the sequences resemble those found in Catarrhini. They can be divided into four groups. The first group contains one sequence, Aotr- DRB3*0601, possessing L10-, VII-specifying codons characteristic of alleles at the DRB3 locus in the Catar- rhini. The two members of the second group, Aotr- DRBl*0301 and *0302, are characterized by codons specifying the motif FQTT at positions 9-12, Y26, Y67, V68, G73, N77, and G86. The third group, repre- sented by Aotr-DRB*W1301 and "1302 is, for the most part, similar to the primate consensus sequence. The two sequences share the codons for F67 and A74. The fourth group, represented by Aotr-DRB*W1801, en- codes the EQAKS primate DRB consensus sequence at codons 9-13.

Cebus apella. The five sequences obtained from two C. apella monkeys fall into two groups. In the first group, sequences Ceap-DRB*W1501 and *W1502 are closely related to each other by the sharing of substitutions at several positions scattered along exon 2; they are distin- guished from the second group by the presence of the Platyrrhini-restricted motif at the 3' end of exon 2. In the second group, the genes Ceap-DRB*W1301 and *W1302 specify identical 131-domain amino acid sequences but differ by a single synonymous nucleotide substitution in codon 44. The third gene, Ceap- DRB*W1303, is more distantly related to these two. All three genes, however, lack the Platyrrhini-restricted motif at the 3' end of exon 2.

Callicebus moloch. In the two individuals of C. moloch, 13 different DRB exon 2 sequences were identified. Five sequences appear to be members of the DRB1 *03 group of alleles. Of these, one allele, Camo- DRB1 "0301, specifies the YST motif at codons 10-12 and is similar to the Catarrhine alleles over the entire length of the exon. Two other alleles, Camo- DRBl*0302 and *0304, also code for the YST motif but carry the Platyrrhini-restricted substitution pattern at the 3' end. The sequences of Camo-DRB1 *0303 and *0305 are distinct from the former sequences in speci- fying the motif HVKH at amino acid positions 10-13. In the dendrogram (Fig. 3), however, they are members of the same clade as the other Camo-DRBI*03 sequences. Four of the sequences we obtained from C. moloch are similar to DRB3. They all lack the Platyr- rhini-restricted motif at posi.tions 84-92. The products of two of them, Camo-DRB3*0501 and *0502, share L10, L l l , R16, Q23, E28, H30, I31, F37, L38, L39, and the cluster of substitutions at positions 56-77 with the products of the Saoe-DRB3*05 alleles. Unlike the lat- ter, however, they seem to be functional. The product of Camo-DRB3*0701 shares L10, L l l , and E28 with the products of the DRB3*05 alleles but differs from them in several positions in the second half of the 131 domain. The last DRB3 allele, Camo-DRB3*0601, specifies the motif HVKA at codons 10-13, which is similar to the one specified by Camo-DRBl*0303, *0305, and -DRBll alleles. Its product shares with the product of Camo-DRB3*0701 the variable amino acids E28, H32, P49, and G86. The product of another sequence, Camo- DRBll*OIO1, which is characterized by the HHKA motif at positions 10-13, shares amino acids A13, H17, L26, E28, and the substitutions at 84-92 with the products of the Saoe-DRBll alleles. Two alleles are distantly related to the DRB*W14 alleles of the squirrel monkey and group with them in the dendrogram (Fig. 3). We therefore name them Camo-DRB*W1401 and *W1402. The Camo-DRB*W1402 nucleotide sequence terminates at a Bam HI site, which was cut during subcloning. Thus, the last five codons of exon 2 are not available for this sequence. The last sequence

K. Trtkovfi et al.: Platyrrhine Mhc-DRB genes 219

we identified in C. moloch does not cluster closely with any other sequence in a dendrogram. It is, therefore, assigned to a new altelic lineage. Camo-DRB*W1701.

Discussion

The available Platyrrhini DRB sequences can be divid- ed into two large groups - those closely allied with Catarrhini DRB sequences and those not. In the former group are sequences at three Catarrhini loci, DRB1 *03, DRB3, and DRBS. The DRBl*03 genes represent one of several allelic lineages characterizing the Catarrhini DRB1 locus. The different lineages are as distantly related to one another as are the different DRB loci (Klein et al. 1991, 1992) and the possibility that some of them are pseudoalMes rather than true alleles has not been excluded. The DRB1 *03 lineage has been found in humans (Marsh and Bodmer 1992), chimpanzee (Br~indle et al. 1992; Mayer et al. 1992; Kenter et al. 1992a), pygmy chimpanzee (Gyllensten et al. 1991), gorilla (Kasahara et al. 1992; Kupfermann et al. 1992; Kenter et al. 1992b), orangutan (Schrnbach et al. 1993), rhesus macaque (Slierendregt et al. 1992), pig- tail macaque (Zhu et al. 1991), hamadryas baboon (Riess et al. 1990), and drill (Gyllensten et al. 1991). Our demonstration of its presence in the cotton-top tamarin, common marmoset, and dusky titi indicates that the lineage emerged before the Platyrrhini-Catar- rhini split. At the amino acid sequence level the lineage is characterized by the YST motif at positions 10-12, as well as by the sharing, by some of the allomorphs at least, of several other residues scattered along the ~1 domain. There are, however, two types of Platyrrhini DRB1 *03 sequences. One type, represented by Camo- DRBI*0301 is closely related to the Catarrhini DRB1 *03 sequences along the entire length of exon 2 (Fig. 4). The other type, represented by the Saoe- DRBI*03 alleles, is related to Catarrhini DRBI*03 genes in most of exon 2, except for its 3' end; there the alleles possess a Platyrrhini-restricted motif (see be- low). For this reason, they do not group with the first type in the dendrogram in Figure 3. The second type might be derived from an ancestral sequence that arose by recombination between a typical DRB1 *03 gene and a gene with the Platyrrhini restricted motif.

We found sequences representing DRB3 genes in six cotton-top tamarins, two dusky titis, and one northern night monkey. The tamarin DRB3 sequences were iden- tified in both cloned (B. Grahovac, unpublished data) and amplified genomic DNA (this communication), so the possibility of their being PCR contaminants is ex- cluded. They are very similar or identical to each other and their association with the DRB3 locus is indicated by their clustering with the DRB3 clade in the dendro- gram and by their sharing of codons specifying charac- teristic amino acid motifs, in particular L10, L11, and

E28, with DRB3. In the Catarrhini, the presence of the DRB3 locus has been demonstrated for humans (Marsh and Bodmer 1992), chimpanzee (Fan et al. 1989; Gyl- lensten et al. 1991; Br~indle et al. 1992; Mayer et al. 1992; Kenter et al. 1992a), gorilla (Kasahara et al. 1990, 1992; Gyllensten et al. 1991; Kupfermann et al. 1992; Kenter et al. 1992b), and the rhesus macaque (Slierendregt et al. 1992), so that the locus must have emerged before the divergence of the two catarrhine superfamilies Cercopithecoidea and Hominoidea more than 23 my ago. The data presented in this communica- tion now date the emergence of DRB3 as prior to the divergence of Catarrhini and Platyrrhini more than 37 my ago. The DRB3 gene is believed to have arisen by duplication from an ancestral DRB1 *03 gene (Rol- lini et al. 1985). The presence of the DRBl*03 and DRB3 genes in the Platyrrhini means that the duplica- tion occurred more than 37 my ago. The close identity of the Saoe-DRB3 genes from different individuals is in keeping with the low divergence of other tamarin genes discussed below. The Aotr-DRB3 gene is quite unre- lated to the Saoe-DRB3 genes, but shows sufficient similarity to homologous gorilla and chimpanzee sequences to justify its locus assignment.

The DRB1 *03 and DRB3 genes show a tendency to occur together in individual monkeys: an individual either has both genes or neither of them, the only excep- tion being the two common marmosets in which we found DRBl*03 but not DRB3. The two genes may therefore reside on the same chromosome forming a DRBI*O3...DRB3 haplotype, otherwise known to occur in humans (Klein et al. 1991), in chimpanzees (Br~indle et al. 1992), and gorillas (Kasahara et al. 1992). Hence, it is possible that after the duplication, the DRBl*03 and DRB3 genes remained together in some haplotypes in both the Catarrhini and Platyrrhinj. One dusky titi appears to possess three DRBl*03 and three DRB3 genes, so that at least two genes of each type would have to be borne by single chromosomes. The DRB1 *03. . . DRB3 pair may have therefore dupli- cated in some of the chromosome. As was mentioned earlier, some of the Platyrrhini DRB1 *03 genes have the Platyrrhini-restricted motif and others do not. On the other hand, all the Platyrrhini DRB3 genes thus far tested lack this motif. This distribution suggests that one of the DRB1 *03 genes acquired the motif after the duplication that produced the DRB3 gene.

The DRB5 gene has been found on one of the clones isolated from a minilibrary which was prepared from a DRB-hybridizing fragment of cotton-top tamarin DNA (Grahovac et al. 1992). The most likely reason it has not been amplified by PCR in the present study is that the introns flanking its exon 2 diverged slightly from those of the human DRB5 gene (B. Grahovac, unpublished data), on which the sequence of the primers used was based. In addition to humans (Kawai et al. 1989), the DRB5 locus has previously been described in the chim-

220 K. Trtkov~ et al.: Platyrrhine Mhc-DRB genes

panzee (Fan et al. 1989; Gyllensten et al. 1991; Br~indle et al. 1992; Mayer et al. 1992; Kenter et al. 1992a), gorilla (Kasahara et al. 1990, 1992; Kupfermann et al. 1992; Kenter et al. 1992b), orangutan (Sch6nbach et al. 1993), and rhesus macaque (Slierendregt et al. 1992). Its presence in the tamarin supports our earlier sugges- tion that DRB5 is an old gene as far as DRB genes are concerned (Klein et al. 1992). Like DRBI*03 and DRB3, it must have emerged before the Catarrhini- Platyrrhini split more than 37 my ago.

Two points need emphasizing regarding the sharing of Catarrhini and Platyrrhini genes: first, other Catar- rhini genes might be present in the Platyrrhini in addi- tion to those detected in our study, some of which would not be amplified because they lack exon 2, others may have diverged too much to be detected by the primers used, and still others may not have been amplified only by chance; second, even genes that ap- pear functional may in fact be pseudogenes that have defects outside the sequenced region. This point, of course, applies also to the genes found only in the Platyrrhini.

In contrast to genes at the DRBl*03, DRB3, and DRB5 loci, Platyrrhini DRB genes in the second group have no close allies among the Catarrhini DRB genes. They cannot be assigned easily to any of the known Catarrhini loci: on dendrograms, they form separate clades and they lack the sequence motifs characterizing the various lineages of the Catarrhini genes. We inter- pret these observations as an indication that the genes from which the sequences are derived emerged in the New World monkeys in all probability after the Catar- rhini-Platyrrhini split. We designate these genes by the next available locus number in those instances in which contig mapping (B. Grahovac, unpublished data) and other evidence justify such a step, or by the DRB sym- bol without a locus number, followed by a W ("Work- shop") lineage number. The W designations indicate that the genes represent a new lineage either at a new locus or at one of the already known loci, and that we are currently not in a position to decide between these two possibilities. Each distinct new lineage receives a different W number (the first two digits) and genes within each lineage are numbered serially (last two digits).

The Platyrrhini-restricted sequences can be sub- divided further into two groups. One group is character- ized by codons specifying the EIL(S)R-L-P(L) motif at positions 84, 85, 86, 88, 90, and 92 as well as H at position 17 (Fig. 2), and by the occurrence of the sequences coding for these motifs in both the Callitri- chidae and Cebidae families of the Platyrrhini (Fig. 3). The second group lacks this motif and is restricted to the Cebidae family. In the dendrogram (Fig. 3), the two groups form separate clades (the first group several, the second group one). The Saoe-DRBll*0105 (formerly DRB1 *01) and DRB1 "0102 (formerly DRB1 *02) are

alleles at a new locus because they occupy correspond- ing positions on cosmid clones derived from a single individual which also carries genes at the DRB1, DRB3, and DRB5 loci (B. Grahovac, unpublished data). By extension, we regard the entire clade of DRBll-related sequences as alleles at the DRBll locus. The same individual also possesses another gene, which we orig- inally designated Saoe-DRB2*01 because it seemed to be an allele of Saoe-DRB2*02 (now renamed to Saoe- DRB1 "0301). Further mapping, however, has rendered the postulated allelism less likely (although it has not excluded it), and we therefore redesignate Saoe- DRB2*01 as Saoe-DRB*W1201, a representative of a new lineage, perhaps at a new locus. We consider con- servatively the entire clade represented by Saoe- DRB*W1201 as a single lineage, although we realize that the addition of new sequences may split it into several separate lineages. The sequences designated DRB*W13 through DRB*W19 are, on the other hand, so distant from *WI2 and from one another that their assignment to separate lineages seems justified.

A feature complicating the assignment of sequences to lineages is that some of the genes appear to be derived by intragenic recombination. Thus, Camo- DRB1 *0303 and Camo-DRB1 *0305 share with Camo- DRB1 *0302, for example, the 3' half of exon 2 but are very different from this gene in their 5' half which includes codons specifying the YST motif characteris- tic of the DRBI*03 protein. Similarly, Camo- DRB*W1401 and Camo-DRB*W1402 resemble Saoe- DRB*W1202 and Sasc-DRB*W1201 in their 5' half of exon 2 but resemble other DRB*14 sequences in their 3' half. These and other examples are responsible for some of the ambiguities and inconsistencies of the den- drogram in Figure 3.

A notable feature of the tamarin sequences is that alleles at the same locus are very closely related to one another. This feature is consistent with the finding of limited restriction fragment length polymorphism as documented in an earlier study (Grahovac et al. 1991). The tamarins and marmosets are obviously not mono- morphic at their DRB loci, but their polymorphism is most likely of relatively recent origin. Although limited variability has also been reported for the tamarin class I loci (Watkins et al. 1990, 1991), its nature is somewhat different from that of the class II DRB loci. In contrast to the Catarrhini, which have at least two highly poly- morphic and quite different class I loci, the tamarin possesses class I loci which are very similar to one another in terms of nucleotide sequence. These special features of Mhc variability in tamarins may be the con- sequence of some special event in the phylogenetic history of this species.

Another interesting feature of the tamarin DRB sequences is the frequent occurrence of defects render- ing the sequences pseudogenes and the indication that functional inactivation seems to be prevalent at old loci.

K. Trtkovfi et al.: Platyrrhine Mhc-DRB genes 221

There are three groups of sequences in our sample in which either all or most of the alleles are pseudogenes - the Saoe -DRB! *03, the Saoe-DRB3 alleles, and the Saoe-DRB5 genes. Most of the sequences have mult iple defects, suggesting that they may have been inactive for a long while during which they accumulated the muta- tions gradually. All three are old genes shared by the Catarrhini and Platyrrhini. The overall picture emerg- ing from these data is one of differential usage of D R B genes in New World and Old World monkeys. Several of the loci found in modern Platyrrhini and Catarrhini apparently already existed in the ancestors of these two infraorders. Some of these (DRBl*03 , DRB3, DRBS) have remained funct ional in the Catarrhini, but were mostly inactivated in the Platyrrhini, while the latter evolved their own set of genes D R B l l , D R B * W 1 2 through D R B * W 1 9 ) on which they placed the burden of protection from parasites.

Acknowledgments. We thank Ms. Lynne Yakes for editorial assis- tance, Ms. Anica Milosev for the preparation of the computer graphics, and Professor Dr. Christian Welker, Universitat Kassel, for tissue samples. The work was supported in part by grant AI 23667 from the National Institutes of Health, Bethesda, MD.

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