Comparative anatomy of the primate major histocompatibility complex DR subregion: Evidence for combinations of DRB genes conserved across species

GENOMICS 14, 340-349 (1992)

Comparative Anatomy of the Primate Major Histocompatibility Complex DR Subregion: Evidence for Combinations of DRB Genes Conserved across Species MASANORI KASAHARA,*'1 DAGMAR KLEIN,* VLADIMIRVINCEK,* DONNA E. SARAPATA,*AND JAN KLEIN*'1*Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101; and tAbteilung Immungenetik, Max.Planck-lnstitut for Biologie, W-7400 T(Ibingen, Germany Received January 31, 1992; revised July 6, 1992

T h e c l a s s II r e g i o n o f t h e h u m a n m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x (HLA) is m a d e u p o f t h r e e m a j o r s u b r e g i o n s d e s i g n a t e d D R , DQ, a n d DP. W i t h t h e a i m o f gaining an insight into the evolution and stability of D R h a p l o t y p e s , a t o t a l o f 6 3 c o s m i d c l o n e s w e r e isol a t e d f r o m t h e D R s u b r e g i o n (Gogo-DR) o f a w e s t e r n lowland gorilla. All but one of these cosmid clones were f o u n d to f a l l i n t o t w o c l u s t e r s . T h e l a r g e r c l u s t e r , A , was defined by 41 overlapping cosmid clones and contained a D R B gene segment made up of exons 4 through 6 a n d f o u r D R B g e n e s , d e s i g n a t e d Gogo-DRB6, GogoDRB5*O1, Gogo-DRB8, a n d Gogo-DRB3*01. T h e total length of this cluster was ~ 180 kb. The second cluster, B, e n c o m p a s s e d a c o n t i g u o u s D N A s t r e t c h o f ~ 1 4 5 kb and was composed of 21 overlapping cosmid clones. Cluster B contained three DRB genes, designated Gogo-DRBI*08, Gogo-DRB2, a n d Gogo-DRB3*02. One cosmid clone (WP1-9) containing a DRB pseudog e n e c o u l d n o t b e l i n k e d to e i t h e r c l u s t e r A or B. N e i ther the organization of cluster A nor that of cluster B w a s i d e n t i c a l to t h a t o f k n o w n H L A - D R h a p l o t y p e s . H o w e v e r , t w o g o r i l l a D R B g e n e s , Gogo-DRB6 a n d Gogo-DRB5*01, t h e h u m a n c o u n t e r p a r t s o f w h i c h a r e l i n k e d i n t h e H L A - D R 2 h a p l o t y p e , w e r e f o u n d to b e l o c a t e d n e x t to e a c h o t h e r i n c l u s t e r A. T h e a r r a n g e m e n t o f t h e Gogo-DRB g e n e s i n c l u s t e r B, w h i c h is p r e s u m e d to b e t h e g o r i l l a D R 8 h a p l o t y p e , w a s s i m i l a r to t h a t o f H L A - D R 3 / D R 5 / D R 6 h a p l o t y p e s a n d to t h a t o f the presumed ancestral HLA-DR8 haplotype. These resuits demonstrate that certain combinations of DRB genes have been maintained since the time before the divergence of human and gorilla lineages more than 5 m i l l i o n y e a r s a g o . © 1992 AcademicPress. Inc.

INTRODUCTION The class II region of the human major histocompatibility complex (MHC), known as the H L A - D region, is To whom reprint requests should be addressed at Department of Biochemistry, Hokkaido University School of Medicine, Sapporo 060, Japan. 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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made up of three major subregions designated DR, DQ, and D P (Korman et al., 1985; Trowsdale et al., 1985; Kappes and Strominger, 1988). Each subregion contains at least one pair of A and B genes, whose products associate noncovalently to form a/~ heterodimers expressed on the surface of B cells, macrophages, and activated T cells. These heterodimeric molecules present peptides to helper T cells and thereby play a central role in the recognition events that lead to T cell activation (Klein, 1986; Brodsky and Guagliardi, 1991). Among the three class II subregions, the H L A - D R subregion is unique in that its structural organization differs between individuals. Although all known HLADR haplotypes have one monomorphic DRA gene, the number of DRB genes varies from haplotype to haplotype (BShme et al., 1985). Accumulated evidence indicates the existence of three major, structurally distinct H L A - D R haplotypes (Fig. 1): HLA-DR3/DR5/DR6, HLA-DR4/DR7/DR9, and HLA-DR2. H L A - D R 3 / D R 5 / DR6 haplotypes contain three DRB genes, DRB1, DRB2, and DRB3, which are arranged in this order from centromere to telomere (Rollini et al., 1985). The DRB1 gene encodes D R B molecules carrying the serologically defined DR3, DR5, and DR6 determinants. DRB2 is a pseudogene that lacks the first domain sequence (Rollini et al., 1987). The supertypic DR52 determinant, characteristic of this group of DR haplotypes, is encoded by the DRB3 (and in some cases, also by the DRB1) gene. HLAD R 4 / D R 7 / D R 9 haplotypes consist of four DRB genes (Spies et al., 1985; Andersson et al., 1987): the DRB1 gene encoding the serologically defined D R 4 / D R 7 / D R 9 determinants; the nonpolymorphic DRB4 gene specifying the supertypic DR53 determinant; and two pseudogenes, D R B 7 (Larhammar et al., 1985) and DRB8 (Andersson et al., 1987). The DR2 haplotype characterized by Kawai et al. (1989) contains three DRB genes, DRB1, DRB6, and DRB5, which are arranged in this order from centromere to telomere. DRB6 is a pseudogene that lacks a leader sequence. Both DRB1 and DRB5 are polymorphic and encode functional D R B molecules. Three additional H L A - D R haplotypes, DR1, DRIO, and DR8,

ORGANIZATION OF THE GORILLA DR SUBREGION

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FIG. 1. Schematic comparison of gorilla and human DR haplotypes, not drawn to scale. Functional genes (and gorilla DRB genes with no apparent deleterious mutations) are shown as solid boxes and pseudogenes as open boxes. The DRB gene segment located at the 5'-end of cluster A (Fig. 2) is not shown. Orthologous genes shared between gorilla cluster A and HLA are boxed in a solid line. Those shared between gorilla cluster B and HLA are boxed in a dashed line. The location of the DRB8 gene in HLA-DR4/DR7/DR9 haplotypes has not yet been established (Spies et al., 1985; Andersson et al., 1987). The figure shows its presumed location.

c a n b e r e g a r d e d as v a r i a n t s o f t h e t h r e e m a j o r h a p l o types. DR1 a n d D R I O h a p l o t y p e s are t h o u g h t to be varia n t s o f t h e D R 2 h a p l o t y p e ( B S h m e et al., 1985; E r l i c h et al., 1989). T h e D R 8 h a p l o t y p e is a s s u m e d t o b e a c o n d e n s e d D R 3 / D R 5 / D R 6 h a p l o t y p e t h a t a r o s e as a r e s u l t o f a c h r o m o s o m a l d e l e t i o n ( G o r s k i , 1989). F u r t h e r s u p p o r t for the existence of the three major H L A - D R haplot y p e s is p r o v i d e d b y r e c e n t r e s u l t s o b t a i n e d f r o m m e g a b a s e m a p p i n g o f t h e H L A c l a s s II r e g i o n ( T o k u n a g a et al., 1988; D u n h a m et al., 1989; I n o k o et al., 1989; L a w r a n c e a n d S m i t h , 1990). A n i m p o r t a n t f e a t u r e o f t h e H L A - D R s u b r e g i o n is that multiple DRB genes carried by a given haplotype are in strong linkage disequilibrium. Several investigators have suggested that the apparent lack of intra-DRB subregion recombination between the three major g r o u p s o f D R h a p l o t y p e s m i g h t b e a c c o u n t e d f o r b y diff e r e n c e s i n t h e i r s t r u c t u r a l o r g a n i z a t i o n s ( G r e g e r s e n et al., 1988; D u n h a m et al., 1989). T o e x a m i n e t h e e x t e n t o f stability of DR haplotypes and to gain an insight into the e v o l u t i o n of the D R subregion, we have c h a r a c t e r i z e d the D R subregion of the gorilla M H C , w h i c h we refer to as Gogo-DR i n a c c o r d a n c e w i t h a r e c e n t p r o p o s a l ( K l e i n et al., 1990).

MATERIALS AND METHODS

Source of gorilla DNA. Genomic DNA was extracted from a fibroblast line established from a skin biopsy specimen of a western lowland gorilla (Gorilla gorilla gorilla), named Sylvia, kept at the Baltimore Zoo, Baltimore, Maryland. This cell line was a kind gift of Dr.

341

Kirby D. Smith, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Construction and screening of the gorilla cosmid library. A cosmid library was constructed using the pWE15 vector as described by Evans and Wahl {1987). Briefly, high-molecular-mass DNA (> ~150 kb) was partially digested with Sau3AI and size-fractionated using sucrose gradient centrifugation. DNA fragments ranging in size from 40 to 45 kb were ligated to the BamHI-cut, dephosphorylated pWE15 vector. The vector-insert concatamer was in vitro packaged using Gigapack Gold (Stratagene, La Jolla, CA). After adsorption of the packaged cosmids to Escherichia coli ED8767, the library was plated onto nylon membranes and kept frozen as described by Little (1987). The library contained ~2.0 × 106 independent clones. Initial screening of the cosmid library was performed as described previously (Kasahara et al., 1987), using a chimpanzee DRB cDNA probe, B3-5, described by Fan et al. (1989). The cosmid library was then screened using two walking probes designated as WP1 and WP4 (see Fig. 2). These probes were chosen on the basis of their locations and absence of repetitive sequences. Restriction mapping. Cosmid clones were mapped using the method of Tartof and Hobbs (1988). Briefly, cosmid DNAs were completely digested with NotI and then partially digested with BamHI or EcoRI. After electrophoresis and transfer to a nitrocellulose membrane, the restriction fragments were hybridized with a 32P-end-labeled T3 or T7 oligonucleotide primer in a solution containing 6X SET (1× SET is 150 mM NaC1, 15 mM Tris-HC1, pH 8.3, 1 mM EDTA), 0.1% SDS, 5× Denhardt's solution, and 100 #g/ml of denatured salmon sperm DNA at 40°C for 12 h. After hybridization, the membrane was washed four times for 5 min each in 6X SSC (1× SSC is 150 mM NaC1, 15 mM sodium citrate, pH 7.0), 0.1% SDS at room temperature, dried, and then exposed to Kodak X-ray film at -70°C for 1-2 h. The sizes of the hybridizing bands were then determined to construct the restriction maps of cosmid inserts. The accuracy of the maps thus obtained was verified by double digestion of cosmid DNAs with BamHI and EcoRI. Individual Gogo-DRB exons in cosmid clones were located by hybridization with exon-specific probes obtained by isolating appropriate restriction fragments from the chimpanzee DRB cDNA clones described by Fan et al. (1989). The BamHI or EcoRI restriction fragments hybridizing with the exon-specific DRB probes were subcloned to the pBluescript II SK + vector for further restriction mapping and DNA sequence determination. DNA sequencing. Double-stranded plasmid DNA was sequenced by the chain-termination method of Sanger et al. (1977) using the Sequenase Version 2.0 sequencing kit (United States Biochemicals, Cleveland, Ohio). Exon sequences of Gogo-DRB genes were determined by using the synthetic oligonucleotides described previously (Klein et al., 1991). Additional oligonucleotides used for sequencing were: 5'-TCTGGCCCCTGGTCCTGTCCT-3'for exon 1; 5'-GTCCTTCTGGCTGTTCCAG-3' and 5'-GCGCTTCGACAGCGACGTGG-3' for exon 2; 5'-GAACCACCTGACTTCAATGCT-3'and5'-CTGCAGCACCACAACCTCCTG-3'for exon 3; 5'-GTATCAAGGAGGAGGTCTTTTCG-3' for exon 4; 5'-TCTCAGGTGGGAGATCTGGGGCT-3' for exon 5; and 5'-CAGGAGCTGAGGAAGCCACAA-3'and 5'-GCTCTTATTCTTCCACAAGAG-3' for exon 6. Construction of phylogenetic trees. Phylogenetic trees were constructed using the PAUP (phylogenetic analysis using parsimony) program (Swofford, 1990).

RESULTS A N D DISCUSSION

Identification of T w o Clusters of Overlapping C o s m i d Clones E n c o d i n g G o g o - D R B Genes A t o t a l o f 63 c o s m i d c l o n e s w e r e i s o l a t e d b y s c r e e n i n g the gorilla library with the chimpanzee DRB cDNA probe B3-5 and the two walking probes WP1 and WP4.

342

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ORGANIZATION OF THE GORILLA DR SUBREGION The result of restriction mapping revealed that all but one of these cosmid clones could be organized into two c l u s t e r s , A a n d B (Fig. 2). C l u s t e r A, d e f i n e d b y 41 o v e r lapping cosmid clones, encompassed a contiguous DNA s t r e t c h o f ~ 180 k b a n d c o n t a i n e d a DRB g e n e s e g m e n t made up of exons 4 through 6 and four DRB genes. On the basis of sequence similarity to known HLA-DRB g e n e s (see b e l o w ) , t h e s e f o u r D R B g e n e s w e r e d e s i g n a t e d a s Gogo-DRB6, Gogo-DRBS*01, Gogo-DRB8, a n d GogoDRB3*01. C l u s t e r B, m a d e u p o f 21 o v e r l a p p i n g c o s m i d c l o n e s , s p a n n e d "---145 k b a n d c o n t a i n e d t h r e e D R B g e n e s . T h e s e g e n e s w e r e d e s i g n a t e d Gogo-DRBI*08, Gogo-DRB2, a n d Gogo-DRB3*02 u s i n g t h e s a m e p r i n c i ple. The cosmid clone, WP1-9, isolated by the WP1 probe, did not overlap with any of the clones belonging t o c l u s t e r A o r B (Fig. 2C). T h e D R B g e n e i n W P 1 - 9 w a s judged to be a pseudogene since it failed to hybridize with an exon 2-specific probe. Southern blot hybridization of genomic DNA isolated from Sylvia showed the p r e s e n c e o f s e v e n B a m H I a n d s i x EcoRI f r a g m e n t s h y bridizing strongly with a 3'-untranslated (3'-UT) reg i o n - s p e c i f i c c h i m p a n z e e D R B p r o b e (Fig. 3). A l l o f these fragments could be accounted for by the cosmid c l o n e s s h o w n i n F i g . 2. T h i s r e s u l t i n d i c a t e s t h a t w e h a v e i s o l a t e d m o s t , i f n o t all, o f t h e D R B g e n e s p r e s e n t i n t h e g e n o m e o f S y l v i a . S i n c e c l u s t e r A, c l u s t e r B, a n d t h e cosmid clone WP1-9 could not be linked to each other or to the DRA gene, it was not possible to determine whether the two clusters and the DRB gene on WP1-9 are located on one chromosome or on two separate, homologous chromosomes.

Characterization o[ Individual Gogo-DRB Genes by D N A Sequence Determination C l u s t e r s A a n d B c o n t a i n a t o t a l o f s e v e n DRB g e n e s a n d o n e D R B g e n e s e g m e n t (Fig. 2). T o e x a m i n e whether the gene organizations in these clusters resemble those of known HLA-DR haplotypes, all of these s e v e n D R B g e n e s w e r e s e q u e n c e d . F o u r g e n e s , GogoDRB6, Gogo-DRB8, Gogo-DRB2, a n d Gogo-DRB3*02, were found to be pseudogenes. The nucleotide sequences o f t w o p s e u d o g e n e s , Gogo-DRB8 i n c l u s t e r A a n d GogoD R B 2 i n c l u s t e r B, w e r e d e s c r i b e d p r e v i o u s l y ( K l e i n et al., 1991; V i n c e k et al., 1992). F i g u r e 4 s h o w s t h e e x o n s e q u e n c e s o f t h e r e m a i n i n g five D R B g e n e s , Gogo-DRB6, Gogo-DRB5*01, Gogo-DRB3*01, Gogo-DRBI*08, a n d Gogo-DRB3*02. A b r i e f d e s c r i p t i o n o f i n d i v i d u a l GogoD R B g e n e s follows. T h e five Gogo-DRB g e n e s s e q u e n c e d

343

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FIG. 3. Southern blot hybridization of Sylvia genomic DNA with a 3'-UT region-specific DRB probe. The genomic DNA isolated from Sylvia was digested with BamHI or EcoRI, electrophoresed, and blotted to a nylon filter. A 3'-UT region-specific DRB probe, which has been shown to be locus-specific in primates (Rollini et al., 1985; Bontrop et al., 1990), was isolated from the chimpanzee DRB cDNA clone B3-5. All of the hybridizing bands can be accounted for by the cosmid clones shown in Fig. 2. The derivation of individual bands is as follows: a 15.0-kb BamHI fragment, cosmid clone 26 (Gogo-DRB6); a 12.5-kb BamHI fragment, cosmid clone 37 (Gogo-DRB2);an 8.8-kb BamHI fragment, cosmid clone 71 (Gogo-DRBI*08);8.2-kb BamHI fragments, cosmid clones 98 (Gogo-DRB3*02) and WP1-9; a 5.1-kb BamHI fragment, cosmid clone 10 (Gogo-DRB3*01);a 3.5-kb BamHI fragment, cosmid clone 43 (Gogo-DRBS*01); a 2.1-kb BamHI fragment, cosmid clone 38 (Gogo-DRB8);an l l - k b EcoRI fragment, cosmid clone 38 (Gogo-DRB8);a 9.8-kb EcoRI fragment, cosmid clone 71 (Gogo-DRBI*08); an 8.4-kb EcoRI fragment, cosmid clone 10 (GogoDRB3*01); 5.2-kb EcoRI fragments, cosmid clones 26 and 98 (GogoDRB6 and Gogo-DRB3*02);a 4.8-kb EcoRI fragment, cosmid clone 43 (Gogo-DRBS*01); and 4.6-kb EcoRI fragments, cosmid clones 37 (Gogo-DRB2) and WP1-9. For the nomenclature of the genes, see text and Fig. 2. in this study were compared to representative HLAD R B g e n e s b y c o n s t r u c t i n g a p h y l o g e n e t i c t r e e (Fig. 5). T h e Gogo-DRB6 g e n e o f c l u s t e r A w a s j u d g e d t o b e orthologous to the HLA-DRB6 pseudogene carried by t h e H L A - D R 2 h a p l o t y p e ( E r l i c h et al., 1989; F i g u e r o a e t al., 1991; C o r e l l etal., 1991). A l t h o u g h a s e q u e n c e h o m o l o g o u s t o e x o n I is p r e s e n t ~ 2 0 k b u p s t r e a m f r o m e x o n 2 o f t h e Gogo-DRB6 gene, t w o o b s e r v a t i o n s s u g g e s t t h a t t h i s s e q u e n c e d o e s n o t b e l o n g t o t h i s gene: F i r s t , i n t y p i c a l h u m a n a n d g o r i l l a D R B g e n e s , e x o n 1 is l o c a t e d

FIG. 2. Organization and restriction map of the gorilla DRB subregion. (A, B) Two clusters of cosmid clones. The cosmid clone WP1-9, which could not be linked to either cluster A or B, is shown in C. Individual cosmid clones are indicated by horizontal bars. Exons are shown as solid boxes and are denoted as I for the leader sequence; 2 for the first domain; 3 for the second domain; 4 for the transmembrane segment; 5 for the cytoplasmic tail; and 6 for the 3'-UT region. Restriction enzymes used for the construction of the map are: EcoRI (abbreviated E) and BamHI (abbreviated B). The locations of two walking probes, WP1 and WP4, are shown by solid boxes directly beneath the restriction map. WP1 is a ~ 1.3-kb EcoRI fragment isolated from the cosmid clone 26. WP4 is a ~2.8-kb EcoRI fragment derived from the cosmid clone 65. The cosmid clones isolated with these walking probes are marked by prefixes WP1 or WP4. WP4 was not specific for the Gogo-DRB1 08 gene. The precise locations of two leader sequences in cluster A, one upstream from the DRB6 gene and the other downstream from the DRB3*01 gene, were not determined. These leader sequences are indicated by hatched boxes.

344

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

35

AAC

45

CTC T ....

CTG

TAC

ATC T--

TAC

AGA

20 CAT

-A ..........

CAG

DRB3*OI

TGT

A ....

DRB6

Exon

ATG

C . . . . . . . . . . . . . . . . . . .

G. . . . . . . . .

AG-

DRB5*OI

Exon

GTG

.........

GGG

9O AGC

3 95

100

DRB6

TC

CAA

CCT

DRB5*OI

-T

G-G

.......

DRB3*OI

....

T

---

AAG C--

GGG

ACT

105 GTG

Ii0

TTA

AAG

ATC

T .......

T .....

GC-

-G-

-C ....

CAG

-T .......

T .....

GC .....

CT

.....

C

CAC

CAC

AAC

DRBI*08

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

T .......

T ......

C .....

C-

--A

-TC

....

A .............

DRB3*02

....

T .......

T ......

C .....

C ......

C

....

A .............

C

.......

115

120

125

CTC

130

DRB6

CTG

CTC

DRB5*OI

---

G ..........

DRB3*OI

---

G ............

G .....................................

CA-

DRBI*08

---

G ............

G .....................................

C .....

DRB3*02

---

G ..................................................

DRB6

GGC

DRB5*OI

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

G . . . . . . . . . . . . . . . .

DRB3*OI

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

G .......

DRBI*08

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

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DRB3*02

--A

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DRB6

TTC

DRB5*OI

.......

DRB3*OI

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

DRBI*08

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

G . . . . . . . . . . . . . . . . . . . . . . . . .

DRB3*02

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

G . . . . . . . . . . . . . . . . . . . . . . . . .

DRB6

GTG

DRB5*OI

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

TGC

TCT

GT/ G

135

///

GGT

TTC

TAT

CCA

AAT

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

140 CAG

GAA

GAG

AAG

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

GCT

A--

155 ACC TT

A--

GTG

/--

ATG

TCC

GAA

CCA

AGT

GTG

C

....

GTC

ACA G

A

AGG

TGG

TTC

TGG

AAT

C ....

C

--C

C ..... 150

GGC

CTG

ATC T

CAG

AAT

GGA

GAC

TGG

ACC

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

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

165

170

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

180 CAC

ATT

CTG GTG ATG CTG GAA ACA GTT CCT CAG AGT GGA ...................................................

175 GAG

AGC

145 GGG

160 CAG

GGC

GAG

GTT

G . . . . . . . . . . . . . . .

TAC T

ACC

TGC

CAA

.........

185 AGG C-

AGC

CCT

CTC

ACA

GTG

CAA

TGG

-T . . . . . . . . . . . . .

G ......

-C . . . . . . . . . . . . . . . .

G ......

DRB3*OI

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

C

--A

DRBI*08

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

C

....

CA

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

G ......

DRB3*02

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

C-

-T . . . . . . . . . . . . .

G ......

A

F I G . 4. Nucleotide sequences (exons i through 6) of five gorilla DRB genes. The coding region of the genes is numbered from - 2 9 to - 1 for the leader sequence and from 1 to 237 for the mature protein"--" and "/" signify identity with the top sequence and absence of residues, respectively. The stop codon located at codon 94 of the Gogo-DRB6 gene is underlined. The sequences of the 5'-UT region were not determined.

ORGANIZATION OF THE GORILLA DR SUBREGION Exon

345

4 190

DRB6

195

200

205

GA GCA CAG TCT GAA TCT GCA CAG AGC AAG ATG CTG AGT GGA GTC GGG GGC TTT GTG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DRB5*OI DRB3*OI

. . . . . .

DRB1

*08

A . . . . .

G . . . . . . . . . . .

DRB3

* 02

A . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

210

DRB6

GGC

DRB5*OI

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 CTC

TTC

CTT

GGG

220 CTG

TTC

ATC

225

ACA

GGG

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

G-C

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

T-

DRB3*OI

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

G-C

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

T. . . . .

DRBI*08

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

G-T

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

T. . . . . . . . . . . . . .

DRB3*02

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

G-C

. . . . . . . . . . .

T. . . . . . . . . . . . . .

CCA

ACA

Exon

CTG

CTG

TAC

G

TGC

....

AGG

AAT

CAG

AAA

G

-A . . . . . . . . . . . G . . . . . . . .

5 230

DRB6 DRB5

GA

*01

CAC

TCT

GGA

CTT

CAG

DRB3*OI

-G . . . . . . . . . . . . . . C ....... . . . . . . . . . . . . . . . . . . . . . . . .

DRBI

*08

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

DRB3

* 02

........

Exon

A . . . . . .

DRB5

GA

*01

C . . . . . . . .

6 235

DRB6

G

CTC

237 TTG

AGC

TGA

AGTGCAGATG

A/CCACATTC

AAGGAAGAAC

.....

G . . . . . . . . . . . . . . . . . . .

/ ............

DRB3*OI

--

T--

C . . . . . . . . . . . . . . . . . . .

/-/-/---T

DRB1

*08

--

T--

C . . . . . . . . . . . . . . . . . . .

DRB3

* 02

.....

DRB6 DRB5

/ .................

GATGATAAGC

*01

TTTCCCACTT

.....

A .........

DRB3*OI

.....

A ..........

DRBI

*08

T

GGCTCTTATT

/-/T

.........

.....

A ..........

T-C ....

DRB3*02

.....

A ..........

G-C-

.......

CTTCCACAAG

DRB6

GGTTCAGCAA

DRB5*OI

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

DRB3*OI

..........

DRBI*08

CTCTGCAGAA

AG ....

C

AG////CTTT

T

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

G

T ..................

G

.......

CTCAGGACCA

..........

GGTTGCTACT

C--T

.........

C

G

..........

G .......

G .............

T

..........

G .............

T

..........

CTTGTTGCTT

CCTCAGCTCC

TGCTCTTGGC

CTGAAATCCC

G .................

C .....

// .......................

G .................

C ...........

G ....

..........

// .......................

G .................

C ...........

G ....

DRB3*02

..........

// .......................

G .................

C ...........

G ....

DRB6

AGCACTGATA

DRB5*OI

....

T ....

G

....

DRB3*OI

....

T ....

G

.......

DRBI

*08

....

T ....

G

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

DRB3

* 02

....

T ....

G

....

~AC-CGCCTC

AT ......

G

G .....................

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

AATGTCCTCC

T

A .......

AGGA

G ............

A---G

AGCTTTGCAA

-A .....................................

TG ......................... T-C .....

CTTCTGCCCC

GG .......................

ATCTTCAACT

TTTGTGCTTC

T ................. ///

T-T

A .....

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

...........

G---C

.....

CCTTTACCTA

AACTTTCCTG

.....

G .......

CC-ATG

C-

.....

G .......

CC-ATG

......

C-

.....

GTG .....

CC-ATG

...........

/-CC-ATG

.........

/

......

DRB3*OI

.........

C

A ......

A//

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

A ......

-A .......

DRBI*08

..........

A ......

A//

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

A ......

-A--

DRB3*02

.... C .....

A ......

A//

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

A ......

-A .............

TG-

CCTCCCGTGC

C-

G ......

ATCTGTACT/

CCACAAACAC

G ....

....

DRB5*OI

T ..........

.....

C-

DRB6

CCCCTG/GTG

A

ATTGCATTAT

TCAATGTTTC

-C-A-G

-A---T

....

TCAAACATGG

.......

A---

........... T ....

AGTT

....

G ........

FIG. 4--Continued ~ 1 0 - 1 5 kb upstream from exon 2. Second, this particular exon 1 sequence is located ~ 10 kb downstream from exon 6 of the gorilla DRB gene present at the 5'-end of cluster A. This distance is similar to that found between Gogo-DRB3 (or HLA-DRB3) and a lone exon I sequence located downstream from them (Fig. 2). Therefore, the overall organization of Gogo-DRB6 and HLA-DRB6 (Kawai et al., 1989) is identical, in the sense that they lack only exon 1. These two genes also cluster together to the same branch in the phylogenetic tree (Fig. 5). The Gogo-DRB6 gene has a nonsense mutation (TGA) in exon 2 (codon 94) and a 4-bp deletion in exon 3 (codons 119-120), which shifts the reading frame (Fig. 4). The absence of these mutations in HLA-DRB6 suggests that they have taken place after the separation of human and gorilla lineages. The Gogo-DRB5*01 gene of cluster A is assumed to be the gorilla counterpart of the HLA-DRB5 gene carried by the DR2 haplotype (Fig. 5). The structural similarity

between these genes extends to all the exons. Virtually all polymorphic sequences, w h i c h characterize HLADRBS, are present in Gogo-DRB5*01. The GogoDRB5*01 gene has no apparent deleterious mutations precluding its expression and is therefore assumed to be functional. Like the HLA-DRB8 gene carried by the HLA-DR4 haplotype (Andersson et al., 1987), Gogo-DRB8 lacks exons 1 and 2 (Fig. 2). In addition to this structural similarity, the exon and intron sequences of these two genes are very similar ( ~ 9 3 % nucleotide sequence identity), and their introns share two Alu repeats at corresponding positions. For these reasons, the Gogo-DRB8 gene was judged to be a pseudogene orthologous to HLA-DRB8 (Klein et al., 1991). The Gogo-DRB3*01 gene of cluster A is most closely related to HLA-DRB3 alleles carried by HLA-DR3/ DR5/DR6 haplotypes (Fig. 5). However, exon 1 of this gorilla gene does not share polymorphic sequences char-

346

KASAHARA E T AL.

IDRB]*OIO! DRBI*1502

I

IDRBI*0301] IDRBI*1101]

IDRBI*1201] Gogo-DRBI~O8 Gogo-DRB3*02

I

F III

DRB3*0201 I 6ogo-DRB3*O! DRBI*0401 DRBI*0701

I

DRBI*0901 DRB4*OIO1 Gogo-DRB6

I

DRB5 DRB5*0201 Gogo-DRBS*OI DRBI*IO01

F I G . 5. A phylogenetic tree of primate DRB genes obtained by the maximum parsimony method. The tree was constructed using the nucleotide sequences of exons 2 through 6 (a total length of 924 bp). The sequences were aligned using the method of Needleman and Wunsch (1970) before tree construction. The tree shown in this figure is a strict consensus tree (Sokal and Rohlf, 1981) made from three equally parsimonious trees. The identical tree was obtained by the semistrict consensus method (Bremer, 1990). The Gogo-DRB genes sequenced in this study are shown in boldface letters. HLA-DRB genes carried by HLA-DR3/DR5/DR6 haplotypes are boxed. The sources of the HLA-DRB sequences are as follows: DRBI*0101, Tonnelle et al. (1985); DRB1*1502 and DRB5*0201, Wu et al. (1987); DRB I*0301 and DRBI*IO01, Gustafsson et al. (1984); DRBl*1101, Tieber et al. (1986); DRB1*1201, Navarrete et al. (1989); DRBI*0802, Jonsson et al. (1989); DRB3*0201, Long et al. (1983); DRBI*0401 and DRB4*0101, Andersson et al. (1987); DRBI*0701 and DRBI*0901, Gregersen et al. (1986); and DRB6, Figueroa et al. (1991).

acteristic of HLA-DRB3. Since its sequence does not resemble closely that of any known HLA-DRB genes, this exon may have been contributed by an ancestral DRB gene, the descendants of which no longer exist in the human population. The Gogo-DRB3*01 gene has no apparent deleterious mutations and is therefore assumed to be functional. The Gogo-DRBI*08 gene of cluster B is most closely related to HLA-DRBI*08 (Fig. 5). Virtually all polymorphic sequences characteristic of HLA-DRB1 *08 are present in Gogo-DRBI*08, indicating an orthologous relationship between these two genes. The nucleotide sequence of the Gogo-DRBI*08 gene reveals no features incompatible with its expression. The Gogo-DRB2 gene of cluster B was considered to be orthologous to the HLA-DRB2 pseudogene carried by HLA-DR3/DR5/DR6 haplotypes on the basis of three observations (Vincek et al., 1992): First, both genes lack exon 2; second, both genes contain a 20-bp deletion in exon 3, which shifts the reading frame; and third, the nucleotide sequences of the two genes are ~95% identical. The Gogo-DRB3*02 gene of cluster B does not appear to be orthologous to any known HLA-DRB alleles. A close inspection of this gorilla gene, however, suggests

that it has numerous features characteristic of HLADRB3 alleles, as indicated by the dendrogram in Fig. 5. The exon I sequence of the Gogo-DRB3*02 gene is identical to that of HLA-DRB3 alleles. The nucleotide sequences of exon 2 (in particular, codons 9-14 and 26-30) and exon 6 also suggest a close evolutionary relationship between Gogo-DRB3*02 and HLA-DRB3. However, exon 2 of Gogo-DRB3*02 contain a sequence stretch at codons 69-71 that resemble more closely that of DR52associated HLA-DRB1 alleles. Unlike HLA-DRB3 alleles encoding the supertypic DR52 determinant, GogoDRB3*02 is a pseudogene; it has two single-basepair deletions that shift the reading frame, one at codon 142 and the other at codon 236 (Fig. 4). Additional evidence for the close evolutionary relationship between GogoDRB3*02 and HLA-DRB3 (or Gogo-DRB3*01) is provided by similarity in the distribution of EcoRI restriction sites flanking these genes (compare Gogo-DRB3*01 and Gogo-DRB3*02 in Fig. 2). The results presented above show that, with the possible exception of Gogo-DRB3*02, all the Gogo-DRB genes characterized in this and previous studies (Klein et al., 1991; Vincek et al., 1992) have their orthologous human counterparts. Therefore, not only the gene duplication events that gave rise to individual DRB genes (and pseudogenes) but also the subsequent allelic diversification must have taken place before the divergence of human and gorilla lineages. A growing body of evidence obtained from sequence analysis suggests that MHC polymorphisms predate speciation (Mayer et al., 1988; McConnel et al., 1988; Figueroa et al., 1988; Lawlor et al., 1988; Fan et al., 1989; Gyllensten and Erlich, 1989; Kasahara et al., 1990). The sequences of the Gogo-DRB genes described here support this suggestion.

Structural Comparison o[ Gorilla and Human DR Haplotypes The structural organizations of clusters A and B (Fig. 2) were then compared to those of known HLA-DR haplotypes (Fig. 1). In cluster A, two DRB genes, GogoDRB6 and Gogo-DRB5*01, were found to be located adjacent to each other. The human counterparts of these genes are linked on the HLA-DR2 haplotype (Kawai et al., 1989). Thus, this combination of DRB genes must have been maintained since the time before the divergence of human and gorilla lineages. However, the arrangement of the other genes in cluster A differs from that of DRB genes in known HLA-DR haplotypes. The human counterparts of Gogo-DRB3*01 and Gogo-DRB8, HLA-DRB3 and HLA-DRB8, are located on HLA-DR3/ DR5/DR6 and HLA-DR4/DR7/DR9 haplotypes, respectively. Therefore, these results show that reshuffling of DRB genes took place during primate evolution, as suggested previously by Southern blot analysis (Bontrop et al., 1990). It is not clear whether such reshuffling took place before or after the divergence of human and gorilla lineages. At the time when ancestral DR haplotypes were created, they might have still been able to recom-

ORGANIZATION OF THE GORILLA DR SUBREGION bine with each other, and numerous DR haplotypes may have been generated. By the time the two species diverged, they might have become frozen, and ancestors of humans and gorillas might have inherited distinct DR haplotypes. Alternatively, certain combinations of DRB genes may have become frozen only after the divergence of h u man and gorilla lineages. Two lines of evidence suggest t h a t the D R B 1 - D R B 2 - D R B 3 organization in human D R 3 / D R 5 / D R 6 haplotypes is probably more ancestral th an the Gogo-DRB8-Gogo-DRB3*01 arrangement in cluster A. First, as described below, the gorilla cluster B has the HLA-DR3/DRS/DR6-1ike organization, indicating t ha t this type of organization existed before the separation of hum a n and gorilla lineages. Second, a group of HLA-DRB1 and -DRB3 alleles carried by H L A - D R 3 / D R 5 / D R 6 haplotypes (boxed in Fig. 5) are more closely related to each other t han they are to other DRB alleles. This observation is consistent with the idea t h a t a duplication event t h a t gave rise to an ancestral DRB1*03,05,06 gene and an ancestral DRB3 gene took place after the divergence of other DRB alleles, and that, since then, the duplicated DRB1 and DRB3 genes have stayed on a single chromosome. In the H L A - D R 2 haplotype, DRB5 is located most telomeric among the three DRB genes (Kawai et al., 1989). Available evidence does not allow us to determine whether the DRB5*O1-DRB8 arrangement in cluster A or the HLA-DR2-1ike organization is more ancestral. T h e structural organization of cluster B is similar to t hat of H L A - D R 3 / D R 5 / D R 6 haplotypes (Fig. 1). In these H L A - D R haplotypes, the DRB2 pseudogene is flanked by the DRB1 gene at the centromeric side and by the DRB3 gene at the telomeric side. In cluster B, the gorilla counterpart of the human DRB2 pseudogene, Gogo-DRB2, is flanked by the Gogo-DRBI*08 gene (the gorilla counterpart of the HLA-DRBI*08 gene) and the Gogo-DRB3*02 gene (assumed to be related to the HLADRB3 gene). Although the H L A - D R 8 haplotype contains only one DRB gene designated as DRBI*08, the sequence (exons 1 through 5) of this gene is related to t h at of the DRB1 genes carried by D R 3 / D R 5 / D R 6 haplotypes. It has been proposed t ha t the original HLADR8 haplotype had the same gene organization as t h a t of H L A - D R 3 / D R 5 / D R 6 haplotypes and t ha t the present-day H L A - D R 8 haplotype arose as a result of a deletion involving exon 6 of the DRB1 gene, the entire DRB2 gene, and exons 1 through 5 of the DRB3 gene (Gorski, 1989). T h e fact t h a t the arrangement of DRB genes in cluster B is identical to t hat of the presumed ancestral H L A - D R 8 haplotype suggests t h a t the gorilla genome still maintains the ancestral form of the DR8 haplotype. T h e exon 6 sequence of the HLA-DRB1*08 gene is identical to t h a t of the HLA-DRB3*01 gene (Gorski, 1989). Hence, the presumed deletion took place relatively recently in the human lineage. It is interesting to note t hat Gogo-DRB3*02 is a pseudogene, and that, like the HLADR8 haplotype, cluster B contains only one potentially functional DRB gene. This finding raises the possibility that, in both h u m a n and gorilla DR8 haplotypes, selec-

347

tive pressures might have been at work to keep only one DRB gene functional (assuming t hat clusters A and B are located on two separate, homologous chromosomes). T h e deletion in the ancestral H L A - D R 8 haplotype might have actually taken place after the inactivation of the DRB3 gene. T he structure and the restriction map of the DRB pseudogene carried by the cosmid clone WP1-9 (Fig. 2C) resemble closely those of the Gogo-DRB2 pseudogene in cluster B. This unlinked pseudogene might be located upstream from the Gogo-DRBI*08 gene. If this were the case, cluster B could be viewed as a duplication product of the ancestral D R B 2 / D R B 3 (or WP1-9/ DRB1) pair. CONCLUSIONS We have shown t h a t the structural organization of the gorilla DR subregion differs considerably from th a t of known human DR haplotypes, indicating th a t reshuffling of DRB genes took place during primate evolution. Nevertheless, certain combinations of DRB genes appear to be quite stable. Our results indicate t h a t such combinations can persist beyond the lifespan of species. Since hum an and gorilla lineages are assumed to have diverged about five million years ago (Pilbeam, 1984), the D R B 6 - D R B 5 and DRB1-DRB2-DRB3-1ike organizations must be at least this old. Whet her the maintenance of particular combinations of DRB genes has any functional significance or is simply an accident (perhaps due to the absence of recombination hot spots a n d /o r the presence of inversions) is an open question. T h e identification of the Gogo-DRB gene cluster, the organization of which resembles t hat of the presumed ancestral H L A - D R 8 haplotype, demonstrates t ha t the characterization of nonhum an primate M H C is an effective approach to reconstructing the evolutionary history of the H L A complex. Finally, analysis of additional Gogo-DR haplotypes should provide more detailed information on the stability and evolution of DR haplotypes in primates. ACKNOWLEDGMENTS The authors thank Mayra G. Oberto for her technical assistance and Dr. Kirby D. Smith and Ms. O. Colin Stine, The Johns Hopkins University Schoolof Medicine,for the gorillacell line. This work was supported by research Grants AI 23667 (J.K.) and HD 25493 (M.K.) from the National Institutes of Health, Bethesda, Maryland. The sequence data presented in this article have been submitted to the DDBJ/EMBL/GenBank Nucleotide Sequence Databases under Accession Nos. M77151-77155. REFERENCES

Andersson, G., Larhammar, D., Widmark, E., Servenius,B., Peterson, P. A., and Rask, L. (1987).Class II genes of the human major histocompatibilitycomplex.Organization and evolutionaryrelationship of the DR/~genes. J. Biol. Chem. 262: 8748-8758. BShme, J., Andersson, M., Andersson, G., MSller,E., Peterson, P. A., and Rask, L. (1985). HLA-DRflgenes vary in number between different DR specificities,whereas the number of DQ/3 genes is constant. J. Immunol. 135: 2149-2155.

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