Gene 259 (2000) 75–79
www.elsevier.com/locate/gene

Gene diversity of chimpanzee ABO blood group genes elucidated
from exon 7 sequences
Kenta Sumiyama a,1, Takashi Kitano a, Reiko Noda a, Robert E. Ferrell b,
Naruya Saitou a, *a Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima 411-8540, Japanb Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA
Received 17 April 2000; received in revised form 6 July 2000; accepted 25 August 2000
Received by T. Gojobori

Abstract
Human and non-human primate ABO blood group genes show relatively large numbers of nucleotide differences. In this study,
we determined exon 7 sequences for 10 individuals of common chimpanzee and for four individuals of bonobo to estimate
nucleotide diversities among them. Sequence data showed the existence of chimpanzee specific 9-base deletion in the beginning of
the exon 7 coding region. From a phylogenetic network of exon 7 sequences of ABO blood group genes for human, common
chimpanzee, bonobo and gorilla, effects of parallel substitutions and/or some kinds of convergent events are inferred in thechimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes, andthese values were 0.4% and 0.2%, respectively. These values are higher than that of most human genes. © 2000 Elsevier Science
B.V. All rights reserved.
Keywords: Glycosyltransferases; Hominoids; Nucleotide diversity; Phylogenetic network

1. Introduction
The ABO alleles A and B code for terminal glycosyltransferases, which transfer N-acetylgalactosamineand galactose, respectively, to a common precursor (Hsubstance). Yamamoto et al. (1990) determined thecDNA sequences of the three common human alleles
A1, B, and O, and Yamamoto et al. (1995) determined
the genomic organization of the gene. Critical sites forthe distinction between A and B activities of the glycosyltransferase have been identified ( Yamamoto andHakomori, 1990) in exon 7, and many non-human ABOblood group exons 7 were sequenced (e.g. Kominatoet al., 1992; Martinko et al., 1993; Kermarrec et al.,1999). Saitou and Yamamoto (1997) compared nucleotide sequences of primate ABO blood group genes, andAbbreviations: PCR, polymerase chain reaction; SSCP, single strand
conformational polymorphism.
* Corresponding author. Tel.: +81-559-81-6790;
fax: +81-559-81-6789.
E-mail address: nsaitou@genes.nig.ac.jp (Saitou Naruya)
1 Present address: Department of Biology, Yale University, New
Haven, CT 06520, USA.

relatively large numbers of nucleotide differences werefound among them. The number of differences is unusually large for human allelic divergence under neutral
evolution.
In this study, we determine and analyze the nucleotide
sequences of exon 7 for two Pan species to compare andelucidate the gene diversity among the hominoid ABO
blood group genes.

2. Materials and methods
2.1. Sequencing of genomic DNA
Genomic DNAs of 10 unrelated common chimpanzees (Pan troglodytes) and four unrelated bonobo (Panpaniscus) were used for DNA sequencing. PCR reaction
was performed containing 1×Gene Taq Universal
Buffer (Mg2+ free) (Nippon Gene), 1.5 mM MgCl ,20.2 mM dNTP, 10 pmol of each primer, and 1 unit of
AmpliTaq Gold (PE Biosystems). PCR primers weredesigned based on database sequences including human,chimpanzee and gorilla as follows. SN-1: 5∞-GT-0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 44 0 - 6

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K. Sumiyama et al. / Gene 259 (2000) 75–79

TAACCCAATGGTGGTGTTCT-3∞, SN-2: 5∞-GAGACGGCGGAGAAGCACTT-3∞, SN-3: 5∞-TGGGTCGCGGAACTCCATGTC-3∞, SN-4: 5∞-ACGTCCTGCCAGCGCTTGTA-3∞, SN-5: 5∞-AAGCGCTGGCAGGACGTGT-3∞, SN-6: 5∞-GTGGGCGTGGAGATCCTGACT-3∞, SN-7: 5∞-CCCTCGTCCTTGGGGATGTAG-3∞, SN-8: 5∞-GGGCCGGCGCTCGTAGGT-3∞,SN-9: 5∞-CCCCAGTCCCAGGCCTACAT-3∞, SN-10:5∞-CGATGCCGTTGGCCTGGTC-3∞, SN-11: 5∞-GGTGGCAGGCCCTGGTGAG-3∞.
Amplification was carried out in DNA GeneAmp
PCR System 2400 (PE Biosystems) with the following
temperature parameters: 10 min at 95°C followed by 40
cycles of 95°C for 30 s, 60–65°C for 15 s, and 72°C for
1 min. PCR products were purified using MicroSpin
Columns S-300 HR (Amersham Pharmacia Biotech).
At first, PCR products were sequenced in both strands

using the direct sequencing method. Heterogeneous PCR
products were then cloned in the TA cloning vector
pCRII (Invitrogen). DNA sequencing was performedon double-stranded plasmid DNA and PCR products
using Dye Terminator Cycle Sequencing Kit and ABIprism 377 DNA sequencer (PE Biosystems).

2.2. PCR-SSCP analysis
PCR-SSCP analysis following the method of Hayashi(1991) was used to identify heterozygous sites. PCRprimers were end-labeled by gamma-32P-ATP, and PCR
was done using the conditions described above. We used
0.5×Tris–Borate–EDTA buffer and 5% acrylamide gel
containing 5% glycerol for electrophoresis. During electrophoresis, the temperature was always kept at 16°C.

Table 1
Variant positions of ABO blood group genes in exon 7aa 1: Yamamoto et al. (1990), 2: Kominato et al. (1992), 3: Martinko et al. (1993). Dots, asterisks, and hyphens mean identical nucleotides as
the human-A1 (Hu-A1) sequence, unavailable positions, and deletions, respectively.

K. Sumiyama et al. / Gene 259 (2000) 75–79

2.3. Sequence analyses
CLUSTAL W version 1.6 ( Thompson et al., 1994)
was used for multiple alignments. Phylogenetic networks
were constructed following the procedure of Bandelt(1994) and Saitou and Yamamoto (1997). The nucdiv
program of the ODEN package (Ina, 1994) was used
for estimation of nucleotide diversities.

3. Results and discussion
3.1. Comparison of sequences
We sequenced exon 7 for five ABO blood groupalleles for common chimpanzee and three alleles forbonobo(DDBJ/EMBL/GenBankInternational
Nucleotide Sequence Database accession numbers are
AB031368–AB031372). Table 1 shows the variant sitesof the exon 7 ABO blood group alleles. Type Ch-I andCh-II are identical to reported chimpanzee A allele
sequences, while others (types Ch-III, Ch-IV, and Ch-V )are novel. A 9-base deletion was found in position 518
to 526 of the type Ch-V sequence. This deletion does
not cause frame shift mutation, and has previously beenreported by Kermarrec et al. (1999).
We used PCR-SSCP analysis to test for heterozygosity
in two overlapping fragments covering the entire exon
7 sequence. Fig. 1 shows a typical result of PCR-SSCPanalysis, and shows the two distinct bands caused byheterozygosity of the amplified region. Each chimpanzee
genotype gave a unique PCR-SSCP banding pattern,

77

and those typings were used to verify the results obtained
from sequencing of the cloned exon 7 PCR product.
3.2. The phylogenetic network of hominoid ABO bloodgroup genes
Fig. 2 shows the phylogenetic network of exon 7
sequences of ABO blood group genes for human,common chimpanzee, bonobo, and gorilla. This phylogenetic network was constructed from sequence data ofTable 1. The HCG node indicates possible ancestral
position for all human–chimpanzees–gorilla ABO bloodgroup genes. Sites 796 and 813 separate B type allelesof human and gorilla from others.
This network contains several parallelograms whichindicate incompatible sites. Those structures can begenerated by parallel substitutions and/or by some typesof convergent events. For example, position 777 separates [Ch-II, Ch-III, Bo-1, Bo-2, Bo-3] and [Ch-I, Ch-IV,Ch-V ], but this partition is incompatible with that for
position 791 which separates [Ch-I, Ch-II, Ch-V, Bo-1,Bo-2, Bo-3] and [Ch-III, Ch-IV ]. This incompatibility
is possibly the result of recombination between chimpanzee ABO alleles.
Allele Ch-V has G in position 589, while all otherchimpanzee alleles have C in this position. Because allother primates also have G in this position, it is possiblethat this is a reflection of ancestral polymorphism, orparallel mutation. A similar situation was also observedin position 467 for Ch-A4.
Positions 468 and 474 make a partition between[Ch-A3, Ch-A4, Ch-A5] and others. This means thatFig. 1. Two examples of PCR-SSCP. Lane 1: CH-36, lane 2: CH-46, lane 3: CH-206, lane 4: CH-75, lane 5: CH-83, lane 6: CH-80, lane 7: CH-76,and lane 8: CH-86. In the case of the fragment amplified by using SN-5 and SN-8, lanes 1, 3 and 6 share the same sequence, while lane 1 andothers share another sequence. In the case of the fragment amplified by using SN-9 and SN-11, lanes 1 and 6 show typical heterozygous pattern.
Lane 7 was not amplified effectively (a weaker band appeared here).

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K. Sumiyama et al. / Gene 259 (2000) 75–79

Fig. 2. The phylogenetic network of ABO blood group gene exon 7 sequences for common chimpanzee, bonobo, gorilla, and human. Numbers arenucleotide positions responsible for corresponding edges, and edge lengths are proportional to number of nucleotide differences. Abbreviations ofalleles are the same as in Table 1. A star indicates human–chimpanzees–gorilla (HCG) common ancestral node.

there are two kinds of variations in the first part ofexon 7 (positions 375 to 489). Positions 533 and 589
characterize allele Ch-V. The 9-base deletion is anotherunique feature for allele Ch-V. Those create anothervariation in the middle part of exon 7 (positions 513 to695). The last part of exon 7 has more complicatedpolymorphism, because position 777 is incompatible
with positions 702, 813, and 791. This might be aconsequence of recombination, and if this is the case, atleast one recombination has occurred between positions
777 and 791.
There is no separate branch for the bonobo specificcluster, but the three bonobo sequences are closelyrelated.
3.3. Nucleotide diversities in chimpanzee ABO bloodgroup genes
We computed nucleotide diversities for the commonchimpanzee and bonobo ABO blood group exon 7
sequences. Table 2 shows the genotype at the ABOblood group locus for each individual. Chimpanzee
CH-90 and bonobo BO-8 are heterozygous, but only
one allele could be identified. Nucleotide diversities ofcommon chimpanzee and bonobo were 0.384% and0.204%, respectively (see Table 3). Li and Sadler (1991)
estimated nucleotide diversity in humans to be 0.110%

Table 2The list of chimpanzees and their genotypes for ABO blood groupgene exon 7Individual nameCommon chimpanzee (Pan troglodytes)
CH-46
CH-75
CH-76
CH-80
CH-83
CH-86
CH-90
CH-206
CH-220
CH-235
Bonobo (Pan paniscus)
BO-3
BO-4
BO-5
BO-8

Genotype

Ch-III/Ch-III
Ch-I/Ch-I
Ch-I/Ch-II
Ch-II/Ch-V
Ch-II/Ch-II
Ch-II/Ch-II
Ch-IV/–aCh-V/Ch-V
Ch-V/Ch-V
Ch-III/Ch-III
Bo-1/Bo-1
Bo-2/Bo-3
Bo-2/Bo-3
Bo-3/–aa CH-90 and BO-8 are heterozygous, but another allele could not bedetermined.

by using 49 loci. Nucleotide diversities among alleles ofchimpanzee ABO blood group genes seem to be higherthan that of alleles of most human genes. Kaessmannet al. (1999) showed that the nucleotide diversity among
alleles of chimpanzee for chromosome X non-coding isK. Sumiyama et al. / Gene 259 (2000) 75–79

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Table 3
Nucleotide diversities in chimpanzee ABO blood group genes

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Number of sites
compared (bp)Common chimpanzee (Pan troglodytes)
19468
Bonobo (Pan paniscus)
7468

Nucleotide
diversity (%)

0.384
0.204

higher than that of most human genes, and our resulton the ABO gene agrees with their study. Kitano et al.
(2000) estimated nucleotide diversity values of 0.219%
and 0.208% for intron 6 sequences of the common
chimpanzee and bonobo ABO blood group gene. This
result suggested that the nucleotide diversity of exon 7
is higher than that of intron 6 for common chimpanzee
ABO blood group genes.
Saitou and Yamamoto (1997) estimated allelic differences of the coding region of human ABO blood groupgene to be 0.8–1.3% per nucleotide site. Those values
are much larger than those of two chimpanzee species.
The higher level of nucleotide diversity and the literaturesuggesting association between allelic variation at theABO blood group locus and a variety of infectious
diseases raises the possibility that the higher nucleotide
diversity in humans is driven by position selection(Mourant et al., 1978). In chimpanzee, this kind ofselection may be weak or neutral.

Acknowledgement
This study was partially supported by grants-in-aid
for scientific studies from the Ministry of Education,
Science, Sports, and Culture, Japan to N.S.