Immunogenetics (2000) 51 : 856±862
Digital Object Identifier (DOI) 10.1007/s002510000202

ORIGINAL PAPER

Takashi Kitano ´ Naruya Saitou

Evolutionary history of the Rh blood group-related genes
in vertebrates

Received: 13 January 2000 / Revised: 17 April 2000 / Published online: 17 June 2000
 Springer-Verlag 2000

Abstract Rh and its homologous Rh50 gene products
are considered to form heterotetramers on erythrocyte
membranes. Rh protein has Rh blood group antigensites, while Rh50 protein does not, and is more conserved than Rh protein. We previously determinedboth Rh and Rh50 gene cDNA coding regions frommouse and rat, and carried out phylogenetic analyses.
In this study, we determined Rh50 gene cDNA coding
regions from African clawed frog and Japanese
medaka fish, and examined the long-term evolution of
the Rh blood group and related genes. We constructed
the phylogenetic tree from amino acid sequences.
Rh50 genes of African clawed frog and Japanese
medaka fish formed a cluster with mammalian Rh50
genes. The gene duplication time between Rh andRh50 genes was estimated to be about 510 million
years ago based on this tree. This period roughly corresponds to the Cambrian, before the divergence
between jawless fish and jawed vertebrates. We also
BLAST-searched an amino acid sequence database,and the Rh blood group and related genes were foundto have homology with ammonium transporter genesof many organisms. Ammonium transporter genes can
be classified into two major groups (amt a and amt b).
Both groups contain genes from three domains (bacteria, archaea, and eukaryota). The Rh blood group andrelated genes are separated from both amt a and bgroups.
Key words Rh blood group genes ´ Evolutionary
history ´ Gene duplication

T. Kitano ´ N. Saitou ())
Laboratory of Evolutionary Genetics,
National Institute of Genetics, Mishima 411-8540, Japan
E-mail: nsaitou@genes.nig.ac.jp
Phone: +81-559-816790
Fax: +81-559-816789

Introduction
The human Rh blood group plays important roles intransfusion and clinical medicine, being involved inhemolytic diseases of newborns, autoimmune diseases,
and mild hemolytic anemia. Rh polypeptides wereobserved to be phosphorylated Mr 30±32,000 membrane proteins using SDS-PAGE and immunoprecipitation (Gahmberg 1982; Moore et al. 1982), and theRh blood group system is composed of two closely
linked D and CE loci (Mouro et al. 1993). A protein
was obtained together with the Rh gene product on
immunoprecipitation with anti-Rh antibodies fromhuman, and named the 50kD glycoprotein (Moore
and Green 1987). This glycoprotein was considered toform a heterotetramer with Rh blood group geneproducts on erythrocyte membranes (Eyers et al.
1994). The nucleotide sequence of the human 50kD
glycoprotein (Rh50) was determined, and its aminoacid sequence was homologous with that of the human
Rh gene (Ridgwell et al. 1992). That protein was also
predicted to have the 12 transmembrane domains
which are similar to those of the Rh blood group geneproduct (Avent et al. 1990). There are several names
for this gene, such as RHAG, but we call this gene
Rh50 and the Rh blood group gene Rh hereafter for
simplicity.
Nucleotide sequences of Rh genes in nonhumanprimates have been reported (Mouro et al. 1994; Salvignol et al. 1994, 1995). We have also determined thenucleotide sequences of Rh and Rh50 genes in mouseand rat and that of the Rh50 gene for the crab-eating
macaque, and considered their evolutionary relationships (Kitano et al. 1998; see also Matassi et al. 1999).
In this paper, we examined the long-term evolutionof the Rh blood group and related genes throughnewly determined cDNA sequences for Japanese
medaka teleost fish and African clawed frog and857

analyzed the gene duplication time of Rh and Rh50
genes.

Materials and methods
Animals and RNA preparation
Total RNAs were extracted from femora of African clawed frog
(Xenopus laevis) and from whole body of Japanese medaka fish(Oryzias latipes), using ISOGEN (Nippon Gene). Reverse transcription was performed using SuperScript II reverse transcriptase and oligo dT-adaptor primer (Gibco-BRL).
Methods for degenerate PCR and rapid amplification of cDNA
ends
We first performed degenerate PCR for the Rh50 genes. Degenerate primers DP0065 (59-TIGGIYTIGGCTTCCT-39) andDP0077 (59-GCYACIGCYARICCAAAGTA-39) were used for
the Xenopus Rh50 gene, and degenerate primers DP0065(59-TIGGIYTIGGCTTCCT-39) and DP0081 (59-CCACIGCAACWCCTCCTGCIA-39) were used for the Japanese
medaka Rh50 gene. We then performed 59 rapid amplificationof cDNA ends (59RACE) using the 59RACE System for Rapid
Amplification of cDNA Ends version 2.0 (Gibco-BRL).
39RACE was also carried out. To amplify the complete cDNA
sequence, PCR was performed using gene-specific primers.
These primers were designed on both noncoding regions determined by 59 and 39RACE methods. Primers DP0114(59-TCAGTCCCCTGCAGGTCTGAGAT-39)
and
DP0112(59-TGCAGTTGTTGAAATGCCATTTAC-39) were used for
the Xenopus Rh50 gene, and DP0107 (59-CAAGAGTGATC
AAGCATCGTACC-39) and DP0109 (59-GGTCTCCCCAGTG
CAGCTTTAG-39) were used for the Japanese medaka Rh50
gene. PCR was performed in a 20-l reaction containing 0.5±1 lof first-strand cDNA, 1Gene Taq Universal Buffer (Mg2+ free)(Nippon Gene), 1.5 mm MgCl2, 0.2 mm dNTP, 10 pmol of eachprimer (designed on sites of 59 and 39 ends), and 1 unit of
AmpliTaq Gold (PE Biosystems). Amplification was carried out
in a DNA GeneAmp PCR System 9700 (PE Biosystems). PCR
products were purified using MicroSpin Columns S-300 HR(Amersham Pharmacia Biotech). DNA sequencing was performed on PCR products using the BigDye Terminator Cycle
Sequencing Kit and ABI PRISM 377 DNA sequencer (PE Biosystems). A progressive sequencing strategy was carried out withfurther primers to complete the sequence for the coding regionof both strands of the cDNA.
Sequence analyses
CLUSTAL W version 1.6 (Thompson et al. 1994) was used for
multiple alignment. Numbers of amino acid substitutions were
estimated using Kimura©s (1983) method. The dista program of
the ODEN package (Ina 1994) was used to estimate the numberof amino acid substitutions. The PredictProtein server (EMBL)
was used for analyses of transmembrane helix location. The program protml of MOLPHY version 2.2 (Adachi and Hasegawa
1994) was used for the maximum-likelihood analyses.

Results and discussion
Sequence comparison of Rh, Rh50, and related genes
We sequenced Rh50-like genes in Xenopus and Japanese medaka (DDBJ/EMBL/GenBank International

Nucleotide Sequence Database accession numbers
AB036510 and AB036511, respectively). Nucleotide
sequence lengths of these genes are 1275 bp for Xenopus and 1467 bp for Japanese medaka. GC contents of
those genes for Xenopus and Japanese medaka are
45.9 and 48.9%, respectively. These values are similar
to other Rh50 genes (Kitano et al. 1998; Matassi et al.
1999).
Figure 1 shows the multiple alignment of aminoacid sequences of Rh, Rh50, and related genes. Aminoacid sequences of Rh genes for human (Avent et al.
1990; Cherif-Zahar et al. 1990; their amino acidsequences are identical), crab-eating macaque (Salvignol et al. 1994), mouse and rat (Kitano et al. 1998),
and those of Rh50 genes for human (Ridgwell et al.
1992), crab-eating macaque, mouse, and rat (Kitano etal. 1998) were used. We also included the Drosophila
Rh-like gene (AF172639, AF181624, and AF193812;
their amino acid sequences are identical), two Rh-likegenes for Caenorhabditis elegans (Wilson et al. 1994),
and a sponge Rh-like gene (Seack et al. 1997).
The 12 predicted hydrophobic membrane-spanning
regions are surrounded by boxes (Fig. 1). These membrane-spanning regions did not include gaps and are
relatively conserved.
The phylogenetic tree of Rh, Rh50, and related genes
Because membrane-spanning regions did not include
gaps and are relatively conserved, we used only the
222 amino acid sites for membrane-spanning regions(Fig. 1) for tree construction. Figure 2 shows the maximum-likelihood tree for Rh, Rh50, and related genes
using the JTT (Jones et al. 1992) model (the program
protml of MOLPHY version 2.2 was used). The root
was located by assuming the Rh-like gene of sponge
as an outgroup. There are three clusters in this tree:(1) Rh50 genes of mammals and Rh50-like genes of
Xenopus and Japanese medaka, (2) Rh genes of mammals, and (3) genes of Drosophila and C. elegans. This
tree suggests that Xenopus and Japanese medaka
Rh50-like genes are orthologues of other Rh50 genes
and duplication of Rh and Rh50 genes occurred before
the speciation of teleosts and other vertebrates.
We also considered three possible tree topologies,
using the maximum-likelihood method (Fig. 3A±C).
Tree A shows the same topology as Fig. 2. In thiscase, we have to assume that frog Rh and fish Rh
genes were lost (or the genes have not yet been identified). In tree B, duplication of Rh and Rh50 genes
occurred after speciation of fish and other vertebrates,
and we have to assume that the Xenopus Rh gene waslost (or the gene has not yet been identified). In tree
C, duplication of Rh and Rh50 genes occurred after
speciation of amphibians and other vertebrates.
We tried to obtain Rh orthologous genes for Xenopus and Japanese medaka using several degenerateprimers, but could not obtain these cDNA sequences.

858

Fig. 1 Multiple alignment of amino acid sequences of Rh bloodgroup and related genes. Amino acid sequences of Rh genes forhuman (M34015 and X54534; their amino acid sequences are
identical), crab-eating macaque (L37054), mouse (AB015189),
and rat (AB015191), and of Rh50 genes for human (X64594),
crab-eating macaque (AB015467), mouse (AB015192), and rat(AB015194) are shown. Amino acid sequences of Rh-like genes
for Drosophila (AF172639, AF181624, and AF193812; theiramino acid sequences are identical), Caenorhabditis elegans 1(Z74026-B0240.1), C. elegans 2 (U64847-F08F3.3), and sponge(Y12397) are also depicted. Twelve membrane-spanning regions
are boxed

Among the three possible trees in Fig. 3, tree A
had the highest likelihood value. Interestingly, thehuman Rh gene locus is located on Chromosome(Chr) 1p34±p36 (Cherif-Zahar et al. 1991; Ruddle etal. 1972) and the human Rh50 locus is located on Chr
6p11±21.1 (Cherif-Zahar et al. 1996; Ridgwell et al.
1992). These locations are close to two paralogous

regions involving the major histocompatibility complex
in the human genome (Kasahara 1999; Kasahara et al.
1996). Therefore, duplication between Rh and Rh50
genes plausibly occurred by chromosomal or genome
duplication in an ancestral vertebrate.
The branch lengths of Rh genes are much greaterthan those of Rh50 genes, indicating a higher evolutionary rate in the Rh than in the Rh50 gene. This pattern is consistent with the dN result (Kitano et al.
1998). The ratios of Rh-dN and Rh50-dN were 2.0, 1.7,
and 2.0 for human-macaque, mouse-rat, and primaterodent comparisons, respectively. It is interesting thatafter the duplication which produced Rh and Rh50
genes, the Rh gene lineage started to evolve more rapidly than the Rh50 lineage. Kitano and Saitou (1999)
have suggested that some kind of positive selection
occurred on the primate lineage of Rh genes.

859
Fig. 2 The maximum-likelihood tree of Rh blood groupand homologous genes generated using the 12 membranespanning regions. The closed
diamond denotes the duplication event producing Rh andRh50 genes. The open diamond denotes another gene
duplication in the nematodelineage. The root was locatedby assuming the Rh-like protein of sponge as an outgroup

Fig. 3A±C Three possible
tree topologies of Rh bloodgroup and homologous genes.
A likelihood difference fromthe maximum likelihood (ML)by 12 membrane-spanning
regions is shown for each tree.
Values of the maximum-likelihood analysis using all aminoacid sites are shown in parentheses. The program protml of
MOLPHY version 2.2 (Adachi
and Hasegawa 1994) was used

Comparison of the number of amino acid substitutions
We estimated the number of amino acid substitutions(Table 1). The phylogenetic tree of Fig. 2 was usedand number of amino acid substitutions of a single
lineage was estimated applying Ishida and co-workers©
(1995) method. We then estimated the evolutionary
rates of Rh and Rh50 genes using regression throughthe origin. Because the branch length of the Xenopus
Rh50 gene is much shorter than that of other genes,
we did not use it for calibration.
Divergence times between human and macaque,between mouse and rat, between primates androdents, and between tetrapods and fishes wereassumed to be 23.3 (Kumar and Hedges 1998), 30 (Kitano et al., 1999), 112, and 450 (Kumar and Hedges
1998) million years ago, respectively, and they wereused for calibration of the molecular clock. Numbersof amino acid substitutions were thus obtained as
83.87 and 41.24 for Rh and Rh50 genes, respectively(Table 1). If we use these rates, the time of duplication (node indicated by the closed diamond in Fig. 2)

producing Rh and Rh50 genes was estimated to beabout 372 or 514 million years ago from the data for
Rh or Rh50, respectively. The estimate (372 million
years ago) from the data for Rh is lower than thedivergence time between tetrapods and fishes (450
million years ago). This lower estimate suggests thatthe evolutionary rate of Rh genes accelerated after
Table 1 Number of amino acid substitutions and divergencetimes. Node: H-M between human and macaque, M-R between
mouse and rat, P-R between primates and rodents, T-F between
tetrapods and fishes, Rh/Rh50 between Rh and Rh50 genes
Node

Rha
Rh50a
Million years ago
a

H-M

M-R

P-R

T-F

Rh/Rh50

11.93
14.13
23.3

17.40
12.80
30

123.67110.25112±
135.67450

183.87141.24514b

Amino acid substitutions of a single lineage based on the phylogenetic tree in Fig. 2b
Estimated from numbers of amino acid substitutions of Rh50
genes

860

the gene duplication. Therefore, a more plausible timeof duplication is 514 million years ago based on the
Rh50 gene data. This period roughly corresponds to
the Cambrian, before the divergence between jawlessfish and jawed vertebrates lineages.
At least two genome duplications are suggested tohave taken place in a common ancestor of jawed vertebrates (Kasahara 1999; Kasahara et al. 1996). The
first genome duplication might have taken place after
the divergence between jawless fish and other vertebrates. The gene duplication of Rh (1p34±p36) andRh50 (6p11±21.1) genes almost corresponds to thisperiod. If true, another two duplicated Rh/Rh50-likegenes exist (or existed) in jawed vertebrates.
The relationship to ammonium transporter proteins
Because products of Rh and Rh50 genes are predicted
to have 12 transmembrane domains, Rh and Rh50
proteins have been suggested to be related to ammonium transporter proteins (e.g., Marini et al. 1997b).
We BLAST-searched (Altschul et al. 1990) the DDBJ
amino acid sequence database (DAD) for homologous
proteins. Two protein sequences of the human Rh50
gene and the Rh-like gene of sponge were used toquery sequences. Table 2 shows the list of protein
sequences obtained. Seventeen ammonium transporter
proteins were found. No ammonium transporter gene
was found examining the complete genomic sequencesof Haemophilus influenzae and Mycobacterium genitalium, whose natural environment is human tissue (Marini et al. 1997a). Ammonium transporter proteinscarry the ammonium ion (NH4+) into cells (Marini etal. 1994). The ammonium ion is a nitrogen source supporting optimal growth for primitive organisms suchas yeast.
Figure 4 shows the multiple alignment of transmembrane domains for ammonium transporter protein
sequences compared with Rh, Rh50, and related protein sequences. Because the 1st, 2nd, 3rd, and 12th
Table 2 Ammonium transporter proteinspredicted membrane regions did not align well, theseregions were eliminated.
Figure 5 shows the neighbor-joining tree of the Rh
blood group-related genes and ammonium transporter
genes based on the multiple alignment of Fig. 4. Bootstrap values greater than 90% are shown. There areroughly three clusters in this tree; the a group ofammonium transporter genes (amt a group), the bgroup of ammonium transporter genes (amt b group),
and the Rh blood group and related genes (Rh genes
group). Both a and b groups of the ammonium transporter cluster include three kingdoms of life (bacteria,
archaea, and eukaryota). However, the relationshipsof the three kingdoms in both the a and b groups areunclear. Because most bootstrap probabilities onbranches in both a and b groups are not so high, statistical errors may cause these unclear relationships
for the three kingdoms in both a and b groups.
Although the root point of the Rh gene group is not
appropriate (see Fig. 2), members of the Rh genegroup form a cluster.
Marini and co-workers (1997a) and Van Dommelen
and co-workers (1998) created phylogenetic trees forammonium transporter proteins, and those trees also
indicated two major groups, but they were not named.
Therefore, we propose to call these two groups ofammonium transporter genes the a and b groups.
Because most of the ammonium transporter genes are
predicted from genome sequences, however, functional differences between gene products from the aand b groups are not known.
The universal ancestor of all the organisms might
already have had an ammonium transporter gene, inwhich case the gene duplication occurred before thedivergences of the three kingdoms. Therefore, most of
the organisms may have amt a and/or amt b groups.
For example, Arabidopsis thaliana has genes for both
amt a and amt b, while Saccharomyces cerevisiae has
only genes for the amt a group. Because we cannot
define the root point of this tree, defining the divergence pathway of the Rh blood group-related genes is

Accession No.

Organism

Taxon

AE000674
ACJ225126
AF005275
L03216
U82664
D90901AE001036
AE000846
U67574AC003028
X75879
U53338 (C05E11.5)
X95098AF001505
Z72906
Z71418
U40829Aquifex aeolicus
Azorhizobium caulinodans
Azospirillum brasilense
Bacillus subtilis
Escherichia coli
Synechocystis sp.
Archaeoglobus fulgidus
Methanobacterium thermoautotrophicum
Methanococcus jannaschii
Arabidopsis thaliana
Arabidopsis thaliana
Caenorhabditis elegans
Lycopersicon esculentum
Oryza sativa
Saccharomyces cerevisiae (MEP1)
Saccharomyces cerevisiae (MEP2)
Saccharomyces cerevisiae (MEP3)

Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Archaea
Archaea
Archaea
Eukaryota
Eukaryota
Eukaryota
Eukaryota
Eukaryota
Eukaryota
Eukaryota
Eukaryota

861

Fig. 5 The phylogenetic treeof ammonium transporter
genes and the Rh bloodgroup-related genes. Midpoint
rooting was used. Bootstrap
values greater than 90% areshown on each branch. B, A,
and E in parentheses indicate
taxa for bacteria, archaea, andeukaryota, respectively

862
Fig. 4 Multiple alignment of transmembrane domains forammonium transporter proteins compared with Rh, Rh50, andtheir related proteins. This figure was made using BOXSHADE
3.21 (http://www.ch.embnet.org/software/BOX_form.html). Bars
with numbers designate transmembrane domains

difficult. However, the Rh blood group and related
genes appear to exist as essential membrane proteinsin animals and, probably, other eukaryotes. Expressionof Rh and Rh50 genes is restricted to the erythrocyte
in humans and, probably, in other vertebrates, anderythrocytes evolved in the vertebrate lineage. Therefore, the location of expression must have changed
from some other cell type(s) to the erythrocyte membrane before duplication of the Rh and Rh50 genes.
Acknowledgements This study was partially supported by
grants-in-aid for scientific studies from the Ministry of
Education, Science, Sport, and Culture, Japan to N.S.

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