BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 249, 78–85 (1998)
ARTICLE NO. RC989074

Conserved Evolution of the Rh50 Gene Comparedto Its Homologous Rh Blood Group Gene
Takashi Kitano,* Kenta Sumiyama,*,1 Toshihiko Shiroishi,† and Naruya Saitou**Laboratory of Evolutionary Genetics and †Laboratory of Mammalian Genetics,
National Institute of Genetics, Mishima 411-8540, Japan

Received July 3, 1998

clear evidence of positive Darwinian evolution (Kitano and Saitou, unpublished). Therefore, it is interesting if a similar evolutionary pattern also exists
in other mammalian groups.
A protein was obtained together with the Rh geneproduct on immunoprecipitation with anti-Rh antibodies from human, and named as 50 kD glycoprotein (10).
This glycoprotein was considered to form heterotetramer with Rh blood group gene products on erythrocyte membranes (11). The nucleotide sequence of thehuman 50 kD glycoprotein was determined, and its
amino acid sequence was homologous with that of thehuman Rh gene (12). That protein was also predicted
to have the 12 trans-membrane domains which aresimilar to those of the Rh blood group gene product.
There are several names for this gene such as RHAG,
but we call this gene as Rh50 and the Rh blood groupgene as Rh hereafter for simplicity. It has been shownthat the Rhnull regulator and the Rhmod phenotypes aresuppressed by the Rh50 product (13), and a splicing
mutant of this gene was shown to cause an Rhnull phenotype (14). These observations clearly indicate that
the Rh50 gene is essential for expression of Rh antigens
on erythrocytes.
These Rh gene and Rh related gene products seemto play an important role for erythrocytes. In thisstudy, we determined nucleotide sequences for Rh
genes and Rh50 genes of two mouse subspecies and rat
as well as the Rh50 gene for crab-eating macaque, andcompared these evolutionary relationships.

We have sequenced the complete coding region of
the Rh blood group gene for mouse and rat and thatof Rh-related 50 kD glycoprotein (Rh50) for mouse,rat, and crab-eating macaque. Phylogenetic analyses of Rh and Rh50 amino acid sequences indicatethat the Rh50 gene has been evolving about two
times more slowly than the Rh blood group gene inboth primates and rodents. This conservative nature of the Rh50 gene suggests its relative importance to the Rh blood group gene. The time of gene
duplication that produced the Rh and Rh50 genes
was estimated to be about 240-310 million years ago.
We also conducted window analyses of synonymousand nonsynonymous nucleotide substitutions for
those two genes. Some peaks where nonsynonymous
substitutions are higher than synonymous ones
were located on outer membrane regions. This suggests the existence of positive Darwinian selection
on Rh and Rh50 genes through host-parasite interactions. q 1998 Academic Press
Key Words: evolutionary rate; phylogenetic tree; window analysis; positive selection.

The human Rh blood group plays important roles
in transfusion and clinical medicine, including
haemolytic diseases of newborns, autoimmune diseases, and mild haemolytic anemia. Nucleotide sequences of human Rh genes were determined (1-5),
and their products were estimated to have 12 transmembrane domains through hydropathy analysis (2)and immunological studies using an antipeptide antibody (6). Nucleotide sequences of Rh blood group
genes in nonhuman primates were also reported (79). We recently analyzed those published Rh bloodgroup genes of primates, and found a higher nonsynonymous substitutions than synonymous ones, a

MATERIALS AND METHODS
PCR-direct sequencing of cDNA. Total RNAs were extracted
from bone marrow of two mouse (Mus musculus) subspecies (M.
m. domesticus and M. m. brevirostris), rat (Rattus norvegicus), andcrab-eating macaque (Macaca fascicularis), using the AGPC (Acid
Guanidinium-Phenol-Chloroform) method. After DNase reactions,reverse transcription was performed by using AMV (Avian Myeloblastosis Virus) reverse transcriptase and oligo dT-adaptor primerof RNA PCR Kit AMV Ver. 2.1 (TaKaRa). Degenerate PCR was
performed and a partial product was obtained. We then performed

1
Present address: Department of Biology, Yale University, New
Haven, CT 06520-8103.

0006-291X/98 $25.00

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5* RACE (rapid amplification of the 5* cDNA ends) using 5*RACE
System for Rapid Amplification of cDNA Ends version 2.0 (GibcoBRL). PCR was performed in a 20 ml reaction containing 0.5-1 mlof the first-strand cDNA, 1 1 Gene Taq Universal Buffer (Mg2/
free) (Nippon Gene), 1.5 mM MgCl2, 0.2 mM dNTP, 10 pmol ofeach primer (designed on sites of 5* and 3* ends), and 1 units of
AmpliTaq Gold (Perkin-Elmer). Amplification was carried out in
DNA GeneAmp PCR System 2400 (Perkin-Elmer) with the following temperature parameters: 10 min at 957C followed by 40 cyclesof 957C for 30 sec, 657C for 15 sec, and 727C for 1 min. PCR products
were purified using MicroSpin Columns S-300 HR (Pharmacia Biotech) and cloned in the TA cloning vectors pCRII (Invitrogen).
DNA sequencing was performed on double-stranded plasmid DNA
and PCR products using Dye Terminator Cycle Sequencing Kit
and ABI prism 377 DNA sequencer (Perkin-Elmer). A progressive
sequencing strategy was carried out with design of further primers
to complete the sequence for coding region of both strands of the
cDNA.

nucleotides around positions 100-150 (see Fig. 2), andits consensus sequence is AATGCTTCCCAGCAG. Rat
and mouse have 5 and 3 repeats, respectively, while
the two primate species have single repeat. Because
all gaps were multiple of 3, they did not alter codon
frames.
Sequence similarities (both for nucleotide and aminoacid) are shown in Table 1. Nucleotide and amino acidsequence similarities between Rh genes and Rh50
genes are 47-49 and 34-38%, respectively. The GC contents of Rh and Rh50 genes were 53-55 and 45-47%,
respectively (shown on the diagonal of Table 1). These
values were similar to those previously reported (21),and may be related to gene locations on gnomes; theRh gene is located on chromosome 1p34-36 (22-23),
while the Rh50 gene is on chromosome 6p21-qter (12).
We would like to note that similar sequencing results
for mouse and macaque were recently obtained independently by G. Matassi et al. (personal communication).

Sequence analyses. CLUSTAL W version 1.6 (15) was used for
multiple alignments and tree analyses. The neighbor-joining method(16) was used for constructing phylogenetic trees. Numbers of aminoacid substitutions were estimated by using Kimura’s method (17).
The PredictProtein server (EMBL) was used for analyses of transmembrane helix location of these proteins. ODEN package (18) wasused to estimate numbers of synonymous and nonsynonymous substitutions (19), and the WINA program (20) was used for window
analyses.

Evolutionary rates and the phylogenetic tree. We estimated numbers of synonymous (dS) and nonsynonymous (dN) substitutions for Rh and Rh50 genes (Table2). dS and dN values between primates and rodents were
estimated by averaging pairwise values. Numbers ofsynonymous substitutions (dS) were similar between
Rh and Rh50 genes, and they are more or less similar
to those for other genes (24). Branching pattern of the
Rh and Rh50 genes are also compatible with the established mammalian phylogeny. This indicates that we
did orthologous comparison both for Rh and Rh50
genes.
Numbers of nonsynonymous substitutions (dN) areabout two times higher for the Rh gene than for the
Rh50 gene; the ratios of Rh-dN and Rh50-dN are 2.0, 1.7,
and 2.0 for human-macaque, mouse-rat, and primatesrodents comparisons, respectively (we neglected thecomparison of the two mouse subspecies, for standard
errors are so large). This evolutionary conservation of
the Rh50 gene suggests that it may have more important function than the Rh gene. A relatively uniform
ratio of Rh-dN and Rh50-dN for three different levels ofdivergence also suggests that a molecular clock (constancy of evolutionary rate) exists both for Rh and Rh50
genes.
Majority of genes are known to undergo neutral
evolution, and number (dS) of synonymous substitutions are expected to be higher than those (d N) fornonsynonymous substitutions under this situation(17). We compared dS and d N values to see if there is
any unusual pattern deviated from neutrality in Rh
and Rh50 genes. dN of both Rh and Rh50 genes werehigher than d S when human and macaque sequenceswere compared, while the situation is reversed forother comparisons (Table 2). We previously foundthat many branches of a phylogenetic tree of primate

RESULTS AND DISCUSSION
PCR-cloning and sequencing of Rh and Rh50
cDNA coding regions. We sequenced Rh and Rh50
gene cDNAs for two mouse subspecies and rat. Those
newly determined rodent sequences (DDBJ/EMBL/
GenBank international nucleotide sequence database accession numbers are AB015189 - AB015194)
were compared with the human Rh gene (1, 2) andthe human Rh50 gene (12). Figure 1 shows the multiple alignment of nucleotide sequences of Rh genes.
Nucleotide sequence lengths of human, crab-eating
macaque, mouse, and rat are 1254, 1254, 1257, and1269 bp, respectively. Four gaps (3, 15, 3, and 6 nucleotide long) were observed between primate androdent sequences, and the rat Rh gene had extra 12
nucleotides (positions 337-348). Lengths of all gaps
were multiplication of 3 and there is no frame shift.
We also obtained an incomplete sequence for the rat
Rh cDNA which lacks sites 149-661. These sites correspond to exons 2-4 of the human Rh gene, and thisincomplete cDNA were probably produced by a splicing error.
Figure 2 shows the multiple alignment of nucleotide
sequences of Rh50 genes. We obtained a Rh50 gene
cDNA for crab-eating macaque (DDBJ/EMBL/GenBank accession number is AB015467), and it was also
compared. Nucleotide sequence lengths of human,crab-eating macaque, mouse, and rat are 1230, 1287,
1317, and 1353 bp, respectively. The location of thestop codon of the human Rh50 gene is different fromthat of others, and its protein is 19 amino acids shorter
corresponding to this region. There are repeats of 15
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FIG. 1. The multiple alignment of nucleotide sequences of Rh genes. Nucleotide sequences for human RhcE (M34015 or X54534; they
are identical) and for crab-eating macaque (L37054) were also included for comparison. Gaps are denoted by hyphens, and only nucleotides
different from those of the human sequence are shown. MMD, MMB, and CEM denote M. m. domesticus and M. m. brevirostris, and crabeating macaque, respectively.

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FIG. 2. The multiple alignment of nucleotide sequences of Rh50 genes. Human Rh50 (X64594) was also included. Gaps are denoted byhyphens, and only nucleotides different from those of the human sequence are shown. Equal signs surrounded with angled brackets designate
the repeat unit of 15 nucleotides. Abbreviations of species are the same as Fig. 1.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSTABLE 1

Similarities (%) of Rh and Rh50 Nucleotide Sequences (above Diagonal) and Amino Acid Sequences (below Diagonal) and
GC Content (%) of Each Gene (on the Diagonal in Parentheses)

1. Human RhcE
2. CEM Rh
3. MMD Rh
4. MMB Rh
5. Rat Rh
6. Human Rh50
7. CEM Rh50
8. MMD Rh50
9. MMB Rh50
10. Rat Rh50

1

2

3

4

5

6

7

8

9

10

(53.7)
79.1
57.9
58.1
56.7
35.2
35.4
34.4
34.7
33.8

90.4
(52.5)
59.1
59.3
58.6
35.7
37.7
35.4
35.7
35.1

71.4
71.9
(55.2)
98.3
81.6
37.1
37.8
35.0
34.8
34.7

71.3
71.8
99.1
(55.3)
81.8
36.8
37.5
35.0
34.8
34.7

70.8
70.9
88.3
88.3
(54.0)
35.3
36.3
35.5
35.3
34.4

48.6
48.8
48.6
48.5
48.8
(46.7)
88.8
77.0
76.8
75.8

48.5
48.9
48.9
48.8
48.5
94.6
(47.4)
74.3
74.1
73.1

47.3
48.4
47.2
47.1
47.7
80.0
79.6
(45.0)
99.8
88.8

47.4
48.5
47.1
47.0
47.6
79.8
79.4
99.8
(45.0)
89.0

47.3
48.2
46.9
46.8
47.4
79.4
78.6
91.6
91.7
(45.4)

Note. MMD, MMB, and CEM denote M. m. domesticus, M. m. brevirostris, and crab-eating macaque, respectively.

Rh genes showed higher dN than dS (Kitano and Saitou, unpublished), and this is compatible with ahigher d N for human and macaque Rh gene shownin Table 2. It is interesting that the Rh50 gene also
showed a similar evolutionary pattern for primates,
but not for rodents. If the heterotetramer structureof the Rh and Rh50 gene products is correct, it ispossible that this erythrocyte membrane protein
complex is under some kind of positive selection inprimates but not in rodents.
We constructed a multiple alignment of amino acidsequences of Rh, Rh50, and their related genes (alignment not shown). Two genes of C. elegans (25) and an
Rh-like gene of sponge (26) found by database searches
were also included. The 12 predicted hydrophobic membrane-spanning regions did not include gaps and are
relatively conserved. We thus used only the 216 aminoacid sites for membrane-spanning regions for construction of the neighbor-joining tree (Fig. 3). The root was
located by assuming the Rh-like protein of sponge as
an outgroup. There are three clusters in this tree; Rh50
genes of mammals, Rh genes of mammals, and two
genes of C. elegans. The branch lengths of Rh50 genes
is much shorter than those of Rh genes, indicating alower evolutionary rate in the Rh50 gene than in the
Rh gene. This pattern is consistent with the result ofdN in Table 2 where all the coding regions were compared. It is interesting that after the gene duplication(node D in Fig. 3) which produced Rh and Rh50 genes,
the Rh gene lineage started to evolve more rapidly thanthe Rh50 lineage.
Numbers of amino acid substitutions (dA) are also
estimated (Table 3). The phylogenetic tree of Fig. 3
was used and single-lineage dA values were obtained
applying Ishida et al.’s (27) method. Because dA values
for Rh were consistently two - three times higher thanthose for Rh50, a rough molecular clock exists for bothgenes. This is consistent with the result of Table 2.
Therefore, we estimated evolutionary rates of Rh andRh50 genes by using the regression through origin. Divergence times between human and macaque, between
mouse and rat, and between primates and rodents wereassumed to be 23.3, 40.7, and 112 million years (28),and they were used for calibration of the molecular
clock. The rate of amino acid substitutions (per site peryear) were thus obtained as 2.12 1 1009 and 0.94 1
1009 for Rh and Rh50 genes, respectively. If we use
these rates, the time of gene duplication (node D in
Fig. 3) producing Rh and Rh50 genes was estimated to
be about 240 or 310 million years ago from the data
for Rh or Rh50, respectively. This period roughly corresponds to the late Paleozoic where the mammalian lin-

TABLE 2

Numbers of Synonymous (dS) and Nonsynonymous (dN) Substitutions

dS of RhdS of Rh50
dN of RhdN of Rh50

MMD vs MMBa

Human vs macaquea

Mouse vs rata

Primates vs rodentsb

0.013 { 0.007
0.007 { 0.005
0.007 { 0.003
0.001 { 0.001

0.071 { 0.016
0.049 { 0.013
0.115 { 0.011
0.057 { 0.008

0.226 { 0.031
0.200 { 0.028
0.098 { 0.011
0.058 { 0.008

0.595
0.620
0.302
0.153

Note. MMD and MMB designate M. m. domesticus and M. m. brevirostris, respectively.
a
Pairwise values with standard errors.
b
Averages of pairwise values.
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FIG. 3. The phylogenetic tree of mammalian Rh blood group genes and their homologous genes. Amino acid sequences of only membranespanning regions (216 sites) are used. Bootstrap probabilities (%) are shown on interior branches. The root was located by assuming the
Rh-like protein of sponge as an outgroup. Accession numbers and gene IDs for C. elegans 1 and 2 are Z74026-B0240.1 and U64847-F08F3.3,
respectively.

eage started to diverge from an ancestral reptilian lineage.
The Rh and its related genes have probably been
existing as essential membrane proteins in many animal phyla. Because its evolutionary rate is lower thanthat for the Rh protein gene, the Rh50 protein may becloser to the ancestral form before gene duplication of
Rh and Rh50 genes. In this context, it may be interesting to note a similarity between the Rh50 protein andNH4/ transporters (29). However, two coding regionsof C. elegans (C05E11.4 in the DDBJ/EMBL/GenBank
accession number U53338 and M195.3 in Z66498) are
more similar to NH4/ transporters. Therefore, the actual function of Rh and Rh50 gene products remain to
be found.

cleotide substitutions to investigate their possible
correlation with the protein structure (Fig. 4). The
twelve predicted hydrophobic membrane-spanning
regions are shown by black boxes with numbers.
There are several peaks (depicted by arrows) where
nonsynonymous substitutions are higher than synonymous ones on putative outer membrane regions
on primate Rh genes (Fig. 4A). One peak (designatedas long arrows) is observed at the cell surface region
between membrane-spanning regions 3 and 4 in all
four comparisons, and four and two peaks were also
observed for other cell surface regions in primate Rh
and Rh50 comparisons, respectively (indicated byshort arrows with asterisks). Whether an intermembrane region is on the cell surface or in the cytoplasm is based on the currently accepted model ofthe Rh and Rh50 protein structure (2, 30).
We examined Rh blood group gene and its homologous Rh50 genes in the present study. Blood groupantigens may play a key role in pathogenesis of diseases (31), and there is a possibility of positive Darwinian selection caused by interaction between organisms(host mammals and parasites such as bacteria). In fact,
Saitou and Yamamoto (32) and Kitano and Saitou (unpublished) found evidences of positive selection in the
ABO and Rh blood group genes of primates, respectively. Comparison of synonymous and nonsynonymous
substitutions for the Rh50 gene also revealed a possibility of existence of positive selection for this gene inprimates. Because primates showed more clear sign ofpositive selection than rodents both for Rh and Rh50
genes, it is possible that the pattern of host-parasite
interaction is different between primates and rodents.

Window analyses of synonymous and nonsynonymous substitutions. We performed window analyses for synonymous (dS) and nonsynonymous (dN) nu-

TABLE 3

Numbers (dA) of Amino Acid Substitutions
and Divergence Times
Human/ Mouse/ Primates/
macaqueratrodents Rh/Rh50

Diverging nodeSingle lineage dA of Rha
Single lineage dA of Rh50a
Divergence time (MYA)

0.120
0.042
23.3b

0.072
0.029
40.7b

0.228
0.104
112b

0.663
0.225
240–310caBased on phylogenetic tree of Fig. 3.
Taken from Ref. 28.
c
Estimated from dA values.
b

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FIG. 4. Window analyses for synonymous (dS: gray lines) and nonsynonymous (dN: black lines) nucleotide substitutions for Rh genes
between human and crab-eating macaque (A), for Rh genes between M. m. domesticus and rat (B), for Rh50 genes between human andcrab-eating macaque (C), and for Rh50 genes between M. m. domesticus and rat (D). The 12 predicted hydrophobic membrane-spanning
regions are shown by black boxes. Horizontal axes indicate numbers of nucleotide sites. Arrows indicate peaks of dN higher than dS, andarrows with asterisks are in the putative cell surface regions. Arrows in the same cell surface region for all the A-D comparisons are drawn
longer than others.
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ACKNOWLEDGMENTS
We thank Dr. Y. Ikehara for preparation of the crab-eating macaque tissues and Dr. T. Sagai for preparation of the mouse tissues.
This study was partially supported by grants-in-aid for scientificstudies from the Ministry of Education, Science, Sport, and Culture,
Japan, to N. S.

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