Download Evolution of RH Genes in Hominoids: Characterization of a Gorilla

Evolution of RH Genes in Hominoids:
Characterization of a Gorilla RHCE-like Gene
A. Blancher and P.-A. Apoil
The human RH locus is responsible for the expression of the Rh blood group antigens. It consists of two closely linked genes, RHD and RHCE, that exhibit 92%
similarity between coding regions. These observations suggest that they are derived from a relatively recent duplication event. Previously a study of nonhuman
primate RH-like genes demonstrated that ancestral RH gene duplication occurred
in the common ancestor of man, chimpanzees and gorillas. By amplification of
intron 3 and intron 4 of gorilla RH-like genes, we have now shown that, like man,
gorillas possess two types of RH intron 3 (RHCE intron 3 being 289 bp longer than
the RHD intron 3) and two types of intron 4 (RHCE intron 4 being 654 bp longer
than the RHD intron 4). Here we report the characterization of a cDNA encoded by
a gorilla RH-like gene which possesses introns 3 and 4 of the RHCE type. A comparison of this gorilla RHCE-like coding sequence with previously characterized
human and ape cDNA sequences suggests that RH genes experienced complex
recombination events after duplication in the common ancestor of humans, chimpanzees and gorillas.
From the Laboratoire d’Immunogénétique Moléculaire,
Université Paul Sabatier, Pavillon Charles Lefebyre,
Hôpital Purpan, Toulouse, France. This work was supported by funds from MESR (contrat Jeune équipe
1966) and from Agence Française du Sang (contract
65001731231). We wish to thank Dr. Francis Roubinet
for critically reviewing this manuscript and for helpful
discussions and advice. We are indebted to Stéphanie
Despiau for her very efficient laboratory assistance. All
experiments described in this article were performed
in accordance with French laws and regulations currently in force. We thank Alejandro Rooney and Masatoshi Nei for helpful discussion and critical review. The
gorilla RHCE-like coding sequence was submitted to
GenBank/EMBL. We are waiting for the accession number. Address correspondence to Antoine Blancher, Laboratoire d’Immunologie, CHU (Centre Hospitalier et
Universitaire) de Toulouse, Hôpital Purpan, 31059 Toulouse CEDEX, France, or e-mail: blancher@mail.
easynet.fr. This paper was delivered at a symposium
entitled ‘‘Genetic Diversity and Evolution’’ sponsored
by the American Genetic Association at the Pennsylvania State University, University Park, PA, USA, June
12–13, 1999.
2000 The American Genetic Association 91:205–210
The human Rh system encompasses five
main antigens—D, C, c, E, and e—that are
present on red blood cells ( Issitt and Anstee 1998). The term ‘‘Rhesus antigen’’ was
introduced by Landsteiner and Wiener,
who found that rabbits (and later, guinea
pigs) immunized with red blood cells
(RBCs) from a rhesus monkey produced
antibodies which agglutinated 85% of Caucasian blood samples ( Landsteiner and
Wiener 1940, 1941). The antibodies were
called anti-Rhesus (anti-Rh), and the two
human groups were defined as Rh positive
and Rh negative. Before the discovery of
the rhesus factor it was suspected that
some cases of grave hemolytic anemia in
newborn babies resulted from an unspecified immunologic incompatibility between the mother and the fetus ( Levine
and Stetson 1939). Several observations
made following the discovery of the Rh
factor firmly established that the Rh factor
was at the root of unexplained reactions
that occurred during transfusion of blood
that was of the same ABO, MNS and P
types as the recipient, and that it was the
cause of hemolytic disease in newborns
( Levine et al. 1941; Wiener and Peters
1940). Because the animal anti-Rh antibodies required extensive absorption before
being able to produce faint Rh-specific agglutination, the use of animal anti-Rh an-
tibodies was abandoned and the definition
of the Rh antigen was based on the use of
human anti-Rh antibodies. Another problem was that it was impossible to demonstrate the presence of the Rh antigen on
macaque red cells by tests with human
anti-Rh reagents. Twenty years after Landsteiner and Wiener’s experiments, Levine
et al. (1961) characterized in the serum of
a guinea pig immunized with human RBCs
a fraction of antibodies (called anti-LW in
honor of Landsteiner and Wiener), which
identified an antigen present on human
Rh-positive and Rh-negative and rhesus
monkey red cells ( Levine et al. 1961,
1963). The antigen LW was clearly distinct
from the Rh antigen, and later it was demonstrated that the LW gene lies on human
chromosome 19 (Sistonen 1984), while the
RH gene is localized on the short arm of
human chromosome 1 (Chérif-Zahar et al.
1991; Marsh et al. 1974). Although the two
genes are distinct, the Rh and LW proteins
are part of a molecular complex called the
Rh complex at the surface of red cells
(Cartron et al. 1998). In conclusion, if the
term ‘‘Rh’’ was coined by Landsteiner and
Wiener because of the source of antigens
(the rhesus monkey) they used to obtain
anti-Rh in rabbits, it is highly probable
that, in fact, they produced anti-LW antibodies. Despite that, the term Rh has con-
205
Table 1. Primer sequences
Name of primers
Sequences
References
Ex.1-dir
Ex10.rev
Ex.4-dir
Ex.5-rev
Int.3-dir
Int.3-rev
RB46-dir
intron4.RHCE-rev
Gor.CE-dir
GCCTGCACAGAGACGGACACAGG
CAACAGCCAAATGAGGAAACTTCC
CGATACCCAGTTTGTCTGCCATGC
TTGGGGTGAGCCAAGGATGAC(C/A)C
(A/G)GGATTACAAGCAAGCATCACC
CACGCAC(C/T)TCACTGATTCCTACTTC
TGGCAAGAACCTGGACCTTGACTTT
CCACCCTTGTTCCTTCACTCCTGG
AAAGGGGGATAAAGGTCAGAG
Apoil and Blancher
Apoil and Blancher
Apoil and Blancher
Apoil and Blancher
Apoil and Blancher
Apoil and Blancher
Matassi et al. 1997
This study
This study
tinued to be used for the most important
human blood group system after ABO for
clinical transfusion.
Cross-reactivity of human anti-Rh reagents with nonhuman primate RBCs indicated that apes, contrary to rhesus monkey, possess counterparts of human Rh
antigens (Masouredis et al. 1967; MoorJankowski et al. 1973). More recently, the
use of monoclonal antibodies confirmed
that some chimpanzees and gorillas express antigens that share epitopes (the
term epitope designate the portion of an
antigen which reacts with the antibody
site of a given antibody) with the human
D antigen ( Blancher et al. 1992a,b; Socha
and Ruffié 1990) and that chimpanzees, gorillas, and gibbons express monomorphic
c-like antigens (Socha and Ruffié 1983).
Chimpanzee and gorilla counterparts of
human antigen D are antigens Rc and Dgor,
respectively ( Blancher et al. 1992a,b;
Roubinet et al. 1993). The expression of
antigens Rc and Dgor was shown to depend
on chimpanzee and gorilla RH-like genes
(Apoil et al. 1999; Salvignol et al. 1993,
1994). Although Rh-like antigen expression
appears restricted to apes, Rh-like polypeptides are present at the surface of
RBCs of rhesus monkey as well as several
nonprimate mammalian taxa (e.g., cat,
cow, rat; Saboori et al. 1989). Moreover,
Rh-like polypeptides were also found in
proteins extracted from RBC membranes
of both Old World monkeys (OWM), New
World monkeys ( NWM), lemur, mouse,
rat, and dog by immunoblotting techniques (Apoil and Blancher 1999; Mouro et
al. 1994; Salvignol et al. 1995).
The human RH locus consists of two
closely linked genes, RHD, responsible for
the expression of the antigen D, and RHCE,
which encodes proteins carrying antigens
C or c and E or e. RHD is present or absent
depending on the RH haplotype (Colin et
al. 1991), and RHCE displays four common
alleles (ce, Ce, cE, CE) responsible for the
expression of the two antithetical (allelic)
series of antigens C or c and E or e (Mouro
et al. 1993). Moreover, it was demonstrat-
206 The Journal of Heredity 2000:91(3)
1999
1999
2000
2000
2000
2000
ed that the four main alleles of the RHCE
gene were derived, by intergenic exchanges and interallele recombinations, from a
few ancestor alleles (Carritt et al. 1997).
Southern blot studies have shown that
chimpanzees and gorillas possess at least
three and two RH-like genes per haploid
genome, respectively, whereas orangutan,
gibbon, OWM, and NWM possess only one
RH-like gene per haploid genome ( Blancher et al. 1992a; Blancher and Socha 1997).
Thus it was inferred that the duplication
event that produced the human RHCE and
RHD genes occurred in the common ancestor of humans, chimpanzees, and gorillas.
In man, intron 3 and intron 4 in the
RHCE gene are longer by 289 bp and 654
bp, respectively, than those of the RHD
gene (Arce et al. 1993; Matassi et al. 1997).
By PCR it was shown that chimpanzees
and gorillas have two types of intron 3
(RHCE-like and RHD-like) homologous in
length with those of human RHD and RHCE
genes, respectively, and two types of intron 4 (RHCE-like and RHD-like) homologous in length with those of human RHD
and RHCE genes, respectively (Apoil et al.
1999; Apoil and Blancher 2000; Westhoff
and Wylie 1996). Moreover, sequence
studies showed that chimpanzees and gorillas have two types of RHD intron 4,
which differ by the absence (type 1) or
presence (type 2) of a 12-mer repeat
(Apoil and Blancher 2000). Apparently
only the RHD type 1 intron exists in humans, because the type 2 intron 4 cannot
be detected by PCR experiments (Apoil
and Blancher 2000). Existence of the two
types of introns in chimpanzees and gorillas suggests that the gene with type 2 intron was lost in the human lineage after
the split from chimpanzees (Apoil and
Blancher 2000).
The Rh-like cDNA sequences of three
chimpanzees and one gorilla (Mabeke)
were shown to be closer to the human
RHD coding sequences than to the RHCE
coding sequences (Salvignol et al. 1995).
However, RHCE-like genes, which possess
Figure 1. Length polymorphism of intron 3 and 4 regions in gorilla RH-like genes. Black rectangles show
the DNA regions present in the introns of the RHCE
type and absent from introns of the RHD type. (A)
Length differences between gorilla RHCE and RHD
types introns 3 (289 bp) and between gorilla RHCE and
RHD types introns 4 (654 bp). (B) A fragment of 1.95
kb amplified from gorilla Kessala genomic DNA. It contains introns 3 and intron 4 of the RHCE type. (C) Primer Gor.CE-dir used for amplication of gorilla RHCE-like
cDNA from exon 4 to exon 10.
intron 3 and 4 of the RHCE type may exist
in chimpanzees and gorillas. Nevertheless,
the existence of functional RHCE-like
genes in chimpanzees and gorillas has to
be confirmed by detecting RHCE-like transcripts. Here we report the results of characterization of a cDNA encoded by a gorilla RH-like gene, which possess introns 3
and 4 of the RHCE type.
Materials and Methods
Data Collection
Fresh blood samples of gorillas were obtained from CIRMF (Centre International
de Recherche Médicale de Franceville), Franceville, Gabon. Frozen blood samples
were from LEMSIP ( Laboratory for Experimental Medicine and Surgery in Primates,
New York Medical Center, Tuxedo, NY).
The intron 4 and intron 3 regions of gorilla
RH-like genes were studied by PCR amplification using two pairs of oligonucleotide
primers described in Apoil et al. (1999)
and Apoil and Blancher (2000). The sequences of primers used here are given in
Table 1. From these preliminary studies,
we opted to select DNA samples from a
gorilla ( Kessala) that has introns 3 and 4
of the RHCE type. Long PCR amplification
from intron 3 (primer RB.46-dir) to intron
4 (primer intron 4.RHCE-rev) was carried
out with the genomic DNA from this gorilla
( Figure 1B). The length of the amplified
fragment was identical to that obtained
from the genomic DNA of a human control
possessing only the RHCE gene (i.e., an
RhD-negative Caucasian). The exon 4 region of the gorilla RHCE-like amplified fragment was sequenced on both strands using automated protocols ( Figure 1B).
The mRNA extracted from the peripheral white blood cells of the previously
mentioned gorilla was submitted to reverse transcription PCR (RT-PCR). The re-
sultant cDNA product was first amplified
from exon 1 to 10 (primers Ex.1-dir and
Ex.10-rev) using cDNAs obtained from total RNA extracted from peripheral blood of
gorilla Kessala as templates. The resulting
amplified fragments were used for a second seminested PCR amplification (from
exon 4 to 10) using primers Gor.CE-dir and
Ex.10-rev. The fragment obtained by the
seminested PCR was sequenced (see Figure 1C). In order to enhance the specificity
of the PCR reaction; a mismatch was introduced into the sequence of primer Gor.CEdir at position 6 from the 5⬘ end. Amplified
fragments were separated by size with
agarose-gel electrophoresis and were purified on columns (Qiagen, Hilden, Germany). Following purification, PCR fragments
were sequenced on a 373A automated
DNA sequencer (PE Applied Biosystems,
Foster City, CA).
DNA Sequence Analysis
An alignment of nucleotide sequences was
conducted by taking into account the deduced amino acid sequence alignment of
the RH-like genes. The alignment was done
by using the computer program CLUSTALW version 1.7 ( Thompson et al. 1994),
with subsequent corrections made after
visual inspection. Phylogenetic analysis
was carried out using the computer program MEGA version 1.03 ( Kumar et al.
1993). Pairwise distances between nucleotide sequences were calculated by using
Kimura’s (1980) two-parameter distances
or the uncorrected p distance. Distances
between amino acid sequences were calculated using a gamma distance with a
shape parameter a ⫽ 2. Phylogenetic trees
were constructed by the neighbor-joining
method (Saitou and Nei 1987) with the
pairwise deletion option. Statistical reliability was assessed using the bootstrap
method ( Felsenstein 1985) with 500 replicates.
Results
Characterization of a Gorilla Gene
Which Possesses Intron 3 and 4 of the
RHCE Type
Figure 1A shows the length polymorphism
in gorilla introns 3 and 4 (Apoil et al.
1999). In the case of intron 4, all gorillas
tested so far (15 unrelated animals) had
an intron 4 sequence similar in length (0.5
kb) to the human RHD intron 4. The second type of intron was similar in length to
the human RHCE intron 4 (1.25 kb), but
was detected by PCR amplification in only
5 of 15 gorillas. In the case of intron 3, 7
of 15 gorillas showed two different PCR
length fragments. One was similar to the
human RHD gene (290 bp) and the other
one was similar to the human RHCE gene
(580 bp). In the remaining eight gorilla
samples, only RHCE-like intron 3 was detected.
To confirm the presence of an RHCE-like
gene in gorillas, we tried to amplify a long
genomic fragment which encompassed
both introns 3 and 4 of this putative gene.
From gorilla Kessala, which was known to
have introns 3 and 4 of the RHCE type, a
DNA fragment approximately 2 kb was amplified using an intron 3 primer (RB46-dir)
and a primer specific to the RHCE intron
4 ( Intron4.RHCE-rev) ( Figure 1B). The
length of this gorilla amplified fragment
was equivalent to those obtained by amplification of human DNA control samples
from individuals having only the RHCE
gene. By means of primers RB46-dir and
Intron4.RHCE-rev, this DNA fragment was
not obtained from a DNA sample of a different gorilla, which did not have intron 4
of the RHCE type. Direct sequencing of the
gorilla 2 kb amplified fragment confirmed
the presence of RHCE-specific elements in
intron 3 and intron 4 (data not shown).
The sequence of exon 4 of these fragments
( hereafter named Gor RHCE exon 4) was
closer to human RHCE exon 4 rather than
to human RHD exon 4 ( Figure 2 and Table
2). Moreover, the Gor RHCE exon 4 sequence differs significantly from all other
gorilla cDNA sequences previously characterized (Salvignol et al. 1995).
Characterization of Gorilla RHCElike cDNA
From the gorilla Gor RHCE exon 4 sequence, we designed a primer (Gor.CE-dir)
for allele-specific PCR amplification (see
Material and Methods; Table 1 and Figure
1C). cDNA obtained from total RNA extracted from peripheral blood of gorilla
Kessala was first amplified from exon 1 to
10 (primers Ex.1-dir and Ex.10-rev). The
resulting amplified fragments were used
for a second seminested PCR (from exon
4 to 10) using primers Gor.CE-dir and
Ex.10-rev. The fragment obtained by the
seminested PCR was sequenced (see Figure 1C). The cDNA region which corresponds to exon 4 (33 bp) was identical to
the genomic Gor RHCE exon 4. The cDNA
region that corresponds to the Gor RHCE
exon 5 sequence differed from the genomic exon 5 sequence obtained in another
study by only one position (Apoil et al.
1999). We concluded from these compari-
sons that the amplified fragment corresponded to the mRNA encoded by RHCE
gene detected in the genome of Kessala.
All our attempts to amplify the RHCE-like
cDNA of gorilla Kessala from exon 1 to
exon 4 failed.
A coding sequence was deduced from
the Kessala genomic exon 4 and from Kessala’s RHCE-like cDNA. This gorilla RHCElike coding sequence (exon 4 to 10) was
aligned to human and primate Rh cDNA
sequences ( Figure 2). Gorilla RHCE-like
cDNA differs from human RHCE cDNA (cE
allele) by 34 nucleotides and from human
RHD cDNA by 35 nucleotides ( Table 2).
However, the phylogenetic tree shown in
Figure 2 suggests that the gorilla RHCE-like
coding sequence is closer to that of human RHCE (cE allele) than to that of human RHD ( Figure 3), although the bootstrap value was very low (34%). The
phylogenetic trees constructed for exons
4 and 5 confirmed this result (data not
shown). For other coding regions, the gorilla RHCE-like cDNA sequence clustered
either with human counterparts [RHD and
RHCE (cE and Ce alleles)] and chimpanzee
Patr 211 sequences (exon 6), or with human RHD cDNA and the gorilla cDNA sequences Gor IC and Gor ID (exon 7), or
with the two gorilla cDNA sequences Gor
IC and Gor ID (exons 8, 9, and 10).
The gorilla RHCE-like coding region was
translated into an amino acid sequence
and compared with the sequences of other primates ( Figure 4). The number of amino acid differences between the gorilla
RHCE-like polypeptide and the human RhD
and RhcE polypeptides were 29 and 25, respectively ( Table 2). Differences between
gorilla RHCE-like polypeptide and two other gorilla Rh-like polypeptides were almost
identical (24 and 25, respectively) ( Table
2). The topology of the phylogenetic tree
obtained from the alignment shown in Figure 4 was identical to the tree reconstructed from nucleotide sequences given in Figure 3.
Discussion
We have shown here that a gorilla gene is
similar to the human RHCE gene with respect to introns 3 and 4. A gorilla cDNA
amplified fragment that is encoded by this
gorilla RHCE-like gene was partially sequenced. Although it is not possible to determine if RHCE-like encoded polypeptides
are expressed at the surface of red blood
cells on the basis of this study, one might
still speculate on the relationship between
gorilla RHCE-like encoded protein and the
Blancher and Apoil • Genes Evolution in Primates 207
Table 2. Number of total and nonsynonymous
nucleotide substitutions observed when the
Gor.CE-like cDNA is compared to human and
gorilla RH sequences
Analyzed
region
RhcE
Exon 4a
Exon 5
Exon 6
Exon 7
Exons 8–10
Exons 4–10
6
7
4
13
4
34
RhD
Gor.IC
Gor.ID
(6)
9 (9) 11 (9)
8
(6)
5 (5)
7 (6)
6
6 (4)
7 (3)
8
(2)
9
(8) 12 (8)
5 (3)
(3)
3 (3)
5 (4)
0
(25) 35 (29) 35 (25) 31
(8)
(6)
(5)
(5)
(0)
(2.4)
Nb
121
165
137
133
180
736
The numbers in parentheses correspond to nonsynonymous substitutions. The lowest numbers of differences are underlined.
a
Only 121 positions were available in exon 4 for comparison.
b
Number of nucleotide positions analyzed.
Figure 2. Gorilla RHCE-like coding sequence in comparison with human and other primate sequences. Nucleotide
positions are numbered according to the RHCE cDNA (⫹1 taken as the first nucleotide of the ATG initiator codon).
The nucleotide sequence of human RHCE (cE allele) cDNA is used as a reference, with a point (.) for nucleotide
identity, a dash (-) for nucleotide deletion, and a question mark (?) when the position is unknown. Only variable
positions are shown. Exon boundaries are those of the human RHCE gene (Chérif-Zahar et al. 1994). The references
of Rh and Rh-like cDNA sequences presented in this figure are human RhD (Homo sapiens): L08429 (Arce et al.
1993); human RhcE and RhCe (Chérif-Zahar et al. 1990, Mouro et al. 1993); Chimp. ⫽ chimpanzee (Pan troglodytes);
gorilla (Gorilla gorilla); cynomolgus (Macaca fascicularis) (Salvignol et al. 1995); baboon (Papio papio) (Apoil et al.,
2000); rhesus monkey (Macaca mulatta) (Mouro et al. 1994); capuchin monkey (Cebus apella) (this study, accession
number AF101479).
Rh-like antigens of gorilla. The single gorilla Rh-like antigen that can be studied is
called Dgor and is detected on red blood
cells by means of certain human anti-D
monoclonal antibodies (Roubinet et al.
1993). The reactivity of one of these human monoclonal anti-D antibodies, LOR15C9, was extensively studied with large
panels of human RhD antigenic variants
(Apoil et al. 1997). It was deduced from
the results that the reactivity of LOR-15C9
208 The Journal of Heredity 2000:91(3)
depended mainly on the sixth external
loop of the Rh polypeptide, and more precisely on a motif of three amino acids
(Asp350-Gly353-Ala354; Apoil et al. 1997). This
was confirmed by tests with erythroleukemia cells transfected with human RHCE
cDNA mutagenized at codons 350, 353,
and 354 ( His350- → Asp, Trp353- → Gly,
Asn354- → Ala) ( Liu et al. 1999). Rh polypeptides that are recognized by the LOR15C9 antibody are expressed on the mem-
branes of these transfected cells (Smythe,
JS personal communication). Moreover,
the mutation Asp350- → His in the human
DIVa variant is sufficient to abolish reactivity with LOR-15C9 (Apoil et al. 1997; Rouillac et al. 1995). Of interest, the gorilla
RHCE-like polypeptide shows the motif
His350, Glu353, Ala354, which is equivalent to
human DIVa. These data suggest that the
gorilla RHCE-like polypeptide is not responsible for the expression of the Dgor antigen in gorilla. In fact, Kessala was Dgor
negative (Apoil et al. 1997). By contrast,
an animal that does not have the RHCElike gene (Mabeke) is Dgor positive and expresses one Rh polypeptide (Gor ID) that
shows the RhD characteristic motif Asp350Gly353-Ala354 ( Figure 4).
The RHCE-like cDNA exhibited a sequence suggestive of recombination between gorilla RHCE-like and RHD-like
genes. Indeed, the gorilla RHCE-like coding
sequence appears to be composite: RHCElike in exons 4 and 6, slightly closer to
RHD-like in exon 5, and closer to other gorilla sequences than to human RH sequences from exon 8 to exon 10 (see Table
2). These observations suggest that the
Figure 3. Neighbor-joining tree of RH and RH-like
coding sequences. Evolutionary distances were calculated by Kimura’s two-parameter method on the basis
of the nucleotide alignment ( Figure 2). Bootstrap values are based on 500 replications. The percentages of
occurrence of each branching are reported on the tree.
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Figure 4. Amino acids alignment of predicted Rh and Rh-like polypeptides. The human RhcE polypeptide is used
as reference, with a point (.) for amino acid identity and a dash (-) for a deletion. Only variable positions are
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gorilla RHCE-like gene might be a product
of a series of complex recombination
events between two gorilla ancestor
genes: one resembling the human RHD
gene and another that was closer to human RHCE. If this is the case, it is possible
that the common ancestor of man and gorilla already had two different RH genes:
the RHD ancestor and the RHCE ancestor.
In this case, humans, chimpanzees, and
gorillas would have RH genes of two clearly differentiated RHD and RHCE types.
This is demonstrated only in a region encompassing introns 3 and 4. The noncoding regions of nonhuman RH-like genes
outside intron 3 and 4 have not yet been
characterized. Moreover, the analysis of
coding sequences does not allow us to determine whether actual RH genes of humans, chimpanzees, and gorillas are closer to the RHD ancestor or to the RHCE
ancestor genes (Apoil and Blancher 2000;
Salvignol et al. 1995). One might evoke another hypothesis in which the differentiation of the resulting genes occurred inde-
pendently in humans, chimpanzees, and
gorillas after the duplication of the ancestor gene, and some parts of the genes
were subsequently subject to homogenization by intergenic exchanges. This type
of evolution is compatible with the mosaic
aspect of the gorilla RHCE-like cDNA,
which is closer to human RHCE in some
exons and closer to human RHD or gorilla
sequences in other exons. However, in humans the RHD and RHCE genes remain differentiated (along 417 codons, 41 nucleotide substitutions in the coding parts 35
being nonsynonymous) despite intergenic
exchanges which are now well documented ( Huang 1997). In gorilla the difference
between the RHCE-like and other gorilla
RH-like genes is similar to that observed
in man (along 245 codons, 33 or 35 nucleotidic substitutions, 24 or 25 being nonsynonymous; see Table 2). This suggests
that the maintenance of such differences
between the two types of genes in humans
and in gorillas could have been favored by
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Corresponding Editor: Masatoshi Nei