Evolution of RPS4Y
Andrew W. Bergen,*† Marina Pratt,* Patrick T. Mehlman,‡ and David Goldman*
*Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland; †Genetic
Epidemiology Branch, Division of Cancer Genetics and Epidemiology, National Cancer Institute, Rockville, Maryland; and
‡LABS of Virginia, Inc., Yemassee, South Carolina
Sequence variation within RPS4Y, a ribosomal protein gene located in the nonpseudoautosomal region of the Y
chromosome, was used to elucidate the origin of this gene in primates. Complete coding and additional flanking
sequences (949 bp) of the RPS4Y locus were determined in four nonhuman primate species. Phylogenetic reconstruction of RPS4 sequence evolution supports the monophyly of mammalian RPS4 and RPS4Y. Molecular evolutionary rate estimation reveals significantly elevated rates of DNA and protein evolution in RPS4Y compared with
its X-chromosome homologs. These rates enable us to estimate the timing of the transposition of RPS4X to the Y
chromosome (95% confidence interval, 32 MYA–74 MYA), and this estimate was verified by Southern hybridization
analysis of prosimian and simian genomic DNA. These data support a transposition event of ancestral primate
RPS4X to the Y chromosome prior to the divergence of Prosimii.
Introduction
Evolution of genes on the Y chromosome and of
pairs of homologous loci found on the X and Y chromosomes have been intensively investigated for the insights they provide into sex-chromosome mutation rates,
chromosome evolution, and the basis of sex determination and infertility. Estimates of evolutionary substitution rates have generally revealed that loci on the Y
chromosome are evolving approximately twice as fast
as their X-chromosome homologs (e.g., Pamilo and
Bianchi 1993; Shimmin et al. 1993, 1994), supporting
the male-driven mutation model of Miyata et al. (1987).
However, synonymous substitution analysis of 238 autosomal, 33 X-linked and 6 Y-linked rat–mouse homologous loci support a significantly reduced rate of rodent
X-chromosomal sequence evolution compared with rodent autosomes and the rodent Y chromosome where
the rate of Y-chromosomal sequence evolution does not
differ significantly from that of autosomes (McVean and
Hurst 1997). X- and Y-linked homologous sequences
have been used to reconstruct sex chromosome evolution, e.g., the present short- and long-arm pseudoautosomal boundaries and their sequence evolution (Ellis et
al. 1990; Kvaloy et al. 1994). Y-chromosome–linked
loci have also been used to investigate the evolution of
the sex-determination locus, Sry (Whitfield et al. 1993),
where high KA/KS ratios and limited evidence for positive selection in great apes have previously been described (Pamilo and O’Neill 1997).
The ribosomal protein small subunit 4 (RPS4) is a
small (29 Kd, 263 amino acid), basic ribosomal protein.
RPS4 is involved in mRNA binding and is located at
the 40S/60S subunit interface of the small ribosomal
subunit (Nygard and Nika 1982). The sequence, function, and position of RPS4 and other ribosomal proteins
are highly conserved (Lake 1985). The small ribosome
Key words: RPS4Y, RPS4X, Hominidae, relative rates, phylogenetic reconstruction.
Address for correspondence and reprints: Andrew W. Bergen,
12420 Parklawn Drive, Park 5 Bldg., Room 451, Rockville, Maryland
20852.
E-mail: [email protected] or [email protected].
Mol. Biol. Evol. 15(11):1412–1419. 1998
q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1412
subunit is responsible for accurate translation, and the
functional interactions among proteins within this subunit are conserved across prokaryotic and eukaryotic
lineages (Alksne et al. 1993). The highly conserved position of RPS4 within the ribosome suggests that protein–protein interactions in the local environment may
have constrained the structural evolution of RPS4.
In the human, two homologous loci, RPS4X (Xq13)
and RPS4Y (distal Yp, 70 kb, proximal to the pseudoautosomal boundary of the Y chromosome), encode proteins with 92.8% amino acid identity (Fisher et al.
1990). Both human RPS4 genes complement a temperature-sensitive mutant of hamster Rps4 (Watanabe et al.
1993), and each is expressed and incorporated into functional ribosomes (Zinn et al. 1994). Human males therefore contain two different species of ribosome, although
the relative importance of the less highly expressed
RPS4Y gene product, expressed at one-tenth the level of
RPS4X, is unknown (Zinn et al. 1994). No other human
ribosomal protein is known to have a homologous copy
(Fisher et al. 1990). Rodent RPS4 cDNA have been
cloned from mouse, rat, and Chinese and Syrian hamster
species (Devi et al. 1989; Zinn et al. 1991; Watanabe et
al. 1993; M. Ebihara, personal communication). The
chicken contains an S4 homolog that is nearly identical
to RPS4X and that is probably autosomal (Zinn et al.
1994). S4-protein homologs have also been identified
and cloned in yeast (Synetos et al. 1992), fruit fly (Yokokura et al. 1993), and tetrahymena (Palm et al. 1995).
Reverse-transcription polymerase chain reaction (RTPCR) products corresponding to RPS4X have been sequenced from six species representing four orders and
six families of mammals (Omoe and Endo 1996). Amino acid identity of X-linked RPS4 protein among all
vertebrates is $98.5%, suggesting severe functional
constraints on structural evolution (table 1).
The Y-chromosome homolog, RPS4Y, has only
been detected in primates. Subsequent to the cloning of
the RPS4Y cDNA in humans (Fisher et al. 1990), RTPCR sequencing and genomic hybridization provided
evidence for the presence of a Y-specific locus within
Japanese macaques (Macaca fuscata, Mfu) (Omoe and
Endo 1996), and a single exon-to-exon PCR product
Evolution of RPS4Y
1413
Table 1
Nucleotide Distances (31,000) and Nonidentical Amino Acids in the RPS4 Gene Homologs of Primates, Rodents,
Chicken and Fruit Fly
HsaY
HsaY . . . .
Ppn . . . . .
Ptr . . . . .
Ggo . . . . .
Ppy . . . . .
HsaX . . .
Rra . . . . .
Mmu . . . .
Cgrc . . . .
Cgrs . . . .
Gga . . . . .
Dme . . . .
Ppn
5.1
1
1
1
8
19
19
19
19
19
16
70
0
2
9
20
20
20
20
20
17
71
Ptr
7.7
2.5
2
9
20
20
20
20
20
17
71
Ggo
10.2
12.8
15.4
7
20
20
20
20
20
17
71
Ppy
21.9
24.5
27.2
21.9
18
18
18
18
18
17
72
HsaX
208
206
206
202
199
0
0
1
0
4
66
Rra
Mmu
Cgrc
Cgrs
Gga
Dme
234
236
238
236
228
95.7
223
225
221
225
218
97.1
47.4
215
217
213
217
209
88.4
53
48.8
211
213
209
213
202
97.1
68.2
65.3
31.2
215
220
220
220
212
185
186
185
182
177
433
438
441
430
437
414
427
419
434
435
360
0
1
0
4
66
1
0
4
66
1
4
66
4
66
63
NOTE.—Nucleotide distances appear above the diagonal; nonidentical amino acids appear below the diagonal. Taxa and RSP4 homolog abbreviations are as
follows: HsaY, human RPS4Y; Ppn, pygmy chimpanzee (Pan paniscus); Ptr, common chimpanzee (Pan troglodytes); Ggo, gorilla (Gorilla gorilla); Ppy, orangutan
(Pongo pygmaeus); HsaX, human RPS4X; Rra, rat (Rattus rattus); Mmu, mouse (Mus musculus); Cgrc, Chinese hamster (Cricetulus griseus); Cgrs, Syrian hamster
(Cricetulus griseus); Gga, chicken (Gallus gallus); and Dme, fruit fly (Drosophila melanogaster).
provided evidence for RPS4Y in the Hominidae and in
gibbons (Hylobates lar, Hla; Samollow et al. 1996). Genomic hybridization and RT-PCR have failed to demonstrate male-specific bands corresponding to a RPS4Y
locus in all other mammalian orders tested (Omoe and
Endo 1996).
Sequence, RT-PCR, hybridization, and phylogenetic analyses of RPS4 sequences have been interpreted as
indicating the following: (1) RPS4 genes homologous to
RPS4X and RPS4Y existed before the mammalian radiation, and RPS4Y sequences were subsequently lost in
mammalian orders, specifically, in Rodentia, Carnivora,
and Artiodactyla, but not in Primates (Omoe and Endo
1996); and (2) avian RPS4 arose as a result of gene
conversion between ancestral RPS4X and RPS4Y homologs (Zinn et al. 1994). A lack of available full-length
RPS4Y sequences has limited tests of phylogenetic hypotheses regarding the origin of RPS4Y and its subsequent evolution. We determined the sequence of the entire RPS4Y gene in four nonhuman primate species, hybridized RPS4Y to Primate DNA, and performed phylogenetic and molecular evolutionary rate analyses to
resolve the uncertain origin, evolution, and phylogeny
of RPS4Y. We address the following issues: (1) the phylogeny and relative evolutionary rates of RPS4Y and
RPS4X and the possible origin of avian RPS4 by conversion between ancestral RPS4Y and RPS4X homologs,
and (2) the origin and evolution of the RPS4Y protein.
Materials and Methods
For sequencing, the nonhuman primate sample consisted of genomic DNA from two common chimpanzees
(Pan troglodytes, Ptr), two pygmy chimpanzees (Pan
paniscus, Ppn), two gorillas (Gorilla gorilla, Ggo), and
one orangutan (Pongo pygmaeus, Ppy) (a gift from S. J.
O’Brien). Exon-specific PCR primers that contained intronic flanking sequences for the seven exons of the
RPS4Y gene were synthesized (GenBank accession
number L24370; Zinn et al. 1994; L. Brown, personal
communication). 59 universal-tagged (221M13 and
M13REV) primer sequences were used for PCR, and
universal primers were used for direct sequencing of
PCR products as previously described (Bergen et al.
1996). Those exon products refractory to direct sequencing (e.g., exon 2) were amplified with intronic flanking
primers, gel purified, cloned, and sequenced as previously described (Bergen et al. 1996). A total of 949 bp
of RPS4Y sequence, including 792 bases of coding sequence, 94 bases of 59 UTR sequence, and 63 bases of
39 UTR sequence, was determined on both strands via
direct sequencing of amplified RPS4Y exons for each
nonhuman primate (fig. 1). The coding sequence only
was used for analysis in this paper, and these sequences
are available as GenBank accession numbers
AF076991–AF076994, for Ppn, Ptr, Ggo, and Ppy, respectively.
Phylogenetic analysis of RPS4 evolution among
vertebrates was performed using these novel sequences;
human RPS4Y, HsaY (M58459); human RPS4X, HsaX
(M58458); Chinese hamster, D26473 (Cricetulus griseus, Cgrc); Syrian hamster, D11087 (Cricetulus griseus, Cgrs); mouse, M73436 (Mus musculus, Mmu); rat,
X14210 (Rattus rattus, Rra); and chicken, L24368 (Gallus gallus, Gga); with the fruit fly RPS4 sequence,
D16257 (Drosophila melanogaster, Dme) as designated
outgroup taxon (table 1 and fig. 1). As all of the additional sequences were derived from cDNA clones that
included variable amounts of nontranslated sequence,
phylogenetic analyses were performed with coding sequence only (792 bases of sequence). Phylogenetic reconstruction of protein evolution among human RPS4X
and Hominidae RPS4Y sequences was performed with
the translated Hominidae RPS4Y and HsaX coding sequence (fig. 2).
Alignment of the published sequences was performed by inspection of the translated nucleotide sequence. The vertebrate sequences code for a 263–amino
acid protein, while the Drosophila sequence codes for a
261–amino acid protein; thus, the nucleotide sequences
were aligned from translation initiation codon to ter-
1414
Bergen et al.
FIG. 2.—RPS4 amino acid evolution. Most parsimonious phylogram of length 89 steps and consistency index 0.97. Asterisks denote
nonconservative amino acid substitutions. Phylogeny within rodents
(one substitution at Chinese hamster) and between fly and vertebrates
is not shown. Abbreviations of taxa and RPS4 homologs are as described in Materials and Methods.
FIG. 1.—RPS4 phylogeny: (a) Maximum-parsimony cladogram of
length 544 steps and consistency index 0.818. The number of base
changes are depicted above the branches. Bootstrap resampling support
is indicated above the branch, where a single asterisk indicates .90%
support and double asterisks indicate .95% support. Jackknife support
was .98% at all branches, except at the ancestral Gcr node. (b) Maximum-likelihood phylogram. The minimum-evolution phylogram had
an identical topology. Scale 5 0.1 divergence. Abbreviations of taxa
and RPS4 homologs are as described in Materials and Methods.
mination codon, with the only gaps required inserted
into the Drosophila sequence at codon 260 and at codon
263 to make all sequences equal in length and to optimize the nucleotide alignment. Gaps were considered to
be missing data for parsimony reconstruction (gaps are
considered to be missing data by default for maximumlikelihood and minimum-evolution reconstruction in
PAUP). While the mammalian RPS4 sequences analyzed derive from a diverse group of species, among
mammalian RPS4 homologs, divergence is moderate
(table 1), base frequencies (mean frequency of A 5
0.276, mean frequency of C 5 0.219, mean frequency
of G 5 0.258, and mean frequency of T 5 0.247) are
homogeneous (x2 5 4.87, df 5 27, P 5 0.999), and
mean transition/transversion ratios are 2.23 6 0.76 (SD).
The Kimura 2-parameter method (Kimura 1980) was
used to construct distance matrices (table 1). All phylogenetic analyses were performed using the beta-test
version 64 of PAUP 4.0 (Swofford 1998) and MacClade
3.0 (Maddison and Maddison 1992). Resampling was
performed with 1,000 repetitions and the bootstrap (all
792 characters) and jackknife (20% deletion) options in
PAUP 4.0.
Numbers of synonymous and nonsynonymous
changes were estimated by Synonym.exe (Pamilo and
Bianchi 1993). Differences in relative rates of substitution in RPS4Y versus RPS4X and Rps4 lineages were
evaluated by the relative-rate test (Wu and Li 1985) with
parameter values calculated from Synonym.exe output
and with chicken RPS4 as reference outgroup. A paired
t-test with pooled variances was used to determine the
significance of differences between rates. To test for nucleotide substitution rate differences, transversions at
fourfold degenerate sites alone (B4) were used because
of moderate transition substitution saturation at fourfold
degenerate sites (transition/transversion ratio 5 0.67 6
0.058), while to test for protein substitution rate differences, nonsynonymous substitution rates (Ka) were used
(Wu and Li 1985; Pamilo and Bianchi 1993). The standard error of the mean rate ratio between RPS4 homolog
lineages was calculated by the delta technique as in
McVean and Hurst (1997). The transposition time of
RPS4X onto the Y chromosome was estimated by the
Poisson model of Kimura (1969) with the calculated
nonsynonymous rates and with the numbers of amino
acid substitutions observed in the protein phylogeny.
Evolution of RPS4Y
Standard errors are given throughout, either obtained
from Synonym.exe or calculated here.
To evaluate the presence of RPS4X and RPS4Y loci
in various primates, Southern blotting of genomic DNA
(10 mg) from female and male Hsa, Tonkean macaque
(Macaca tonkeana, Mto), owl monkey (Aotus trivirgatus, Atr), and ring-tailed lemur (Lemur catta, Lca) was
performed by overnight digestion with EcoRI (Boehringer Mannheim), electrophoresis for 17 h at 2.4 V/cm in
a 0.7% agarose (BRL) horizontal gel with l HindIII
DNA markers (Research Genetics), and overnight capillary transfer to a nylon membrane (Boehringer Mannheim), followed by baking at 808C for 1 h. The nylon
membrane was prehybridized for 1 h at 558C in 0.5 M
NaHPO4, 7% SDS, 1 mM EDTA, pH 7.2 (Church and
Gilbert 1984) with 10 mg/ml of denatured human Cot-1
DNA (Boehringer Mannheim) and hybridized overnight
at 558C with dCTP 32P random-primer labeled (Feinberg
and Vogelstein 1984) RPS4Y probe at ;6 3 108 CPM/
mg. The RPS4Y probe was a XhoI-HindIII fragment of
pcDEBS4Y (a gift of A.R. Zinn; Watanabe et al. 1993),
containing the entire RPS4Y cDNA from pDP1278
(Fisher et al. 1990) and small amounts (;15 bp) of pBS
MCS sequences (M. Watanabe, personal communication). Following hybridization, filters were washed twice
at high stringency (558C washes at 0.2 3 SSC and 0.1%
SDS) and exposed at 2808C to Kodak XAR film. Approximate molecular weights of hybridizing DNA fragments were calculated by comparison with a l HindIIIsize standard.
Results and Discussion
Reconstruction of RPS4 Phylogeny
Phylogenetic reconstruction of RPS4 vertebrate homolog evolution across 12 species representing primates, rodents, and birds with the criterion of maximum
parsimony yielded two trees of equal length and consistency index (fig. 1). The only topological difference was
the presence or absence of a hamster (Cricetulus griseus) ancestor. Resampling analyses provided marginal
support (51.8% with bootstrap, 50.8% with jackknife)
for the monophyly of a Cgr ancestor but significant support (.90% with bootstrap, .95% with jackknife) for
all other internodes (fig. 1a). Phylogenetic reconstruction with the criteria of minimum evolution and maximum likelihood produced single trees with topology
identical to the maximum-parsimony tree shown (fig.
1b). Resampling with these two latter methods provided
increased bootstrap support for the hamster ancestor and
decreased bootstrap support for the ancestor of gorilla,
chimpanzee, and human, while jackknife support remained above 95%. Phylogenetic reconstruction of vertebrate RPS4 homologs thus supports divergence of
monophyletic avian and mammalian RPS4 sequences
from a common vertebrate ancestor and subsequent divergence of mammalian RPS4 into X- and Y-chromosomal homologs.
It has been suggested that the Gga RPS4 gene arose
through conversion of ancestral RPS4Y and RPS4X homologous sequences. This suggestion has been based on
1415
Table 2
Evolution of RPS4 Exon 4 Amino Acid Residues
SPECIES OR
HOMOLOG
HsaYa . . . .
Ppy . . . . . .
HsaX . . . .
Gga . . . . .
Dme . . . . .
CODON/RESIDUE
93
GAG Glu
GAT Asp
GAC Asp
GAG Glu
GAG Glu
98
102
116
CAT His
CAT His
AAT Asn
CAC His
TTC Phe
GTC Val
GTC Val
ATC Ile
GTG Val
GTC Val
GTG Val
GTG Val
CCT Pro
GCT Ala
GCC Ala
NOTE.—Abbreviations of taxa and RPS4 homologs are as follows: HsaY,
human RPS4Y; Ppy, orangutan (Pongo Pygmaeus); HsaX, human RPS4X; Gga,
chicken (Gallus gallus); and Dme, fruit fly (Drosophila melanogaster).
the findings that the only amino acid differences between human RPS4X and chicken RPS4 (residues 93,
98, 102, and 116) are within a single exon and that three
of the four nonidentical amino acid residues are identical
in chicken RPS4 and human RPS4Y (Zinn et al. 1994).
Inspection of the codon sequences from chicken RPS4,
human RPS4X, and RPS4Y reveals that, at amino acid
residues 93, 98, 102, and 116, human RPS4X has undergone the same number of substitutions (six) as human RPS4Y, if one assumes a RPS4 to RPS4X to RPS4Y
phylogeny (table 2). Parsimony reconstruction of amino
acid evolution identifies two changes at each of these
residues, one substitution occurring between chicken
and ancestral mammalian RPS4 and one substitution occurring between RPS4X and primate RPS4Y; that is,
these four residues are homoplasic (table 2 and fig. 2).
Within the single exon (exon 4) proposed to have undergone conversion, the number of nucleotide differences among the three species does support a closer relationship between Gga and RPS4Y (8 differences) than
between Gga and RPS4X (13 differences). However, for
the entire coding region, each of three phylogenetic reconstruction methods supports mammalian RPS4X and
Hominidae RPS4Y as monophyletic sister taxa paraphyletic to Gga (fig. 1), where Gga is closer to HsaX, not
HsaY (table 1). Nor does phylogenetic reconstruction of
RPS4X and RPS4Y homologs support gene conversion
events after the divergence of mammalian RPS4. Thus,
the suggested conversion of exon 4 of ancestral RPS4X
by ancestral RPS4Y is not supported by evaluation of
sequence evolution and phylogenetic reconstruction.
This phylogenetic reconstruction significantly supports monophyly of mammalian RPS4X and primate
RPS4Y, but the divergence of RPS4X and RPS4Y homologs appears to have taken place prior to the divergence
of primate RPS4X and rodent Rps4 (fig. 1). This was
the interpretation of a neighbor-joining analysis performed on 496 bases of RPS4 homolog sequence from
12 species (including five of the primate and rodent species used here; Omoe and Endo 1996). This interpretation made an assumption that nucleotide substitution
rates within RPS4X and RPS4Y sequence lineages are
equal. This assumption was not tested by Omoe and
Endo (1996) but is tested here by the relative-rate test
with multiple species and transversions at fourfold degenerate sites (a measure of synonymous substitution
rates less affected by evolutionary distance than transi-
1416
Bergen et al.
Table 3
Mammalian RPS4 Synonymous and Nonsynonymous
Nucleotide Substitution Rates
RPS4Y
B4a
Var (B4)
HsaY . . .
Ppn . . . .
Ptr . . . . .
Ggo . . . .
Ppy . . . .
t5
0.5697
0.5655
0.5697
0.5868
0.6047
2.38
0.008766
0.008563
0.008766
0.009498
0.010313
RPS4Y
Kab
Var(Ka)
HsaY . . .
Ppn . . . .
Ptr . . . . .
Ggo . . . .
Ppy . . . .
t5
0.0476
0.0492
0.0492
0.0500
0.0462
7.33
0.000049
0.000049
0.000049
0.000049
0.000049
RPS4X/Rps4
B4
Var(B4)
0.38773
0.42095
0.46641
0.42639
0.48769
0.003794
0.004460
0.005596
0.004616
0.006184
RPS4X/Rps4
Ka
Var(Ka)
HsaX
Rra
Mmu
Cgrc
Cgrs
P 5 0.000041
0.00159
0.0192
0.0209
0.0192
0.0192
0.000016
0.000016
0.000016
0.000016
0.000016
HsaX
Rra
Mmu
Cgrc
Cgrs
P 5 0.022
NOTE.—Taxa and RSP4 homolog abbreviations are as follows: HsaY, human
RPS4Y; Ppn, pygmy chimpanzee (Pan paniscus); Ptr, common chimpanzee (Pan
troglodytes); Ggo, gorilla (Gorilla gorilla); Ppy, orangutan (Pongo pygmaeus);
HsaX, human RPS4Y; Rra, rat (Rattus rattus); Mmu, mouse (Mus musculus);
Cgrc, Chinese hamster (Cricetulus griseus); and Cgrs, Syrian hamster (Cricetulus griseus).
a B 5 transversion rate at fourfold degenerate sites (synonymous substitu4
tion rate).
b K 5 nonsynonymous substitution rate.
a
tions), with chicken RPS4 sequence as a reference outgroup (table 3). Relative-rate estimates indicate that
Hominidae RPS4Y is evolving significantly faster than
are primate RPS4X and rodent Rps4 sequences (P 5
0.022).
RPS4Y appears to be evolving significantly faster
than its X-chromosome homologs, but this difference in
rates may be because of multiple factors. The mean synonymous rate ratio between Y- and X-chromosome
RPS4 lineages (1.32 6 0.14) is less than that previously
described for either primate or rodent ratios of approximately 2 for Y- and X-chromosome homologous loci
(Pamilo and Bianchi 1993; Shimmin et al. 1993;
McVean and Hurst 1997), yet significantly greater than
1.0. The synonymous rate ratio of primate RPS4Y to
rodent Rps4 is 1.27 6 0.13 (P 5 0.041), and the synonymous rate ratio of human RPS4Y to RPS4X is 1.35,
again, both are greater than 1 but are not significantly
different from the combined rate. Thus, these results are
concordant with the observed lower rate of nucleotide
substitution on the X chromosome as compared with the
Y chromosome (McVean and Hurst 1997). These results
are also compatible with the hypothesis that the mutation rate of the Y chromosome in males is higher than
the mutation rate of the X chromosome in females because of the greater number of germline mutations in
the male order, although they are significantly less than
the Y/X ratio, 2.23, reported for the ZF genes (Shimmin
et al. 1993). These synonymous rate estimates suggest
that it is inappropriate to assume equal rates of RPS4
evolution across Y- and X-linked lineages.
Transposition and Evolution of RPS4Y
The RPS4Y sequence determined in Hominidae can
be used to calculate amino acid substitution rates in the
FIG. 3.—Hybridization of RPS4Y to primates. Autoradiogram of
Southern blot of EcoRI-digested female and male Hsa (human), Mto
(Tonkean macaque), Atr (owl monkey), and Lca (Lemur catta) genomic
DNA hybridized with a RPS4Y cDNA probe. Arrowheads indicate
bands referred to in Results and Discussion.
RPS4Y protein and permits estimation of the time of
transposition of the ancestral RPS4 locus onto the Y
chromosome from the X chromosome. Transposition of
sequences from the X chromosome to the Y chromosome, followed by subsequent divergence due to lack of
recombination-mediated gene conversion, is accepted as
a major mechanism of mammalian Y-chromosome evolution (Graves 1995). The identical sequence and conserved physical location of the X-linked homolog in
mouse and man (Fisher et al. 1990; Hamvas et al. 1992;
Zinn et al. 1994) suggests that the transposition time of
the RPS4X gene to the Y chromosome can be estimated,
assuming that the RPS4X protein sequence represents
the ancestral Hominidae RPS4Y sequence. Inspection of
RPS4X homologs and comparison of nonsynonymous
substitution rates reveal that there has been nearly no
protein evolution among mammalian RPS4X homologs
(table 1, Omoe and Endo 1996, data not shown).
Nonsynonymous substitution rates between RPS4X
and primate RPS4Y sequences (0.053 6 0.0025) are 3.8
times the RPS4Y nonsynonymous substitution rates
among orangutan and the other Hominidae (0.014 6
0.0014), corresponding to a time period of 48 MYA (3.8
residues 3 12.5 MYA). This rate ratio reflects, in part,
the conservation of RPS4X. Furthermore, this point estimate with nonsynonymous rates considers sequence
differences only and is not capable of quantitating
homoplasic substitutions.
Phylogeny reconstruction indicates that a mean of
8.125 residues has evolved between orangutan and the
other primate RPS4Y sequences in the 12.5 Myr since
Ppy and the Homininae lineages diverged (fig. 3; Andrews 1992). Thus, one amino acid is diverging in the
Evolution of RPS4Y
less conserved RPS4Y protein every 2.2 Myr–4.7 Myr
(mean 6 2 SE; Kimura 1969). Phylogenetic reconstruction of RPS4Y protein evolution via parsimony reveals
that 16 amino acids have evolved between RPS4X and
the Hominidae RPS4Y ancestor (fig. 3). Assuming constancy of primate substitution rates (Easteal 1991), the
interval from the time of transposition of RPS4 from the
X to the Y chromosome to the radiation of the Hominidae 12.5 MYA is then approximately 36 Myr–75 Myr
(16 residues 3 2.2 Myr–4.7 Myr). However, assuming
that the reported twofold to fourfold hominoid reduction
in mutation rates (Li et al. 1987; Bailey et al. 1991)
results in an overall primate slowdown of perhaps 30%,
the interval from transposition of RPS4X to the Hominidae radiation would correspond to 24 Myr–50 Myr
(16 residues 3 1.5 Myr–3.1 Myr). A smaller reduction
in primate mutation rates would give a larger range, but
some reduction in rates seems to be necessary to be
consistent with the differences in rates observed in primate lineages alone. Averaging the constant and minimally reduced ranges, which is a conservative assumption, corresponds to a transposition date of the ancestral
RPS4X gene from the X chromosome to the Y chromosome of approximately 42 MYA–74 MYA.
This estimate of the divergence date of RPS4Y and
RPS4X indicates that RPS4 transposed from the X to the
Y chromosome after the mammalian radiation event 85
MYA but does not indicate whether RPS4 transposed
before or after the Anthropoidea–Prosimii divergence 55
MYA (Gingerich 1985). In order to place the transposition event into the appropriate era of mammalian evolution, a physical method comparing extant species for
the presence or absence of RPS4Y was used to provide
critical information on the timing of the transposition.
As assessed by both cDNA and genomic hybridization
and by RT-PCR analysis, other mammalian orders do
not demonstrate a Y-linked RPS4 locus (Hamvas et al.
1992; Zinn et al. 1994; Omoe and Endo 1996). However, Southern hybridization analysis of members of
four primate families (Lemuridae, Cebidae, Cercopithecidae, and Hominidae) representing prosimians, New
World and Old World monkeys, and great apes, respectively, in all cases revealed male-associated bands hybridizing to RPS4Y sequence. Male-specific RPS4 restriction fragments identified were of different sizes: approximately 5.0 kb and 6.0 kb in the Hsa male, 2.8 kb
and 4.8 kb in the Mto and Atr males, and 1.2 kb, 2.4
kb, and 16 kb in the Lca male.
Restriction fragments appearing in the male that
also appear in the female should represent the RPS4X
locus, whereas restriction fragments appearing in the
male that do not appear in the female represent either
the RPS4Y locus or a RPS4X restriction fragment length
polymorphism (RFLP). While the possibility of RPS4X
RFLPs potentially complicates the interpretation of
male-associated restriction fragments, here the interpretation of the presence of male-specific restriction fragments in the lemur male is of interest. The striking increase in the number of restriction fragments observed
in the Anthropoidea compared with the Prosimii species
may represent RPS4 pseudogenes that have accumulated
1417
in simian genomes since divergence of the primate suborders. The reduced or absent pseudogenes in the lemur
simplify interpretation of male-specific restriction fragments in the lemur. The male lemur has each of the
restriction fragments present in the female lemur but
also has three additional restriction fragments that total
nearly 20 kb. The presence of these restriction fragments
in the lemur male indicates that the RPS4Y locus was
present in the ancestor of this Prosimii species. Therefore, it is most parsimonious to conclude from molecular
evolutionary rate analyses and from the available RTPCR and molecular hybridization data that the RPS4 locus transposed from the X chromosome to the Y chromosome prior to the divergence of the Prosimii lineage
but subsequent to divergence of the ancestral primate
from the placental mammalian ancestor. The DNA hybridization results reveal that phylogenetic reconstructions of mammalian RPS4 homologs (fig. 2) that are
based on the constant-rate assumption are thus incorrect
in placing the divergence of RPS4X and RPS4Y homologs before the mammalian radiation. The error apparently arises because of the significant differences in both
synonymous and nonsynonymous substitution rates between RPS4X and RPS4Y loci.
This interpretation predicts that all primates will be
found to have a RPS4Y locus, excepting those species
in which RPS4Y may have undergone secondary loss or
evolution into a pseudogene, as has been suggested for
the STS-P locus (Yen et al. 1988). The alternative conclusion, that RPS4 transposed from the X chromosome
to the Y chromosome in the placental mammalian ancestor and was subsequently lost in all other placental
mammalian orders (Omoe and Endo 1996), is less parsimonious, requiring at least four events (one transposition in the placental mammalian ancestor and three
deletions in the ancestors of Rodentia, Artiodactyla, and
Carnivora). Molecular evolutionary rate analysis indicates that this alternative is not likely because the estimated divergence time of Rodentia from other mammalian orders is approximately 80 MYA–100 MYA (Li
et al. 1990). If the sequence of a single transposition
event and single or multiple deletions of RPS4Y necessary to explain the observed distribution of X-chromosome– and Y-chromosome–linked loci is hypothesized
to have happened in the ancestor of non-Rodentia Mammalia, multiple events (minimum two, maximum three)
are required, again rendering this a less likely phylogeny
than the single event proposed here.
Sequence identity comparisons reveal that RPS4Y
has diverged more from its homolog, RPS4X, than has
Hsa RPS4X from its homolog, Rps4, in other species.
There is complete sequence identity between human
RPS4X and mouse, rodent, and Syrian hamster Rps4
proteins but only 92.8% identity between human RPS4Y
and vertebrate RPS4X homologs (table 1). This remarkable degree of mammalian RPS4X sequence conservation (99.92% mean identity, Cgrc varies at one residue,
table 1) is presumably due to severe structural constraints that have acted on this functional ribosomal protein. However, despite RPS4Y’s role as a functional ribosomal protein (Watanabe et al. 1993), it has evolved
1418
Bergen et al.
as if less structurally constrained than RPS4X. Under
the assumption that the primate and rodent RPS4X sequence is identical to the ancestral RPS4X sequence that
transposed to the Y chromosome, phylogenetic reconstruction of mammalian RPS4X and Hominidae RPS4Y
with maximum parsimony shows 16 residues to have
evolved between RPS4X and the Hominidae ancestral
RPS4Y and shows 9 to have evolved among Hominidae
RPS4Y (fig. 3). Both conservative and nonconservative
amino acid substitutions (using side-group classifications of neutral–hydrophobic, neutral–polar, basic, and
acidic) occurred after the origin of RPS4Y by transposition and after the divergence of the Hominidae (fig.
3). The wide disparity in the ratio of conservative to
nonconservative amino acid substitutions between mammalian RPS4X and the Hominidae RPS4Y ancestor (6/
10) and this same ratio among Hominidae RPS4Y (7/2)
suggests that functional constraints may have restricted
further nonconservative substitutions, supporting the
functional role for RPS4Y (Watanabe et al. 1993). However, this is a small sample of amino acid substitutions
upon which to make the case for constrained evolution
due to selection.
The chromosomal locations of RPS4X and RPS4Y
support the general model of Y-chromosome rearrangements proposed to have occurred in primate evolution.
These rearrangements were a pericentric inversion involving illegitimate recombination between two pseudoautosomal boundary-like sequences to create the present Xp–Yp pseudoautosomal boundary, followed by another pericentric rearrangement to reinvert a smaller portion of the Y chromosome (Ellis et al. 1990, 1994;
Fukegawa et al. 1996; Sargent et al. 1996). The chromosomal distribution of several homologous gene pairs
supports the first rearrangement, e.g., on Xp/Yq, STS
(Yen et al. 1988), KAL (Incerti et al. 1992), and PBDX/
XGPY (Weller et al. 1995), and, on Xq/Yp, SOX/SRY
(Graves 1995), while some homologous gene pairs support the later rearrangement, e.g., on Xp/Yp, AMG (Bailey et al. 1992) and PKR (Schiebel et al. 1997). We
propose here that the location and history of the homologous gene pair RPS4X and RPS4Y support and help
to date this general model, in which RPS4Y arose
through a transposition from Xq to Yq prior to the prosimian–simian divergence, then was inverted to distal
Yp in the first pericentric inversion proposed to have
created the short-arm pseudoautosomal boundary after
the prosimian divergence (Fukagawa et al. 1996). The
observed protein evolution at the RPS4Y locus represents the combined effects of differential selection on
RPS4X and RPS4Y proteins and the differences in synonymous mutation rate between the X and Y chromosomes. The rate of sequence evolution of RPS4Y, its
presence in Prosimii and Anthropoidea, and its absence
in every other mammalian order support transposition
of RPS4X to the Y chromosome in early primate evolution and suggest that RPS4Y should be evaluated as
an ancestral locus in future studies of the evolution of
the Y chromosome of Primates (e.g., Glaser et al. 1997).
Acknowledgments
This publication was in partial fulfillment of the
Ph.D. requirements of A.W.B. at the George Washington
University Program in Genetics. We thank Andrew R.
Zinn for the gift of cDNA, Laura Brown for intronic
flanking sequence, and Stephen J. O’Brien for primate
DNA.
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Accepted July 10, 1998