articles

DNA sequence and comparative analysisof chimpanzee chromosome 22
The International Chimpanzee Chromosome 22 Consortium**A list of authors and their affiliations appears at the end of the paper...........................................................................................................................................................................................................................

Human–chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition ofunique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report
the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with thehuman counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition tonearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of
the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level.
Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting
different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their
biological consequences seem more complex than originally hypothesized.
To understand the genetic basis of the unique features of humans, anumber of pilot studies comparing the human and chimpanzee
genomes have been conducted1–5. Estimates of nucleotide substitution rates of aligned sequences range from 1.23% by bacterial
artificial chromosome (BAC) end sequencing3 to about 2% by
molecular analysis1,6–8, whereas the overall sequence difference wasestimated to be approximately 5% by taking regions of insertions ordeletions (indels) into account9. Chromosomal rearrangements
including duplications, translocations and transpositions havealso been identified10,11. However, owing to technological limitations there is not an integrated picture of the dynamic changesof the genome, thus a gold standard is required to evaluate theoverall consequence of these genetic changes on human evolution.
To address these issues and to be able to detect molecular
blueprints that have shaped the two genomes, we have conducteda human–chimpanzee whole-chromosome comparison at thenucleotide sequence level on human chromosome 21 (HSA21)
and its orthologue chimpanzee chromosome 22 (PTR22). HSA21
is one of the most well characterized human chromosomes7,12–14 andserves as a representative of the human genome by having characteristic features such as uneven distribution of GþC content with ahigh correlation to gene density, and repetitive/duplicated structures, allowing for detailed long-range comparative studies with
PTR22. Moreover, molecular analysis of HSA21 and its genes is ofcentral medical interest because of trisomy 21, the most common
genetic cause of mental retardation in the human population. One
case of trisomy 22 in chimpanzee has been reported, with phenotypic features similar to human Down’s syndrome15. Therefore, ouranalysis of these chromosomes should reveal dynamic changes that
may reflect general evolutionary events occurring throughout thehuman genome.

Mapping, sequencing and overview of PTR22
We used three different BAC libraries prepared from genomic DNA
originating from three male chimpanzees (Pan troglodytes).
Sequence coverage of the euchromatic portion of the long arm ofchromosome 22 (PTR22q) is estimated to be 98.6% (33.3 megabases (Mb)). Accuracy was calculated as 99.9983% from theoverlapping clone sequences and $99.9981% on the basis ofPhrap scores16. Altogether, these efforts enabled us to produce asequence with the highest possible accuracy to be used forreliable comparative analysis (see Supplementary Information for
details).
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The overall structural features of PTR22q are almost the same as
those of HSA21q. The GþC content of these chromosomes isaround 41% (Table 1). The corresponding regions between
HSA21q and PTR22q, where the extra regions (see Methods) areexcluded, show a roughly 400-kilobase (kb) or 1.2% difference insize, with HSA21q being larger than PTR22q. The difference ismainly due to interspersed repeats (ISRs) and simple repeats,
representing 63.2% (53.7% and 9.5%, respectively) of the regions
corresponding to the gaps in PTR22q. The pericentromeric copy of
a 200-kb region found duplicated in HSA21q is missing in PTR22q,
as reported previously17 . We also detected human-specificsequences that are neither repetitive nor low complexity and are
unique in the nr data set of NCBI (http://www.ncbi.nih.gov/). For
example, a 1,245-base-pair (bp) insertion found in the first intron of

Table 1 Statistics on HSA21q and PTR22q
Genome characteristic

HSA21q

PTR22q.............................................................................................................................................................................

Size (bp)*
Unaligned sites†
Sequencing gaps
Clone gaps‡
Estimated total clone gap sizeGþC content (%)
CG dinucleotides
CpG islands
Nucleotide diversity (%)
Repeats

33,127,944
25,24214
373,10840.94
361,259
950
0.072
Bp

32,799,845
101,70922
2
74,311
41.01
358,450885
0.14

HSA21qNumberBp

PTR22qNumber.............................................................................................................................................................................

SINEs
Young Alu elements§
LINEs
Young L1 elementsk
LTRs
Transposons
RNAs{
Satellites
OthersTotal

3,649,153
21,557
5,853,821
82,4933,621,501
949,215
8,830
19,32730,452
14,132,299
42.7%

15,137
75
8,737
48
7,282
3,363
100
21
38

3,614,825
2,606
5,736,911
78,6573,550,807
945,129
8,722
14,773
34,776
13,905,943
42.4%

15,048
10
8,673
55
7,180
3,350
99
18
43

.............................................................................................................................................................................
* Size of the contig data after the site where the first base of the PTR22q contig is aligned.
† Regions extended into HSA21q clone gaps and subtelomeric unmatched regions.
‡ Excluding pericentromeric and subtelomeric gaps.
§ AluYa5, AluYa8, AluYb8 and AluYb9.
k L1Hs and L1PA2.
{ Small nuclear RNA, small cytoplasmic RNA, 5S ribosomal RNA, transfer RNA, 7SL RNA and othersmall RNA genes.

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PFKL in HSA21q was confirmed to be human-specific and evenlocus-specific by searching for similar sequences in the nr database.
Four expressed sequence tag (EST)/complementary DNA (cDNA)
sequences (BQ711940, BQ706616 and AA453553 on the PFKL
strand and AJ003358 on the complementary strand) are mapped
specifically onto this region.
We did not observe significant correlations between the frequencies of base substitution and indels. Two large indel hotspots were
found at around 9.5–11.5 Mb and 16.5–17.5 Mb from the centromere, as previously suggested3,14 (Fig. 1b). In addition, we foundlarge human insertions/chimpanzee deletions in the first introns of
the NCAM2 (,10 kb) and GRIK1 (,4 kb) genes, which are both
related to neural functions.
One of the largest structural changes identified is a 54-kb region
located 11.4 Mb from the centromere in HSA21q but that is absent
in PTR22q. This region is flanked by HSAT5 satellite repeats andconsists of 164 fragments from 64 different long terminal repeat(LTR) elements with inversions of small portions at even intervals,
suggesting rearrangement driven by interspersed repetitiveelements. The most distal 25,242-bp region of HSA21q ending
with a TTAGGG telomeric repeat did not align to the last 80,879 bp
of PTR22q, which instead shares high similarity to human chromosomes 2, 9 and 10. This suggests that the subtelomeric region of
PTR22q might be larger than that of HSA21q. The pericentromericregions of these chromosomes have complex structures: those of
PTR22q show a high degree of similarity to human acrocentricchromosomes 13, 15, and 18 (submetacentric only in humans but
acrocentric in all great apes) as well as HSA21 (data not shown).

is shown in Fig. 1a. The frequency of base substitution is distributed
around 1.44% along the chromosome, except for elevated regions in
the pericentromeric and subtelomeric regions. The most conserved
region was at about the 12.5-Mb region (0.87%, over 100 kb),
corresponding to the distal boundary region of the gene desert12.
Notably, no protein-coding genes have been identified in this highly
conserved region. The correlation between the GþC content andthe base substitution rate increases along the chromosome, and is
especially high in the last 5 Mb of the telomeric region of PTR22q(data not shown). The frequency of base substitution in repetitive
sequences also tends to vary, increasing from CR1/long interspersed
element (LINE) (divergence, 1.13%; GþC percentage, 39.0%) andL1/LINE repeats (1.38%; 35.6%) to Alu/short interspersed element(SINE) (1.81%; 51.7%), ERVK/LTR (1.88%; 44.6%), CpG islands(2.26%; 65.1%) and simple repeats (4.06%; 44.8%), as discussed
previously19. The CG dinucleotide frequencies are significantly(P , 0.01) different between PTR22q and HSA21q (Table 1).

Repetitive elements

The overall nucleotide substitution level in aligned regions between
PTR22q and HSA21q is about 1.44% (excluding indels), which issignificantly higher than the calculation based on high-quality BAC
end sequence data (1.23%)3. Similar trends have also been observedfor HSA21q based on smaller comparisons with the chimpanzee
genome18.
The distribution of base substitution rate along the chromosome

As described above, HSA21q is about 1.2% longer in size than
PTR22q. This difference can mostly be explained by the fact thatseveral subfamilies of transposable elements, such as L1Hs (11versus 2), MER83B (11 versus 0), AluYa5 (23 versus 3) and
AluYb8 (37 versus 2), are more common in human than inchimpanzee20–23. Five LTR subfamilies (LTR/ERV1) are more abundant in HSA21q. All MER4A1-int and MER83B-int elements are
specific to HSA21q and are clustered in limited regions (positions
21,173,756–21,180,021 bp and 40,651,714–40,657,562 bp, respectively) with overlapping tandem repeats.
According to the sequence alignments, single copies of L1Hs and
AluYa5 and two copies of AluYb8 are found in both HSA21q and
PTR22q, indicating their presence in the genome of the lastcommon ancestor. All of the seven AluYb9 elements found in
HSA21q and the one in PTR22q are lineage-specific, suggestingthat these elements have been integrated after speciation. Although
the AluYa8 subfamily is thought to be a recent derivative of AluYa5(ref. 24), we found a few AluYa8 units in both species.

Figure 1 Overview of the differences between HSA21q and PTR22q. a, Nucleotide
divergence level between HSA21q and PTR22q (bin size ¼ 100 kb) is shown in black andGþC content (%) is in red. Dashed black and red lines indicate average values fordivergence level and GþC content, respectively. Abscissa origin is the centromere–
euchromatin boundary position in HSA21q (11,000,001 bp) and the scale is inmegabases. b, In silico D-loop (a computer simulation of a hypothetical hybridization
experiment using two chromosomal DNAs). The top half represents HSA21q and thebottom half represents PTR22q. Each hypothetical loop-out (regions of DNA that do not
hybridize each other) is shown as a vertical line. The y axis indicates size of loop-outs (kb).
Positions of experimentally verified lineage-specific insertions (blue) and deletions (red)are shown in five lanes along the chromosomes. The lanes, from inside to outside,
represent the content of the indels: Alu elements, LINEs, LTRs, other ISRs (Other) andnon-ISRs. The scale bar indicates the physical position in megabases relative to thetelomere of the short arm (outside track) and to the centromere (inside track).

Base substitutions

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articles
Lineage-specific insertions and deletions
Through alignment of the high-quality chromosomal sequences of
HSA21q and PTR22q we identified about 68,000 indels in total.
Greater than 99% of the indels are shorter than 300 bp, but there is aclear abundance of those around 300 bp in size (Fig. 2). These sites
are probably produced either through human insertions/chimpanzeedeletions or vice versa. Thus the precise identification of thesemolecular events in the two genomes is essential to understand theprocesses underlying human and chimpanzee evolution. For thispurpose, we tested 567 indels larger than 300 bp using DNA samples
from five human, five chimpanzee, one gorilla and two orang-utan
individuals by polymerase chain reaction (PCR) amplification
using the same primer sets to classify in which lineage these indels
arose (see Methods and Supplementary Information). We compared the size of the successfully amplified DNA fragments from 219
indels, of which 193 showed lineage-specific changes in size. Thus, wewere able to distinguish insertion from deletion events independently
in human and chimpanzee lineages, and to estimate the originalstate of these regions in the genome of the last common ancestor.
We then classified the indels based on their contents (Fig. 1b).
Insertions were mostly produced by the integration of Alu and L1
elements, whereas deletions were not related to particular repetitive
structures except in a few cases. We observed different distributionsof newly integrated Alu elements between HSA21q and PTR22q:
56% of new Alu elements in HSA21q are inserted in the half of thechromosome with high GþC content, whereas 70% in PTR22q are
in the half with low GþC content; new LINEs are more frequent in
the half with low GþC content of both chromosomes.
The plots of human insertions and chimpanzee insertions show
different multimodal curves (Fig. 3). On the basis of the positions of
the insertion sites on HSA21q and PTR22q, we found that most (41and 13, respectively) of the insertions (300–350 bp in length) weremembers of the AluY family in both chromosomes. In contrast, onlya smaller number of insertions, mostly L1 and LTR elements, were
found in the 370–1,000-bp size range. Notably, human and chimpanzee deletion plots form a similar linear line, suggesting a
relationship between logarithmic size of deletions and the cumulative frequency in both species (Fig. 3).
We also identified integration of L1PA2 elements after human–
chimpanzee speciation, indicating that L1PA2 has been active inboth human and chimpanzee lineages, although the activity seems
to be lower in the human lineage. Two L1PA2 elements reside indifferent strands but overlap in a single inserted region in PTR22q,
suggesting a single L1PA2 integration event followed by an inversion
event within the same region. We also found that some insertions in
PTR22q lie within Alu elements (mostly AluSx) on the same strand.

Figure 2 Size distribution of indels. All of the indels are calculated as insertions either in
HSA21q (blue) or PTR22q (red). The first two bins are off the scale.
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Unlike the insertions, deletions do not correspond exactly to any
ISR elements, indicating that deletion events are independent of
ISRs. However, one of the deleted regions in HSA21q corresponds
perfectly to a single AluYelement in PTR22q, and a deleted region in
PTR22q corresponds almost perfectly to a single AluYb8 element in
HSA21q. In the former case, there are two identical 10-mer
segments around the deleted AluY element, and in the latter case,
the AluYb8 element is embedded within a single AluSx element at asite in the 14-bp A-rich region in the middle of the Alu element,
generating 14–15-bp poly-A/T stretches around AluYb8. Thus, thedeletion of these elements may also have been generated byhomologous recombination between these relatively short identicalor similar flanking segments.
Calculations from the indels in the 300–5,000-bp range indicatethat both chromosomes have undergone a net loss in size since
speciation despite frequent insertion events: HSA21q has gained
32 kb but lost 39 kb, whereas PTR22q has gained 25 kb and lost
53 kb. This suggests that the ancestral chromosome was larger thanboth HSA21q and PTR22q, and that PTR22q has suffered more
losses than HSA21q since speciation. The large indels (.5 kb)
detected in the sequences, which were experimentally confirmed,
are found in the pericentromeric, 10 Mb, 17 Mb and 29 Mb regions.
HSA21q has more indels greater than 10 kb than PTR22q.
With the knowledge of which Alu family element was inserted
after speciation, we carried out an evolutionary analysis of the AluY
families that have been inserted into HSA21q and PTR22q. A
neighbour-joining analysis revealed that such AluY elements canbe largely separated into chimpanzee and human groups andsuggesting contribution from a few active elements (Fig. 4). Taken
as a whole, these results indicate that the expansion of particularelements was repeated several times during the course of evolution.
Humans seem to have experienced such expansions more frequently
and more recently than chimpanzees. If we could determine theoldest expansion event through genomic comparison, we might beable to identify whether such an Alu burst was the driving force for
speciation between the two species from the common ancestor.
Amplification of Alu elements during the evolution of primates andalteration of gene functions through the insertion of repetitiveelements has been discussed in many previous studies25–34. However,further wide-ranging analyses comparing the chimpanzee and otherprimate genomes is necessary to clarify these points.

Chimpanzee and human single nucleotide polymorphisms
Single nucleotide polymorphisms (SNPs) provide important clues
for detecting ancestral and mutant alleles within the human

Figure 3 Size-dependency of indel frequency. Cumulative counts of experimentally
determined lineage-specific insertions and deletions. The x axis represents the log-scaled
size of indels (bp), whereas the y axis represents the cumulative counts of lineage-specificinsertions and deletions.

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population. The chimpanzee genomic sequence is the best resource
for inferring the ancestral allele of any human SNP locus. We thus
unambiguously reconstructed 19,985 ancestral states from 21,435
human SNP sites in HSA21q7.
Transitional changes are more frequent than transversions, as
expected. Among transitions, G!A and C!T changes (19.6% and20.3%, respectively)35 are more frequent than A!G and T!Cchanges (14.0% and 15.1%, respectively). This substitution patternis compatible with the fact that the GþC content of the humangenome is lower than 50%. We conducted the same analysis for
5,781 chimpanzee SNPs obtained from overlapping BAC sequences(Supplementary Table 9A, B), and found that both A!G and T!Ctransitions (12.6% and 13.2%, respectively) are slightly lower thanthose for human.
Because SNPs have been created relatively recently during humanand chimpanzee evolution (approximately 0.5–1.0 million yearsago), nucleotide substitution patterns predicted from SNP data may
be slightly different than those based on current GþC content (thatis, 41% for both human and chimpanzee). As the estimatedequilibrium GþC content for the human genome is 0.422, versus
0.405 for chimpanzee, modern humans seem to have undergone aslight increase in GþC content compared with their ancestors,

Figure 4 Evolutionary relationships among young AluY elements. Neighbour-joining treeof all AluY families that have been inserted into HSA21q (red circles) or PTR22q (bluetriangles) after speciation. Bootstrap proportions greater than 50% are indicated at thecorresponding branches (1,000 replicates). The scale indicates the evolutionary distanceof 0.01.
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whereas chimpanzees have undergone no clear change, although theequilibrium values for humans and chimpanzees are not far fromthe current GþC contents (Supplementary Table 9C, D).
We then conducted an H test36 (Supplementary Table 10), anddetected 18 10-kb DNA regions as candidates of positive selection.
Three known genes, KCNE1, DSCR2 and B3GALT5, were located inthese regions. KCNE1 and B3GALT5 were also identified as relatively
rapidly evolving genes through analysis of the ratio of the numberof amino acid substitutions per site to silent substitutions per site
(K A/K S), as discussed below (see Supplementary Table 6).

Gene catalogue and characterization of coding sequences
We have annotated 284 protein-coding genes and 98 pseudogenes
for HSA21q12 (http://chr21.molgen.mpg.de), and 272 genes and 89
pseudogenes for PTR22q (the comparative gene catalogue of
HSA21q and PTR22q, including pseudogenes, is given in Supplementary Table 3). We lacked information for six genes and 11
pseudogenes on HSA21q located partly or completely in sequencing
gaps of PTR22q, and one pseudogene, OR7E92P, was absent in
PTR22q (Supplementary Table 3). All of the conserved pseudogenes
were matched in size between human and chimpanzee, except for
KRTAP21P1, which is non-processed in HSA21q but processedin PTR22q.
Six HSA21q genes displaying hallmarks of retrogenes (see ref. 37
for review) were not found in PTR22q and were probably inserted
during human evolution (or less likely, deleted during chimpanzee
evolution). These are H2BFS, a member of the histone family S, andfive members of the keratin-associated protein (KAP) gene clusterin 21q22.1. H2BFS was not found in the syntenic region of mouse
chromosome 16, suggesting that it was inserted in HSA21q. The
KAP gene cluster is too divergent in mouse to establish a conclusivecomparison. Three intronless open reading frames (ORFs) havebeen inserted in chimpanzee PTR22q (or deleted in HSA21q). These
are HNRPA1LK1, a heterogeneous nuclear ribonucleoprotein,
RPLP1LK1, a ribosomal protein, and FAM28ALK1, a gene of
unknown function; all three ORFs are absent in the mouse syntenic
region. All but one of the ORFs found in either human insertions/chimpanzee deletions or human deletions/chimpanzee insertions
are intronless and probably represent retrogenes.
We found a ribosomal protein pseudogene (RPL13AP) on
HSA21q with an intact ORF (RPL13ALK1) in PTR22q, allowing
for possible functionality. Four HSA21q coding sequences(C21orf81 and three intronless genes: C21orf115, C21orf104 andC21orf19) have interrupted ORFs in PTR22q, precluding theirfunctionality, and these were annotated as pseudogenes in chimpanzee (Supplementary Table 3).
All other HSA21q genes are potentially active in PTR22q, eventhough several genes show human-specific transcriptional isoforms
due to alteration or inactivation of some of the transcript isoforms
in chimpanzee (see below). The minimum nucleotide sequence
identity is 83% (KRTAP6-3). Of the 272 annotated chimpanzee
genes, we compared the human and chimpanzee coding sequencesin 231 genes for which we could define a non-ambiguous ORF in
both species. We omitted for that comparison the 41 entries of thechimpanzee catalogue for which we had no ORF in one of the two
species (for example, ambiguous ORFs associated to short ESTs
containing mostly untranslated regions (UTRs)) and genes corresponding to pseudogenes in the other species. Among the 231 genes
associated to a canonical ORF, 179 show a coding sequence ofidentical length in human and chimpanzee and exhibit similar
intron–exon boundaries. For those 179 genes, the average nucleotide and amino acid identity in the coding region is 99.29% and99.18%, respectively. Of these, 39 genes show an identical aminoacid sequence between human and chimpanzee, including seven inwhich the nucleotide sequence of the coding region is also identical(Supplementary Table 3). Examples of biological processes that are
perfectly conserved involve transcriptional regulators (RUNX1,©2004 Nature Publishing Group

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PKNOX1, ERG, GABPA, U2AF1), metabolic enzymes (ATP5O,
CBR1, PFKL, CRYZL1, SOD1, ABCG1), gene products associatedwith signal transduction generally showing patterned expression
during development (GRIK1, CXADR, DSCR5, PCP4, DSCAM,DYRK1A, S100B, C21orf4) and gene products involved in protein
folding and degradation (SMT3H1, UBE2G2)38. A total of 140 ofthese 179 genes show amino acid replacements, but no grossstructural changes are expected.
In contrast, 47 PTR22q genes show significant structural changes
affecting at least one of their transcript isoforms. Fifteen genes haveindels within their coding region yet retain frame consistency in all
but one case (TCP10L) (Supplementary Table 4). Marked changes
are observed in PCNT2, a component of the filamentous matrix ofthe centrosome initiating the nucleation of spindle microtubules,
and in TCP10L, a t-complex protein. The third exon of PCNT2 isshorter in chimpanzee owing to a deletion of 195 bp corresponding
to five highly similar copies of a 13-mer repeating unit. Human
PCNT2 has seven repeats, the orthologous chimpanzee gene has
only two whereas the mouse has none, suggesting that the repeats
were inserted during primate evolution. TCP10L has a deletion of 17
nucleotides within the fourth exon in chimpanzee. This generates atranscript that uses the last 16 nucleotides of exon 4 and the adjacent
23 nucleotides that are intronic in human, the gene product ofwhich is predicted to have a short frame shift in the middle of theprotein (Supplementary Table 4). Thirty-two genes show changes
modifying either the first ATG or the stop codon in at least one of
their associated transcripts (Supplementary Table 5).
Five chimpanzee genes could not be classified because they
displayed structural changes caused by indels (SH3BGR, SYNJ1,
C21orf96 and TMPRSS3) or a substitution in the ATG codon(C21orf18). These changes correspond to polymorphisms inhuman and may not be specific to chimpanzee.
Our data suggest that indels within coding regions represent oneof the major mechanisms generating protein diversity and shaping
higher primate species. We observed an additional level of functional diversity generated through the occurrence of multiple
alternative transcripts for a single gene, some of the isoforms
being structurally modified or not functional in chimpanzee. We
are currently lacking cDNA information to define precisely the
structure of these genes but we provide here a framework allowing
for experimental verification of the transcript isoforms.
Taken together, gross structural changes affecting gene products
are far more common than previously estimated (20.3% of the
PTR22 proteins, as listed in Supplementary Tables 4 and 5). In
addition, 87 genes in the catalogue show mutations in at least one of
the splice sites. However, in all cases the modified sequence still fits
the consensus sequence and is therefore expected to be functional.
More subtle effects, such as changes in transcript stability orprocessing, cannot be ruled out.

K A/K S analysis
The neutrality of sequence differences found between orthologous
pairs of human and chimpanzee genes can be assessed using K A andK S values (K A/K S ø 1 indicates neutral evolution; K A/K S . 1
indicates positive selection, and K A/K S , 1 indicates negative
selection; that is, purifying selection). K A, K S and other related
values were calculated for 231 genes on PTR22q, with 10% of thegenes having a K A/K S ratio .1, the highest value being 3.37 for thehuman hair keratin-associated protein 23-1 gene (KRTAP23-1; data
are summarized in Supplementary Table 6), although these values
were not statistically confirmed (Supplementary Fig. 1).
Relatively rapidly evolving genes may be estimated from K A,
K A þ K S , or just nucleotide divergence values. Three KAP
genes, KCNE1 (human cardiac delayed rectifier potassium channel
protein), TCP10L (t-complex protein 10A-2), B3GALT5 (UDPGal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5),
IGSF5 (immunoglobulin superfamily-5 like) and several other386

genes are found in this category (Supplementary Table 6). Genes
showing statistically significant low K A/K S values are thought to be
evolving under purifying selection; for example, C21orf113, PFKL,
AIRE, ITGB2, TMPRSS3 and AGPAT3. PFKL has a K A/K S ratio of 0,
and is likely to be under strong purifying selection over the entire
gene region.
Recently, Clarke et al.39 reported a similar analysis against 7,645
chimpanzee gene sequences including 70 PTR22q gene sequences,in which KRTAP23-1 was not included. Furthermore, we founddiscrepancies with a previous analysis reporting K A/K S values forsome of the HSA21q genes40. One explanation is that the previous
work used only PCR-amplifiable exons for the analysis.

Comparative gene expression analysis
Using Affymetrix HG U95 arrays, we compared the gene expression
profiles of HSA21q genes between humans and chimpanzees in two
tissues: 202 genes in brain (cingulate cortex, HG U95Av2, B, C, D
and E) and 96 genes in liver (HG U95Av2). We detected 60 genes
expressed in brain (this study) and 40 in liver41 in at least one
species. Of these, 9 in the brain and 12 in the liver showed asignificant change in expression level between humans and chimpanzees in the range of a 1.5–10-fold difference (Supplementary Table
7). Overall, the proportion of genes showing changes in expression
level on HSA21 was not significantly different from the rest of the
genome (liver: x2 ¼ 1.87, P ¼ 0.172; brain: x2 ¼ 1.12, P ¼ 0.290)41.
We explored whether the sequence divergence in the different
gene regions can predict how a gene is differently expressed between
the two species using a Mann–Whitney U-test. The data show that
there is a trend for regions with a high divergence in the 5 0 UTR todiffer in messenger RNA levels between humans and chimpanzees(N ¼ 18, 47; P ¼ 0.043). Notably, genes in which the sequence
divergence of an associated CpG islands is high are also more likely
to have changed their expression (N ¼ 13, 39; P ¼ 0.046). However,as CpG islands often stretch into the first exon, the correlations of
CpG island and 5 0 UTR divergence with expression changes are notindependent. In contrast, we found no significant association
between the divergence of non-degenerate sites, 3 0 UTRs, intergenic
and intronic regions and variation in expression levels (Supplementary Table 8). It has been proposed that 5 0 UTRs in humans might
have been under positive selection42, possibly due to their involvement in the regulation of gene expression levels.
Some of the genes displaying significant changes in protein
sequence or differences in expression between human and chimpanzee might be correlated with physiological or disease susceptibility differences exhibited between the two species43. For instance,
IFNAR2, IFNGR2, CXADR, ITSN1 and CRYZL1 are directly or
indirectly involved in the immune response against various pathogens. SH3BGR is strongly expressed in the developing heart, C21orf2
is expressed in the peripheral nervous system, SYNJ1 and ANKRD3
are signalling molecules acting in early brain development,
MCM3AP is associated with cell cycle progression, ETS2 is atranscription factor essential for embryonic development and
COL18A1 is a collagen gene that is mutated in human Knobloch
syndrome associated with encephalocele44.

Promoter analysis
We analysed the upstream region of genes that showed significant
expression changes in liver and brain between human and chimpanzee, as well as the upstream region of each corresponding mousegene. Computational analysis of the transcription-factor-bindingsites within the 1-kb upstream region of each gene is summarized in
Supplementary Table 11. Transcription-factor-binding sites werecompared in the three species, and those specific to either human or
chimpanzee were found in most genes. All of the specific transcription-factor-binding sites were caused by base substitutions in either
human or chimpanzee, but these may not clearly account for the
expression changes observed in this study. To assess precisely the©2004 Nature Publishing Group

NATURE | VOL 429 | 27 MAY 2004 | www.nature.com/naturearticles
expression changes, further analysis of promoter regions as well asother factors, such as enhancers and suppressors located outside thepromoter region and the effects of 5 0 and 3 0 UTRs on mRNAstability, are needed.

against the complete nr-db, and cDNA matches were retained as potential PTR22-specificgenes. Gene orthology with mouse, rat, zebrafish, pufferfish and Ciona was calculated
from the HSA21 genes, and conceptual translations and multiple alignments wereconstructed using CLUSTALW. Detailed information is provided in the Supplementary
Information.

Conclusion

Comparative gene expression analysis

This study shows a chromosome-wide comparison between humanand chimpanzee based on high-quality sequences, and provides thefirst integrated picture of genetic changes during human evolution.
The data presented here suggest that the biological consequences
due to the genetic differences are much more complicated thanpreviously speculated. We hope that our work offers a framework
for the design of future studies to examine differences between thetwo species.
A

Methods
Mapping, sequencing and data availability
The details concerning mapping and sequencing are summarized in Supplementary Tables
1 and 2 (see also http://chimp22pub.gsc.riken.jp). Briefly, three BAC libraries—PTB1(ref. 3), RPCI-43 and CHORI-251 (http://bacpac.chori.org), constructed from three maleindividuals—and a chimpanzee chromosome 22 fosmid library—PTF22 (12-foldcoverage)—were used to isolate clones for this analysis. The first set of the seed clones was
selected from BAC end data3 and PCR screening of expanded BAC libraries using highdensity human STS primers placed at roughly 20-kb intervals. Only the clones having
multiple STS markers and sharing common STSs with neighbouring clones wereconsidered as a member of a clone contig. These clones were then subjected to partial
sequencing, from which new chromosome-walking primers were designed for further
screening. Clone overlaps were then examined before choosing a set of minimum-tilingpath clones for full-scale high-quality sequencing. These steps were repeated until all gaps
were closed or no additional clones could be identified. Some representative clones incontigs were also examined cytogenetically to confirm their localization on the particularchromosome; however, not all such clones were included in the minimum tiling path.
Most of the pericentromeric and subtelomeric regions are not included in this study
because of the difficulty in identifying clones with good sequence matches. Two PTR22
clones, PTB-242K04 and CH251-010A09, have a terminal region that extends into one of
the three clone gaps in HSA21q by 11,546 bp in total. Another clone, PTB-190I13, spans
another HSA21q gap with a 9,284-bp region for the gap where the GþC content is high(54.2%,) and a sequence gap of 1,165 bp in length still remains.
All of the clone data have been released from DDBJ/EMBL/GenBank (accession
numbers are listed in Supplementary Table 1).

Alignment between HSA21q and PTR22q and contig construction
Each PTR22 clone sequence was aligned to the HSA21q data using NCBI BLAST2. From
the BLAST hits, we chose the best match for each site of the corresponding region in
HSA21q. Details of the alignment procedure can be seen in Supplementary Information.
The clone contig was generated by aligning the overlapping regions of each neighbouring
clone. We chose to include the sequence of the clone for the overlap with which thealignment with the counterpart region in HSA21q showed a lower divergence level. On thebasis of this clone contig map, the region represented in the contig is extracted from eachclone alignment to produce the whole chromosome alignment. To fix any small
inconsistencies around the clone boundaries, the final alignment was checked manually.
The nucleotide divergence level was calculated from the regions that aligned best with
HSA21q.

Detection of lineage-specific insertions and deletions
We selected 567 apparent insertions $300 bp from both the human and chimpanzee
sequences and designed PCR primers from their flanking sequences. Using genomic DNA
samples from five chimpanzees, five humans, one gorilla and two orang-utans as atemplate, the size of the PCR products was examined. After amplification, reaction
products were separated through 1% agarose gel electrophoresis for comparison of the
product sizes. We used only the indels that showed a significant size difference between
chimpanzee and human, and one of them was of equivalent size to gorilla and orang-utan,
for the analysis (t-test).

SNP analysis
The HSA21q SNPs7 in dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) were compared to thecorresponding PTR22q sites to infer the ancestral states of the two human alleles. We
developed a modified version of the H test36 based on the computer program at
http://crimp.lbl.gov/htest.html so as to detect positive selection disregarding singletons(details in Supplementary Information). The pattern of nucleotide substitutions was also
estimated using the human SNP data and chimpanzee BAC sequence overlap data
generated in the present study. We estimated the substitution pattern35 and equilibrium
frequencies from the nucleotide substitution matrix45,46.

Genomic annotation
Protein-coding genes in the PTR22q sequence were annotated by extracting theorthologous regions on HSA21q (in the gene catalogue) from the genomic sequence. In
addition, PTR22 sequences with no match to the HSA21 gene catalogue were blasted
NATURE | VOL 429 | 27 MAY 2004 | www.nature.com/nature

For all arrays only the oligonucleotides that matched perfectly between the human andchimpanzee sequences were used for analysis. Gene expression levels were comparedseparately for brain and liver in all nine possible pairwise comparisons between the three
individuals of each species.
Received 16 February; accepted 14 April 2004; doi:10.1038/nature02564.
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Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements We are grateful to T. Ito, C. Kawagoe, T. Kojima, X. Son, A. Beck, K. Borzym,
S. Gelling, V. Gimmel, K. Heitmann, A. Kel, S. Klages, N. Lang, I. Mueller, M. Sontag,
R. Yildirimman, J. Wickings, C. Baumgart, O. Mueller, T. T. Liao, H. Tsai, Y. Huang, Y. Liu and all
the technical staff of the contributing genome centres. This work was supported in part by aSpecial Fund for RIKEN Genomic Sciences Center and Grant-in-Aid for Scientific Research on
Priority Areas ‘Genome Science’ from the Ministry of Education, Culture, Sports, Science andTechnology, Japan; the Ministry of Education and Research, Germany; the Chinese International
Science and Technology Cooperation Project, Ministry of Science and Technology, China; theChinese High-Tech Research and Development Program, Shanghai Commission for Science andTechnology; the National Research Program for Genomic Medicine of National Science Council,
Taiwan; and the Ministry of Science and Technology, Korea.
Authors’ contributions H. Watanabe, A. Fujiyama, M. Hattori, N. Saitou, H.-S. Park, S.-Y. Wang,
M.-L. Yaspo and Y. Sakaki contributed to the consortium leadership.
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to Y.S. (sakaki@gsc.riken.jp). The
accession numbers of the BAC clone sequences have been deposited in DDBJ/EMBL/GenBank
and can be found in Supplementary Table 1.

H. Watanabe1,2*, A. Fujiyama1,3, M. Hattori1,4, T. D. Taylor1, A. Toyoda1, Y. Kuroki1, H. Noguchi1, A. BenKahla5, H. Lehrach5, R. Sudbrak5,
M. Kube5, S. Taenzer6, P. Galgoczy6, M. Platzer6, M. Scharfe7, G. Nordsiek7, H. Blöcker7, I. Hellmann8, P. Khaitovich8, S. Pääbo8, R. Reinhardt5,
H.-J. Zheng9, X.-L. Zhang9, G.-F. Zhu9, B.-F. Wang9, G. Fu9, S.-X. Ren9, G.-P. Zhao9, Z. Chen9,10, Y.-S. Lee11, J.-E. Cheong11, S.-H. Choi11,
K.-M. Wu12, T.-T. Liu13, K.-J. Hsiao13,14, S.-F. Tsai12,13, C.-G. Kim15, S. OOta15, T. Kitano15, Y. Kohara15, N. Saitou15, H.-S. Park11, S.-Y. Wang9,
M.-L. Yaspo5 & Y. Sakaki1
Affiliations for authors: 1, RIKEN, Genomic Sciences Center, Yokohama 230-0045, Japan; 2, Nara Institute of Science and Technology, Ikoma 630-0101, Japan;
3, National Institute of Informatics, Tokyo 101-8430, Japan; 4, Kitasato University, Sagamihara 228-8555, Japan; 5, Max-Planck-Institut für Molekulare Genetik,
D-14195 Berlin-Dahlem, Germany; 6, Institut für Molekulare Biotechnologie, Genomanalyse, D-07745 Jena, Germany; 7, German Research Centre for Biotechnology(GBF), D-38124 Braunschweig, Germany; 8, Max-Planck-Institut für Evolutionäre Anthropologie, D-04103 Leipzig, Germany; 9, Chinese National Human Genome
Center at Shanghai, Shanghai 201203, China; 10, State Key Laboratory of Medical Genomics, Rui Jin Hospital, Shanghai 200025, China; 11, Genome Research Center,
Korea Research Institute of Basic and Biotechnology, Daejeon 305-333, Korea; 12, National Health Research Institutes, Taipei 115, Taiwan; 13, National Yang-Ming
University, Taipei 112, Taiwan; 14, Taipei Veterans’ General Hospital, Taipei 112, Taiwan; 15, National Institute of Genetics, Mishima 411-8540, Japan*Present address: Hokkaido University, Sapporo 060-0814, Japan388©2004 Nature Publishing Group

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