Genes Genet. Syst. (2005) 80, p. 225–232

Evolutionary Conservation of 5’ upstream Sequence of Nine
Genes between Human and Great Apes
Takashi Kitano† and Naruya Saitou*
Division of Population Genetics, National Institute of Genetics Mishima, 411-8540, Japan(Received 26 October 2004, accepted 4 July 2005)

Nucleotide sequences of nine 5’ upstream gene regions for human, chimpanzee,
gorilla, and orangutan were determined. We estimated nucleotide differences (d)for each region between human and great apes. The overall d was 0.027 (ranged
from 0.004 to 0.052). Rates of nucleotide substitution were estimated by using d
and divergence times of human, chimpanzee, gorilla, and orangutan. The overall
rate of nucleotide substitution between human and other hominoids was estimatedto be 0.52–0.85 × 10–9. This rate in 5’ upstream regions was lower than that ofsynonymous sites, suggesting that 5’ upstream regions have evolved under somefunctional constraints. Because lower rates have been reported for coding
sequences in primates compared to rodents, we also estimated the rate (1.17–1.76
× 10–9) of nucleotide substitutions for the corresponding 5’ upstream regions inrodents (mouse/rat comparison). Thus the primate rate was lower than rodent
rate also for the 5’ upstream regions.
Key words: human, great apes, rodents, upstream region, evolutionary rate

INTRODUCTION
Comparison of human and other hominoids is definitely
necessary to understand human evolution (e.g., see Saitou 2005). Chimpanzee chromosome 22, orthologous tohuman chromosome 21, have been fully sequenced
recently for human-chimpanzee comparative genomics(The International Chimpanzee Chromosome 22 Consortium, 2004), however, we need outgroup species to infer
human specific changes. There are such gene comparison studies of human and other hominoids (Chen and Li
2001; O’hUigin et al. 2002; Wildman et al. 2003; Kitano
et al. 2004). Those studies mainly used protein-coding
sequence data. Some studies (Chen and Li 2001; Yi et
al. 2002) compared human and other hominoid non-coding sequence data. However, data from 5’ upstream noncoding regions are much less abundant than data fromcoding regions. 5’ upstream non-coding regions of genes
have important role for controlling gene expression.
Accumulation of analyses of these regions between
human and other hominoids is important to understandhuman evolution.
The nucleotide substitutional difference between thehuman and chimpanzee genomes was estimated to be

1.23% based on a total of 19,813,086 bp BAC end sequence comparison (Fujiyama et al. 2002), while the wholechromosome comparison between human chromosome 21
and chimpanzee chromosome 22 showed somewhat
higher value, 1.44% (The International Chimpanzee Chromosome 22 Consortium, 2004). Many of those differences are probably caused by mutations occurred inso-called junk DNA (Ohno 1972), and have no effect onphenotypic difference between human and chimpanzee.
Some proportion of nucleotide changes, however, must be
responsible for human-specific characters, such as large
brain size, bipedalism, etc. King and Wilson (1975) proposed that genetic changes at the gene expression control
region are more important than changes in the proteincoding region.
We thus estimated the rate of nucleotide substitution of
5’ upstream gene region in hominoids in this study. We
then compared it with corresponding synonymous substitution rates. These rates were also compared with thosefor mouse (Mus musculus ) and rat (Rattus norvegicus ).
Finally, implication of human specific changes in the 5’
upstream region is discussed.

MATERIALS AND METHODS
Edited by Yoko Satta* Corresponding author. Email: nsaitou@genes.nig.ac.jp†
Present address: Department of Experimental and Forensic
Pathology, Yamagata University School of Medicine, Yamagata, 990-9585, JapanWe determined nine 5’ upstream gene regions (seeTable 1) for human (Homo sapiens ), chimpanzee (Pantroglodytes), gorilla (Gorilla gorilla), and orangutan(Pongo pygmaeus ) in this study. Six genes (DDC, DMD,

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T. KITANO and N. SAITOU
Table 1.
SymbolList of genes whose 5’ upstream regions were compared

Name

Chromosomal location

DDC

dopa decarboxylase

7p11

DMD-1

dystrophin

Xp21.2DMD-2dystrophin

Xp21.2

INMT

indolethylamine N-methyltransferase

7p15.3-p15.2

MAOA

monoamine oxidase A

Xp11.4-p11.3

NEF3

neurofilament 3

8p21

NGFB

nerve growth factor, beta polypeptide

1p13.1

PLP2

proteolipid protein 2

Xp11.23

POU3F2

POU domain, class 3, transcription factor 2

6q16

We used description in the NCBI database on gene symbols, names, andchromosome locations.

MAOA, NEF3, NGFB, and POU3F2) are brain and/or
neuron related genes. Two alternative 5’ regions weresequenced for the DMD gene. The 5’ upstream regions
for two non brain-neuron specific genes (INMT and PLP2)
were also sequenced. INMT is known to be expressed inseveral human tissues such as adult thyroid, adult adrenal gland, adult lung, and fetal lung (Thompson et al.
1999), and PLP2 is expressed in colonic epithelial cell
(Olivia et al. 1993).
DNA was extracted from peripheral blood sample ofJapanese individuals with informed consent for humansamples. DNA samples for chimpanzee, gorilla andorangutan were also used, and those samples were thesame as used by Kitano et al. (2004). We sequenced two
human samples and two chimpanzee samples for each
DMD-1, NGFB, and POU3F2 gene, and these twosequences were identical with each other (data notshown). Hot-start PCR was applied to each selected segment (PE GeneAmp PCR system 2400 and 9700). All
PCR primers were designed based on human sequences inthe database. A list of primers used in this study isavailable from TK. PCR products were confirmed by
1.5% agarose gel electrophoresis, and purified using
Micro Spin Columns (Amersham Biosciences). The purified PCR products were sequenced by using BigDye Terminator Cycle Sequencing Kit and ABI PRISM 377/310
DNA sequencer (PE Biosystems). Both strands wereread using PCR primers and inner primers when longsequences were read. Overlapping two peaks on a site
was regarded as heterozygous and the site was designated by following Nomenclature Committee of the International Union of Biochemistry (NC-IUB).
CLUSTAL W ver.1.8 (Thompson et al. 1994) was used
for multiple alignments. Aligned regions without gaps
were used for following analyses. Numbers of nucleotide
substitutions per site (d) were estimated by using oneparameter method (Jukes and Cantor 1969). Numbersof synonymous substitutions per synonymous site were
estimated by using Nei and Gojobori’s (1986) method.

Figure 1 shows 5’ upstream regions of eight genes
whose sequences were determined in this study. Because
the first exons of two genes (DDC and NGFB) were noncoding exons, start codons were not shown in the figure.
Both alternative non-coding exons of DMD were alsosequenced. DMD-1 and DMD-2 correspond to the brain
type (Boyce et al. 1991) and the Purkinje cell type (Holder
et al. 1996), respectively. For simplicity, we used terms‘DMD-1’ and ‘DMD-2’ in this paper.
RESULTSWe determined nine 5’ upstream regions for four hominoid species (human, chimpanzee, gorilla, and orangutan), and their DDBJ/EMBL/GenBank International
Nucleotide Sequence Database accession numbers arelisted in Table 2. A total of 4157 nucleotide sites could
be compared, and 108 variant sites were detected. Variant nucleotide sites are shown in Table 3. Two sites (120of NEF3 and 218 of NGFB) had three character
states. Therefore, a total of 110 changes are necessary
to explain these variant sites.
Because numbers of nucleotide substitutions on eachbranch were low, we added nucleotide substitutions
occurred on all branches of Fig. 2. The total number ofnucleotide substitutions among human, chimpanzee,
gorilla, and orangutan (d) were estimated from the total
number of nucleotide changes (m) among human, chimpanzee, gorilla, and orangutan. The total d for hominoids was 0.027 ± 0.003. Divergence times of human,
chimpanzee, gorilla, and orangutan are shown in Fig. 2
following Chen and Li (2001). By adding divergencetimes of all branches, the total time was 34.8–46.6 MY(million years), and this value was used to estimate rates
of nucleotide substitution. The total rate ranged from
0.52 × 10–9 to 0.85 × 10–9. There were no specific rate differences between brain-neuron expressed genes (DDC,
DMD-1, DMD-2, MAOA, NEF3, NGFB, and POU3F2)
and the remaining genes (INMT and PLP2).

Evolutionary Conservation of 5’ upstream Sequence between Human and Great Apes

227

Fig. 1. Genome structures of the nine sequenced regions. Coding and non-coding exons are shown by close and open
boxes, respectively. Thick bars show sequenced regions. 5’ upstream region of MAOA overlaps with 3’ region of
LOC352786 indicated by a grey box.

We used BLAST homology search system to retrieve
homologous sequences from rat and mouse from thegenome database. However, we could retrieve mouse andrat counterparts only for DMD-1, DMD-2, and POU3F2.
Because the regions around translation and/or transcrip-tion start sites of those six remaining sequences had
homology between human and mouse-rat, we retrieved
same length of sequences with hominoids from regions
just upstream of these homologous regions, and used themas possible mouse and rat counterpart sequences (see228

T. KITANO and N. SAITOU
Table 2.

List of DDBJ/EMBL/GenBank accession numbers* and NCBI gene numbers whose
nucleotide sequences were compared(A) 5’ upstream region
SymbolHuman, Chimpanzee, Gorilla, Orangutan, Mouse, Rat

DDC

AB037497, AB037498, AB037499, AB037500, NT_039515, NW_047430

DMD-1

AB037487, AB037489, AB037491, AB037492, NT_039709, NW_044437

DMD-2

AB037493, AB037494, AB037495, AB037496, NT_039709, NW_044437

INMT

AB041362, AB041363, AB041364, AB041365, NT_039343, NW_047693

MAOA

AB042829, AB042830, AB042831, AB042832, NT_039700, NW_048034

NEF3

AB042833, AB042834, AB042835, AB042836, NT_039606, NW_047454

NGFB

AB037481, AB037483, AB037485, AB037486, NT_039239, NW_047627

PLP2

AB041366, AB041367, AB041368, AB041369, NT_039700, NW_048035

POU3F2

AB037475, AB037477, AB037479, AB037480, M88300, NW_043830(B) CDS region
SymbolHuman, Chimpanzee, Mouse, Rat

DDC

M76180, XM_519096, AF071068, BC087032

INMT

AF128847, XM_527713, M88694, XM_347233

MAOA

BC008064, #, BC029100, XM_343764

NEF3–, –, X05640, Z12152

NGFB

X52599, AY665265, M17298, M36589

PLP2

L09604, AC149133, AK003522, BC061844

POU3F2–, –, M88300, XM_345510*DDBJ/EMBL/GenBank accession numbers starting with AB listed above were
determined in this study. All of them are 5’ upstream regions for hominoids. #: Thirteen
entries (AY684852, AY684860, AY685683, AY685686, AY685688, AY685690, AY685692,
AY685694, AY685696, AY685698, AY685700, AY685702, and AY685704) were used for
chimpanzee MAOA. –: not analyzed.

Table 3.

Variant nucleotide sites of nine 5’ upstream regions of human, chimpanzee, gorilla, and orangutan
DD C

DMD – 1

DMD – 2I NM T

11112222333334444456

12

1111111111223

1 489907781334135552557801

5761

2548

1136726666789180

3 584660173485820198161302

8763

3102

0261371345536903

Hum C G C GG T A T A C C A C C C T C A GG T A A C CC C AAC T ATG C C GGGGG A G C G C A C T

Chi

C G C G C T A T A C C A C C T GGGG C T A A G C

C C GACCAT

A T C A GG A A T GGG C G C T

Gor

C G C A G T A T G C C A C T C GGG A C C C A C GC C AAC T GCG C T GG C GG A GGG C G T C

Ora

T A T GGG T C A A T GG C C GGGG C C C G C G

G T GGT T ATG C C G A GGG A A G C GG C C

MA O ANE F 3

NGFB

PLP2

POU 3 F21111222333345666677789

1111222333334

1122

11112222

4

2 62349589007804256946940

336790266566245880

360811345880027

74

0 72536148247203081818936

149452013206377239

346408215234684

47

Hum C C T T C G T A T A T C A T GGGG C G T GG C

A T T T A GG T A T GGG T C C C GG C T GG A

CCCC T CGT C

CC

Chi

T C T T C G T A C A T C A T G A GG C G T GG C

A T T T ACGT A C GAGT CAAGG C T GG C

CCCC T CGT C

CC

Gor

T C T C T G T T C A T C A T T A GG T G C GG C

A T T C A G C T A C GGG A G C C A

G C G GG C

CCCC T CGT C

CC

Ora

T T C C C C C T C G C T GGG A A A C A T C A AG C C T GG A C G C C G A T C C C G

CA T CATT T T AGAACATG

Hum, Chi, Gor, and Ora denote human, chimpanzee, gorilla, and orangutan, respectively. Numbers indicate
nucleotide positions of each DNA region.

Evolutionary Conservation of 5’ upstream Sequence between Human and Great Apes
Table 2 for list of sequence IDs used for comparison). The
total d between mouse and rat was 0.100 ± 0.005 (Table
4). Since the divergence time between mouse and rat isa matter of argument (Wilson et al. 1977; Jacobs. and Pilbeam 1980), we used two kinds of divergence times
between mouse ,and rat to estimate rates of nucleotide
substitutions; 40.7 MYA (million years ago) (Kumar andHedges 1998) and 30 MYA (Kitano et al. 1999). The total
rate ranged from 1.17 × 10–9 to 1.76 × 10–9. The total rateof nucleotide substitution in hominoids was lower thanthat in rodents.
Figure 3 shows relationship of d between hominoids
and rodents for each region. Hominoid d and rodent d
are positively correlated (correlation coefficient is 0.63).
This is mainly because three regions (DMD-1, DMD-2,
and POU3F2; shown in gray circle in Fig. 3) where primates-rodents homology was found low d both in hominoids and mouse/rat, while the remaining six regions, inwhich no clear homology was found between hominoids
and mouse/rat, showed higher d both in hominoids andFig. 2. A species tree of human, chimpanzee, gorilla, and orangutan. Numbers of each branch indicate nucleotidechanges. Divergence times for three speciation events are shown on the time scale bar. Ranges of divergence times (MY,million years) are shown in parentheses.
Table 4.

Nucleotide differences and substitution rate for 5’ upstream regions of nine
protein coding genes for Hominoids and Rodents

Gene

Hominoidsmbp

Rodents (mouse vs. rat)
d

m

bp

d

DDC

25

718

0.036 ± 0.007

70

718

0.104 ± 0.013

DMD-1*

4372

0.011 ± 0.005

1

238

0.004 ± 0.004

DMD-2*

4232

0.017 ± 0.009

11

207

0.055 ± 0.017

INMT

16

321

0.052 ± 0.013

35

321

0.118 ± 0.020

MAOA

24

917

0.027 ± 0.005

125

917

0.150 ± 0.014

NEF3

19

469

0.042 ± 0.010

56

469

0.130 ± 0.018

NGFB

7

364

0.019 ± 0.007

42

364

0.125 ± 0.020

PLP2

9

311

0.030 ± 0.010

15

311

0.050 ± 0.013

2453

0.004 ± 0.003

19

437

0.045 ± 0.010

110

4157

0.027 ± 0.003

374

3982

0.100 ± 0.005

POU3F2*
Total

229

m: number of nucleotide changes among human, chimpanzee, gorilla, andorangutan, bp: number of compared sites, d: number of nucleotide substitutions
per site.
*Mouse and rat sequences were found to be homologous to hominoid ones by
BLAST homology search (NCBI mouse and rat genome).

230

T. KITANO and N. SAITOU(see Fig. 3). This suggests that the 5’ upstream region ofthis gene reduced its importance in the primate lineage.
DISCUSSION
The average rate of nucleotide substitution for humanand chimpanzee non-coding DNA has been reported to be
0.99 × 10–9 (Yi et al. 2002) or 0.86 × 10–9 (Wildman et al.
2003). The total rate of the 5’ upstream region of hominoids (from 0.52 × 10–9 to 0.85 × 10–9) estimated by thisstudy was lower than those of previous results. We estimated the rate of nucleotide substitution by using
human, chimpanzee, gorilla, and orangutan data sets.
When we estimate rates of nucleotide substitution byusing only human and chimpanzee, the overall rate forhuman and chimpanzee ranged from 0.41 × 10–9 to 0.82 ×
10–9 (m = 26, d = 0.006 ± 0.001). Divergence time (4.6–
6.2 MYA, see Fig. 2) between human and chimpanzee wasused estimate the rate of nucleotide substitutions. The
overall rate of human and chimpanzee was still lower
than those of previous reports (Yi et al. 2002; Wildman etal. 2003).
Rates of nucleotide substitution of various regions weresummarized as nonsynonymous (0.85 × 10–9), synonymous(4.61 × 10–9), 5’ UTR (1.96 × 10–9), 3’ UTR (2.10 × 10–9),
introns (3.14 × 10–9) and pseudogenes (3.52 × 10–9) in various mammalian genes (Li and Graur 1991). It is shownthat the rate of nucleotide substitution in 5’ upstream
regions is lower than those in other regions except fornonsynonymous sites. Studies of divergence between
human and chimpanzee (Chen et al. 2001; Shi et al. 2003)
also showed similar tendency. 5’ upstream regions do
not code gene products, but these regions probably havefunctional roles to gene expression. Our result showed

Fig. 3. Comparison of evolutionary distances for ten 5’ upstream regions between rodents and hominoids, based on data
shown in Table 4. Three regions designated with gray circles
are those with asterisks in Table 4. Two broken lines represent
evolutionary distances for rodents (horizontal) and for hominoids (vertical) under the pure neutral evolution. The genomic
divergence between mouse and rat (Table 6) was used as thepure neutral evolutionary distance for rodents. The value ofgenomic divergence between human and chimpanzee (0.012)
multiplied by 3.77 was used as the pure neutral evolutionary
distance for hominoids, because the total time of all branches(34.8–46.6 MY, mean 40.7 MY) was 3.77 times higher than thetime between human and chimpanzee (10.8 MY = 5.4 × 2). The
diagonal line represents equal selective constraints between
rodents and hominoids. This line is through the origin and theintersection of the two broken lines.

mouse/rat. This suggests that relative intensity of functional constraints had been more or less constant throughthe mammalian evolution. One 5’ upstream region for
hominoids, INMT, showed the evolutionary distancelarger than that expected under pure neutral evolution
Table 5.

Nucleotide differences and substitution rate for synonymous sites of five protein
coding genes for Hominoids (human vs. chimpanzee) and seven genes for Rodents(mouse vs. rat)

CDS region

Hominoids*s

S

Rodents**
dS

s

S

dS

DDC

5

307.5

0.016 ± 0.007

78.5

349.330.267 ± 0.032

INMT

1

112.420.009 ± 0.009

48

199

0.291 ± 0.045

MAOA

9

371.5

0.025 ± 0.008

41

372.580.119 ± 0.019

NEF3

–

–

–

93.5

547.420.194 ± 0.021

NGFB

5

173.830.029 ± 0.013

24

176.580.150 ± 0.031

PLP2

3

119.670.025 ± 0.015

16117.330.151 ± 0.039

POU3F2

–

–

–

25

320.5

0.082 ± 0.017

Total

23

1084.92

0.021 ± 0.005

326

2082.74

0.176 ± 0.010

s: number of synonymous changes, S: number of synonymous sites, dS: number ofsynonymous substitutions per synonymous site, –: not analyzed genes.
DMD gene coding region sequences were not available neither for hominoids nor murids,
so this gene was not listed in this table.
*Comparison between human and chimpanzee. **Comparison between mouse and rat.

Evolutionary Conservation of 5’ upstream Sequence between Human and Great Apes
Table 6.

Comparison of nucleotide differences of human-chimpanzee and mouse-rat

Regiond(Human-Chimpanzee)d(Mouse-Rat)d(M-R)/d(H-C)

5’ upstream

0.006 ± 0.001

0.100 ± 0.005

16.7

Synonymous

0.021 ± 0.005

0.176 ± 0.010

8.4Genomic

0.012*

0.155**

12.9*From Fujiyama et al. (2002).

Table 7.

**From Abe et al. (2004).

Differences between human and other hominoid on possible transcription factor binding sites

Gene

Site

Human sequence (factor)

Ape sequence (factor)

DDC

451

TCGCCA (F-ACT1)

TCCCCA (AP-2alphaA)

451

GCCAGTC (CP2)

CCCAGTC (–)

219

AAGGAA (c-Ets-2)

AAGGAG (–)

219

GGAAAG (NF-1)

GGAGAG (–)

MAOA

620

CAGCTG (XPF-1)

CAACTG (c-Myb)

NEF3

252

CTGCCT (–)

CTGCCC (Sp1)

NGFB

218

ACCACA (AML1)

ACCACY (–)

218

CAGCCC (–)

CYGCCC (Sp1)

INMT

231

On a site 218 of NGFB, chimpanzee and gorilla showed ‘C’ and orangutan showed‘T’, so ‘Y’ was used.

lower rate of nucleotide substitution in 5’ upstream
regions. This suggests that 5’ upstream regions have
evolved under strong functional constraints.
It has been reported that rates of coding region in hominoids are lower than those of rodents (Wu and Li 1985;
Gu and Li 1992; Kitano et al. 1999). In this study, we
showed that 5’ upstream regions also have lower rates ofsubstitution in hominoids than in rodents. To assess
whether the lower rate of substitution in hominoids thanin rodents is due to generation time effects or stronger
constraint in hominoids, we estimated rate of synonymous substitutions for the same genes. For hominoid
comparisons, we used distances between human andchimpanzee, because data for other hominoid species
were not available. Table 5 shows the comparison ofsynonymous substitutions for hominoids and rodents(mouse vs. rat). We then summarized distance differences between hominoids and rodents as shown in Table
6. In all three comparisons (5’ upstream region distance,synonymous distance, and genomic divergence), the evolutionary distance between mouse and rat is 8–17 times
higher than that between human and chimpanzee. The
ratio of the species divergence time between the two species pair, t(M-R)/t(H-C), should be at most 7 times, if there
is no difference in evolutionary rate in primates androdents. This indicates that somehow the evolutionaryrate has been higher in rodents than in primates.
One good characteristic of simultaneous sequence com-

parison of closely related species is clear deduction of species-specific changes. We thus extracted eleven changes
out of total 110 changes as human lineage specificchanges (see Table 3). We searched putative transcription factor binding sites by using TESS (Transcription
Element Search System) under anticipation that even one
nucleotide substitution at the human lineage mightcaused a significant phenotypic change through change ofgene expression pattern. Only five possible transcription elements differences between human and other hominoids were observed, as listed in Table 7. Those site
differences may affect gene expression differences
between human and other hominoids, however, we needfurther experiments to confirm them.
We are grateful for R. E. Ferrell for providing us chimpanzee
DNA samples and for S. Ueda for providing us gorilla and orangutan DNA samples. We thank H. Kobayakawa for technicalassistance. This study was supported by a grant in aid for scientific studies from Ministry of Education, Science, Sport, andCulture, Japan to NS, Joint Research Project (Soken/K99-1) of
The Graduate University for Advanced Studies, Japan to
NS. TK was supported by COE research fellowship of the
National Institute of Genetics and JSPS postdoctoral fellowship.

ELECTRONIC-DATABASE INFORMATIONNCBI Home Page, http://www.ncbi.nlm.nih.gov/
Project Silver Home Page, http://sayer.lab.nig.ac.jp/

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T. KITANO and N. SAITOU~silver/index.html
TESS, http://www.cbil.upenn.edu/tess/

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