Tissue-specific codon usage and the expression ofhuman genes
Joshua B. Plotkin*†‡, Harlan Robins†§, and Arnold J. Levine§*Harvard Society of Fellows and Bauer Center for Genomics Research, Harvard University, 7 Divinity Avenue, Cambridge MA 02138; and §Institute forAdvanced Study, Olden Lane, Princeton, NJ 08540
Contributed by Arnold J. Levine, July 12, 2004

A diverse array of mechanisms regulate tissue-specific protein
levels. Most research, however, has focused on the role of transcriptional regulation. Here we report systematic differences insynonymous codon usage between genes selectively expressed in
six adult human tissues. Furthermore, we show that the codon
usage of brain-specific genes has been selectively preserved
throughout the evolution of human and mouse from their common
ancestor. Our findings suggest that codon-mediated translational
control may play an important role in the differentiation andregulation of tissue-specific gene products in humans.

W

ith the advent of mRNA expression arrays, researchers
have begun to delineate which genes are selectively expressed in which tissues and, in a fundamental way, distinguish
one tissue from another (1, 2). Although such studies help toelucidate expression patterns, the processes underlying differentiation and regulation of tissue-specific proteins remain outstanding problems in developmental and molecular biology.
Here, we show that genes selectively expressed in one humantissue can often be discriminated from genes expressed inanother tissue purely on the basis of their synonymous codon
usage. In particular, we demonstrate that brain-specific genes
show a characteristically different codon usage than liver-specificgenes; uterus genes differ from testis genes; and ovary genes
differ from vulva genes, as well as other pairs of these six tissues.
Codon Bias Across Taxa
Although it came as a surprise to early neutral theorists (3), it
is now clear that codon usage is not random: Among synonymous codons, some codons are used preferentially. Moreover, taxa differ in their codon usage. For example, various
species of Drosophila each have their own particular codon
biases, and their usage differs significantly from Escherichia
coli or Saccharomyces cerevisiae (4 – 6). The dominant theory ofcodon bias for organisms ranging from E. coli to Drosophila
posits that preferred codons correlate with the relative abundances of isoaccepting tRNAs, thereby increasing translational
efficiency (7–10).
Synonymous codon choice also affects gene expression in
mammals: When nonmammalian genes are to be expressed in
mammalian cells, the replacement of mammalian-rare codons
with more common synonyms greatly increases gene expression(11–13). Nevertheless, there is little evidence in mammals of
selection on synonymous codons for translational efficiency.
Instead, mammalian genomes exhibit large-scale variation in GC
content [e.g., isochores (14)] in both coding and noncoding
regions. The GC content in noncoding regions is correlated with
the GC content at the third position of coding regions from thesame isochore. Thus, codon biases observed in the humangenome have been attributed to neutral processes [such as biased
mutation (15) and gene conversion (16)] rather than to selection(17). [Early studies on cDNA clones derived from a diverse setof vertebrate genes failed to find evidence for tissue-specific ortaxon-specific codon usage (18).]12588 –12591 兩 PNAS 兩 August 24, 2004 兩 vol. 101 兩 no. 34

Comparing Codon Usage Between Genes
The most common measure of codon bias, called the effective
number of codons (ENC), is analogous to the effective numberof alleles in population genetics. ENC does not describe theparticulars of which codons are more frequent than others but
rather measures the overall departure from random synonymouscodon choice. As a result, two genes may exhibit the same degreeof overall bias (ENC value) and yet differ dramatically in their
particular choice of synonymous codons.
For this study, we desire a detailed measure of the ‘‘distance’’
between the synonymous codon usage of two genes. We are notconcerned with degree of codon bias in the usual sense, that is,the departure from random synonymous codon choice, butrather with the degree to which genes differ in their encoding ofamino acids. Given the coding sequences for a pair of genes, we
compare their codon usage by first tabulating the absolute
frequency of each codon in each gene. For each amino acid, we
compute a two-tailed Fisher exact test (19) on the n ⫻ 2contingency table given by the frequencies of the amino acid’s
synonymous codons (e.g., for Ala n ⫽ 4: GCC, GCG, GCA, and
GCT). As a result, for each amino acid we obtain a P value
indicating whether or not the genes use significantly different
codons to encode that amino acid. Table 1 summarizes anexample of this analysis by comparing the codon usage of two
human genes.
The number of amino acids that exhibit a statistically different
encoding is a biologically relevant metric of distance between thecodon usage in two genes. All other things being equal (i.e., RNAfolding, protein–RNA recognition, transport, etc.), for a fixedpool of tRNAs, this metric should naturally correlate with thedifference in translation rates between the two genes. Unlike
metrics such as ‘‘relative synonymous codon usage’’ (4), which
are noisy when applied to individual genes, our measure of codon
usage relies on the Fisher Exact test for small sample sizes, andit can be applied to genes that contain only a few examples ofeach amino acid.
Methods
The uterus- and testis-specific genes used in this study (Table 3,
which is published as supporting information on the PNAS website) were obtained directly from the tissue-specific lists compiled by Warrington et al. (1). The brain, liver, ovary, and vulva
genes (Table 3) were taken from the online expression databaseof Hsiao et al. (2). A gene was considered to be brain-specific if,
according to the Hsiao database (2), its mRNA transcript ispresent in brain but absent from all but at most two other tissue
types tested by Hsiao et al. The criteria for tissue-specificconsideration were the same for liver, ovary, and vulva.
Given a dendogram that represents the codon usage of genes
in a pair of tissues (e.g., Fig. 1), we calculate a P value to test
whether the observed clustering of genes is nonrandom. The P†J.B.P and H.R. contributed equally to this work.
‡To whom correspondence should be addressed. E-mail: jplotkin@fas.harvard.edu.

© 2004 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404957101

CodonGene A, n (%)

Gene B, n (%)

GCC
GCG
GCA
GCT
TGC
TGT
GAG
GAA

9 (20)
3 (7)
17 (38)16 (36)
2 (12)
14 (88)13 (34)25 (66)19 (58)8 (24)3 (9)3 (9)5 (50)
5 (50)
22 (92)2 (8)Ala, P ⫽ 0.000024

Cys, P ⫽ 0.068653
Glu, P ⫽ 0.000006

For each codon, we report its absolute frequency of occurrence in eachgene and its relative frequency compared with synonymous codons. The P
value for each amino acid reflects whether or not the two genes differ in theirencoding of the amino acid (Fisher exact test). A complete comparison of all
61 condons is given as Table 2, which is published as supporting informationon the PNAS web site. The comparison between these genes is typical ofcomparisons between other genes from their respective tissues, testes anduterus. Gene A, testis-specific glycerol kinase (GI 516123); gene B, endometrial
bleeding factor (GI 2058537).

value is obtained by comparing the observed summed squared
distances along the tree between genes of the same tissue against
a null distribution produced by randomly permuting the labels of
the leaves.
For each of the 44 brain-specific genes, the corresponding
mouse orthologs were obtained from the ENSEMBL web-site byusing ENSMART, and they were aligned by using CLUSTALW (20).
The same procedure was used to produce orthologous alignments of the genes specific to ovary, testes, uterus, liver, andvulva.
Results on Tissue-Specific Codon Usage
On the basis of two extensive microarray mRNA expression
studies (1, 2), we have identified genes that are selectively

Fig. 1. A dendogram reflecting the codon usage of 26 genes selectively
expressed in human testis (red) and 16 genes selectively expressed in uterus(blue). Genes are denoted by their GI number. The pairwise distances underlying this tree reflect the degree to which the genes differ in their codon
usage. As this tree demonstrates, testis-expressed genes can generally be
distinguished from uterus-expressed genes purely on the basis of their synonymous codon usage. The observed separation between these two classes ofgenes would not have occurred by random chance (P ⫽ 0.0008)

Plotkin et al.

expressed in six adult healthy human tissues: testis (26 genes),
uterus (16 genes), total brain (44 genes), liver (34 genes), ovary(36 genes), and vulva (42 genes). By analyzing expression
patterns from only two studies, we limited ourselves to fewer
data than are available in large compilations of many expression
studies. On the other hand, the expression data we have used arecomparable (both studies used the GeneChip HuGeneFL microarray), and they provide a consistent, unbiased method ofassigning tissue-specificity. The total number of identified tissuespecific genes is smaller than in previous studies (21) because weuse a conservative, stringent definition of tissue specificity (seeMethods). The genes selectively expressed in each of these six
tissues are distributed throughout the genome (Table 3), andthey have similar distributions of gene sizes (the mean genelength within each tissue is well within one standard deviation of
the means of all other tissues.)
We have compared codon usage between pairs of the six
tissues. When comparing testis to uterus, for example, we
calculate the distance between the codon usage of every pair ofgenes (including pairs from the same tissue), obtaining a 42by-42 symmetric matrix of pairwise distances. The distance
between two genes is given by the number of amino acids thatexhibit significantly different (P ⬍ 0.01) codon usage, as defined
above. Our results are not sensitive to the particular choice of athreshold P value within 0.001 and 0.05. By using the neighborjoining method (PHYLIP v3.5), we produced a dendogram thatgraphically represents the measured pairwise distances between
the codon usage in the study genes.
Fig. 1 shows the dendogram resulting from the codon usage intestis- and uterus-specific genes. Note that virtually all testisassociated genes are clustered in a separate clade from theuterus-associated genes. The observed clustering is the result ofsystematic differential codon usage between the testis- anduterus-specific genes. Fig. 1 indicates that we can generally
discriminate between testis- and uterus-expressed genes on thebasis of their codon usage alone.
The separation of testis and uterus genes seen in Fig. 1 would
not have occurred by random chance (P ⬍ 0.0008, see Methods).
Similarly, Fig. 2 indicates that brain-specific genes are easily
distinguishable from liver-specific genes on the basis of theircodon usage (P ⬍ 0.00018). We also find (trees not shown) thatovary-specific genes are distinguishable from vulva genes (P ⬍0.0032), brain genes are distinguishable from testis genes(P ⬍ 0.0044), brain genes are distinguishable from ovary genes(P ⬍ 0.00008), and vulva genes are distinguishable from testes
genes (P ⬍ 0.0092). All but one of these results remain significant
even after Bonferroni–Holm correction for multiple hypotheses.
Despite the results presented above, many pairs of tissuespecific gene sets do not exhibit significantly different codon
usage (e.g., liver versus uterus). The evolutionary processes that
produce differential codon usage between certain pairs of tissues
but not others pose an intriguing question for further research.
Evolutionary Preservation of Codon Usage
It is tempting to hypothesize that the highly nonrandom, tissuespecific codon usage we have observed serves an adaptivefunction. Although we cannot impute an adaptive function, we
can nevertheless demonstrate that the codon usage of brainspecific genes has been selectively preserved far more than
expected by chance during the evolution of human and mousefrom their common ancestor. For this analysis, we have identified and aligned mouse orthologs for the 44 brain-specific humangenes (see Methods) and for the other study tissues.
We considered only those sites in the alignment of the humanand mouse brain genes that exhibited either identical or synonymous codons. There are 31,050 such codons, which we concatenated into a single sequence for each organism. The resultingaligned mouse and human sequences are fairly similar in their
PNAS 兩 August 24, 2004 兩 vol. 101 兩 no. 34 兩 12589

EVOLUTION

Table 1. Comparison of codon usage between two human genes

usage than expected by random chance (P ⬍ 10⫺4), given themutations that have occurred between them. Although thealigned mouse and human sequences exhibit synonymous differences in 28% of their codons, these differences compensate
in such a way so as to preserve the overall codon usage. This
result suggests that there has been selection to preserve thecodon usage of these brain-specific genes throughout the evolution of mouse and human from their common ancestor.
In addition to brain-specific genes, the genes associated withmost of the other study tissues also show a highly significant
degree of synonymous codon usage preservation compared with
their mouse orthologs (P ⬍ 0.0032 each for liver, uterus, andvulva.) Notably, however, the synonymous codon usage in testesspecific and, to a lesser extent, ovary-specific genes do not show
significant preservation between human and mouse (P ⫽ 0.48
and P ⫽ 0.058, respectively). This result is analogous to thewell-established fact that the protein sequences of reproductive
genes, particularly those related to spermatogenesis, have undergone rapid evolution in primates (22). Apparently, synonymous codon usage in testes-specific genes is also undergoing
relatively more rapid divergence.

Fig. 2. A dendogram reflecting the codon usage of 44 brain-specific genes(red) and 34 liver-specific genes (blue). The observed separation between
these two classes of genes would not have occurred by random chance (P ⫽0.00018).

codon usage. There are only two amino acids that have asignificantly different encoding (P ⬍ 0.01) between the orthologous sequences.
The overall similarity of codon usage between the mouse andhuman brain-specific genes does not in itself imply that codon
usage has been selectively preserved, because the human andmouse sequences are similar by descent. There are only 8,837(synonymous) nucleotide mutations between the two sequences.
We have applied a randomization test to compare the codon
usage of the human and mouse sequences, controlling for theirsequence similarity. In each randomization trial, we started withthe mouse sequence, and we introduced in randomly chosensynonymous locations the observed number of nucleotidechanges (preserving even the number of mutations of each type,
A3C, A3T, A3G, C3A, etc.) to produce a randomized
version of the human sequence. The resulting randomized
sequence has the exact same amino acid and nucleotide composition as the observed human sequence. Moreover, the randomized human sequences contain virtually the same dinucleotide CpG content as the actual human sequence. The mean
number of occurrences of CpG in the codons of the randomized
sequences agrees with the actual number of CpGs in theobserved human sequence (all randomization trials fall within
2% of the observed human CpG content).
Among 10,000 such randomization trials, there were on average 7.53 amino acids that exhibited significantly different encodings between the mouse sequence and the randomized
human sequence. There were no examples in which the mousesequence and the randomized human sequence exhibited fewer
than four amino acids with different encodings. In other words,
even when controlling for their amino acid compositions, their
nucleotide compositions, and their CpG compositions, the human and mouse genes are far more similar in synonymous codon
12590 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404957101

Discussion
Here we have reported a significant difference between genesthat are selectively expressed in several human tissues: Suchgenes exhibit characteristic codon usage that, in many cases,
distinguishes the genes expressed in one tissue from thoseexpressed in another. Moreover, in most cases the tissue-specificcodon usage has been selectively preserved throughout theevolution of human and mouse from their common ancestor.
The biological mechanism and impact of this phenomenon
certainly require further study. Nevertheless, our results suggestthat synonymous codon usage in mammalian genes is not simply
the result of neutral evolutionary processes or isochore structure.
Previous studies have explored GC content at the third
position of coding sequences expressed in different humantissues (21). The GC3 content of the genes studied here does varyby tissue type, but the average GC3 content of one tissue is wellwithin one standard deviation of another tissue’s average: testes,
0.55 ⫾ 0.059; uterus, 0.58 ⫾ 0.014; brain, 0.56 ⫾ 0.053; liver,0.52 ⫾ 0.053; ovary, 0.57 ⫾ 0.19; vulva, 0.67 ⫾ 0.15. As a resultof the variation within each tissue, GC3 content alone is not
powerful enough to reliably separate genes by tissue type. For
example, a dendogram analogous to Fig. 1 based on pairwise
GC3-content distances results in an insignificant separation oftissue-specific genes (P ⫽ 0.53). The tissue-specific genes that wehave identified are characterized by differences in synonymouscodon usage above and beyond their GC3 content.
Our results on differential, tissue-specific codon usage suggest
several hypotheses about the mechanisms of protein regulationand tissue differentiation in humans. Differential codon usage
can impact tissue-specific modulation of proteins at several
levels. Codon usage in mammals is known to have dramaticeffects on translation rate (11–13), especially during cell differentiation (23). The existence of systematic tissue-specific codon
usages raises the important possibility that human tissues may
differ in their relative tRNA abundances and that these differences may modulate the expression of the appropriate proteins.
To our knowledge, detailed studies on relative tRNA abundances across human tissues have not yet been performed. Our
results suggest that such studies may be important for understanding tissue differentiation.
Differential synonymous codon usage has further biologicalconsequences because methylated C-residues in DNA frequently
result in transcriptional silencing (24). mRNA modifications arealso base-specific (e.g., pseudouridines). Furthermore, mRNAfolding into secondary and tertiary structures is sensitive to the
choice of synonymous codons (25). RNA transport, protein
Plotkin et al.

tional, posttranscriptional, translational, and transport processes, is most important to biological function. Our results
suggest that synonymous differences in the encoding of genes
may have been selected for and may be used at several levels of
regulation to reinforce differential protein levels of tissuespecific genes.
We thank Jonathan Dushoff, Alice Chen, Hagar Barak, andGyan Bhanot for their input throughout the preparation of this manuscript. J.B.P. acknowledges funding from the Harvard Society of Fellows.

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EVOLUTION

recognition of palendromic sequences, translational efficiency as
modulated by either tRNA abundance or the secondary structure of the mRNA are all impacted by the differences in thecodon usage of an mRNA. As a result, tissue-specific codon
usage also has implications for the optimal design of genetherapies targeted at specific tissues.
Transcriptional control has been the primary focus of gene
regulatory research, especially since the advent of mRNA expression arrays. Nevertheless, the level of mRNA expression
alone is not directly important to cellular function. Instead, thelevel of protein activity, which is a complex result of transcrip-

Plotkin et al.

PNAS 兩 August 24, 2004 兩 vol. 101 兩 no. 34 兩 12591

Table 2. A comparison of codon usage between two human genes
Codon Gene A, n (%)
gcc
9 (20)
gcg
3 (7)
gca
17 (38)gct
16 (36)
Ala, P = 0.000024
tgc
2 (12)tgt
14 (88)
Cys, P = 0.068653
gac
10 (53)gat
9 (47)
Asp, P = 0.020940
gag
13 (34)gaa
25 (66)Glu, P = 0.000006
ttc
8 (35)ttt
15 (65)Phe, P = 0.025510
ggc
8 (17)ggg
6 (13)gga
21 (45)ggt
12 (26)Gly, P = 0.000018
cac
1 (12)cat
7 (88)
His, P = 0.024818
atc
7 (22)
ata
9 (28)
att
16 (50)Ile, P = 0.008006
aag
8 (30)aaa
19 (70)Lys, P = 0.000258
ctc
6 (11)ctg
6 (11)cta
8 (15)ctt
20 (36)ttg
9 (16)tta
6 (11)
Leu, P = 0.000000

Gene B, n (%)
19 (58)8 (24)3 (9)3 (9)5 (50)
5 (50)
13 (93)1 (7)22 (92)2 (8)9 (82)
2 (18)
13 (52)9 (36)
3 (12)0 (0)7 (70)
3 (30)
6 (86)0 (0)
1 (14)
9 (100)0 (0)10 (21)37 (77)
0 (0)
1 (2)
0 (0)
0 (0)

Codon Gene A, n (%)
aac
8 (38)aat
13 (62)Asn, P = 0.076812
ccc
3 (12)
ccg
1 (4)
cca
13 (54)cct
7 (29)
Pro, P = 0.000009
cag
12
(60%)
caa
8
(40%)
Gln, P = 0.006346
cgc
1 (5)
cgg
0 (0)
cga
3 (14)
cgt
5 (24)agg
3 (14)aga
9 (43)
Arg, P = 0.000000
agc
5 (14)
agt
12 (32)
tcc
2 (5)
tcg
1 (3)
tca
6 (16)tct
11 (30)Ser, P = 0.000000
acc
11 (31)
acg
2 (6)
aca
14 (39)act
9 (25)
Thr, P = 0.036607
gtc
7 (16)gtg
13 (30)gta
11 (26)gtt
12 (28)
Val, P = 0.003838
tac
5 (36)tat
9 (64)
Tyr, P = 1.000000

Gene B, n (%)
5 (83)
1 (17)
16 (53)9 (30)
4 (13)1 (3)
23 (96)1 (4)8 (26)
7 (23)2 (6)0 (0)14 (45)0 (0)11 (46)0 (0)7 (29)
6 (25)0 (0)0 (0)9 (82)
0 (0)
1 (9)
1 (9)
8 (27)
18 (60)3 (10)
1 (3)
2 (50)
2 (50)For each codon, we report the absolute frequency in each gene and the relative frequency
compared to synonymous codons. The P value for each amino acid is calculated by using
a n × 2 Fisher exact test on the frequencies of synonymous codons. There are 11 aminoacids with significantly different encodings (P < 0.01) between these two genes. Gene A,
testes-specific glycerol kinase (GI:516123, 554 residues); gene B, endometrial bleeding
factor (gene B, GI:2058537, 371 residues).