Biochemical and Biophysical Research Communications 315 (2004) 10971103

BBRC
www.elsevier.com/locate/ybbrc

Correlations between nucleotide frequencies and amino acid

composition in 115 bacterial species
D. Bharanidharan, G. Ramya Bhargavi, Kavitha Uthanumallian, and N. Gautham*
Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India
Received 24 January 2004

Abstract

We studied the correlations between amino acid composition and mononucleotide and dinucleotide frequencies in 115 bacterial
genomes of varying G + C content. Observed amino acid frequencies were compared with those expected from the actual mono-
nucleotide and dinucleotide frequencies. Both mononucleotide and dinucleotide frequencies correlate well with the amino acid
frequency, with dinucleotide frequencies doing so better. Despite the strong correlations, some of the observed amino acid fre-
quencies, in particular for Arg, Val, Asp, Glu, Ser, and Cys, were consistently dierent from predicted values in all genomes. We
suggest that this variation from predicted values is a consequence of selection pressure at the level of amino acids, while the close
correspondence to the predictions in residues such as Thr, Phe, Lys, and Asn arises only from mutation and selection pressure at the
level of the nucleic acid sequences.
2004 Elsevier Inc. All rights reserved.

Keywords: Mononucleotide; Dinucleotide; Amino acid composition; Selection pressure

The relationship between the nucleotide base com-
position of a genome and the amino acid composition
of the corresponding proteome has been the subject of
many studies [116]. It is well known that the nucleotide
composition, as measured by the G + C content, shows
extreme variation across dierent genomes. In bacterial
species alone, it ranges from 22.5% to 72.0% [17].
Within any particular genome the bias extends across
all regions of it, both coding and non-coding [2,8,9]. In
the coding regions, one possible consequence of this
bias is variation in the amino acid composition of the
corresponding proteome. To illustrate this using an
extreme example, a genome consisting of only G and C,
i.e., 100% G + C content, can code only for Gly, Ala,
Arg, and Pro (or GARP) amino acids (on the basis of
the universal genetic code). In real genomes, with less
extreme, though still biased base composition, the ef-
fects are not as strong as the above example anticipates,
though they are still noticeable [7,10,11,13]. One of the
reasons for the attenuation in the eect is that the
variation in the nucleotide composition is most pro-
*
Corresponding author. Fax: +91-44-2235-2494.
E-mail address: gautham@unom.ac.in (N. Gautham).
nounced at the third codon position, resulting often in a
synonymous codon [5]. Such variation thus remains
silent. However, not all the variation is of this variety.
There are also smaller compositional dierences in the
rst and second codon positions. Together with the
non-synonymous changes at the third position, these
variations lead to marked dierences in the amino acid
frequencies [4,1215]. Singer and Hickey [13] have
demonstrated a correlation between the nucleotide
composition and the amino acid composition in 21
completely sequenced eubacterial and archaeal ge-
nomes. They have noticed that protein sequences in
G + C rich genomes contain a greater proportion of the
GARP amino acids, all of whose codons are also G + C
rich. Likewise, G + C poor genomes code mostly for the
FYMINK group of amino acids that have G + C poor
codons. Thus, nucleotide composition aects both
codon choice and amino acid composition.
The relationship between nucleotide and amino acid
frequencies is modulated by compositional preferences
in the proteome, arising from structural and functional
constraints. Accordingly, some amino acids occur at less
than the rates expected from a random distribution of
the 20 possible residues, or from a random distribution

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.01.129
1098 D. Bharanidharan et al. / Biochemical and Biophysical Research Communications 315 (2004) 10971103
of the 64 possible codons. Others occur at higher than
expected rates [18]. Based on the theoretically predicted
amino acid compositions derived from the nucleotide
frequencies of a set of 59 bacterial genomes, Lobry [10]
showed that in these genomes the observed frequencies
of Arg, Ser, Pro, and Cys were signicantly less than
expected, while for Ala, Lys, Asp, and Glu it was more
than expected. Others have reported similar, but not
identical, results [3,12]. Some of this variation can be
explained as a consequence of selection pressure at the
level of protein structure and function, i.e., the struc-
tural and functional constraints mentioned above. For
example, both Cys and Pro, strongly inuence protein
tertiary structure, the former through disulphide
bridges, and the latter by terminating helices and in-
troducing turns. However, similar reasons cannot be
easily attributed for the discrepancy from expected
composition in the other amino acids. It has been
speculated [3,19] that this discrepancy is a consequence
of the dinucleotide composition, rather than mononu-
cleotide frequencies. For example, Arg is coded by six
triplet codons, four of which begin with the dinucleotide
CG. This particular dinucleotide is known to be sup-
pressed in many genomes, including some of the bacte-
rial genomes [20]. This behaviour may lead to
suppression of codons for Arg and hence to an under-
representation of that amino acid. Elevation and sup-
pression of dinucleotide frequencies is a well-studied
phenomenon since Bird [21] reported the strong sup-
pression of CG in animal DNA. Other studies since then
[3,20,22,23] have reiterated the dierences between ob-
served dinucleotide frequencies and those expected on
the basis of the nucleotide composition. So much so,
these frequencies are often used as pattern recognition
tools in annotating DNA sequences [24,25]. However,
no previous study has rigorously analysed the link be-
tween dinucleotide and amino acid composition. In this
paper, we explore this relationship in bacterial genomes,
by theoretically predicting amino acid composition gi-
ven a specic dinucleotide and mononucleotide com-
position. A comparison between the theoretical
composition so obtained, and the one actually observed
in dierent bacterial proteomes shows that the observed
amino acid percentages correlate well with those pre-
dicted on the basis of the dinucleotide frequencies, just
as they do with those predicted on the basis of mono-
nucleotide frequencies. However, despite the overall
correlation between observations and predictions, some
of the amino acids, especially Arg, Val, Asp, Glu, Ser,
and Cys occur at quite dierent frequencies from those
predicted. The pattern of variation for these residues is
consistently the same in all the genomes studied, at
dierent G + C content. The other amino acids, in par-
ticular Thr, Phe, Lys, and Asn, follow the predicted
values very faithfully. We suggest that the former eect,
i.e., variation from predicted values, is a consequence of
selection pressure at the level of amino acids, while the
latter, i.e., close correspondence to the predictions,
arises only from mutation and selection pressure at the
level of the nucleic acid sequences.
Materials and methods
Source of the data. The data used in the analysis consisted of 115
bacterial gene sequences and the corresponding protein sequences,
obtained from the KEGG database [17]. Only sequences labelled as
coding sequences (CDS) were used. Hypothetical and putative
protein sequences were eliminated. We analysed 345,388 gene se-
quences, with a minimum of 480 sequences in the case of Mycoplasma
genitalium, and a maximum of 8317 sequences in the case of Brady-
rhizobium japonicum. The average length of the protein sequences is
368 amino acids. The chosen sequences were used to evaluate the
mononucleotide, dinucleotide, and amino acid composition of each
gene, which was then averaged to give the overall relative frequencies
for the genome. The expected relative frequencies were calculated, as
explained below, for each gene individually and the values were av-
eraged for each genome.
Calculation of expected relative amino acid frequencies. Expected
amino acid frequencies based on mononucleotide frequencies of a
genome were calculated as suggested by Lobry [10]. However, instead
of calculating the expected amino acid composition solely as a function
of the G + C content, we have used the actual relative frequencies of all
four nucleotides, PA , PC , PG , and PT , in each gene to calculate the
corresponding expected relative frequencies for the 20 amino acids.
The expected frequency of occurrence of each amino acid was obtained
as the sum of the expected frequencies of occurrence of its codons.
Thus, for example, for Gln
PGln PCAA PCAG 1
PCAA and PCAG are expressed in terms of the independent probability of
the three bases in the codon, normalised to take the three stop codons
TAA, TAG, and TGA into account, e.g.,
PC PA PA
PCAA : 2
1
PT PA PA PT PA PG PT PG PA
Expected amino acid frequencies based on observed dinucleotide
frequencies were calculated in a similar manner. Again, the relative
codon frequencies were rst obtained. For example, the probability of
occurrence of the codon CAA was calculated as the product of three
numbers: the probability of C at rst position, the conditional prob-
ability of A in the second position given C in the rst, and the con-
ditional probability of A in the third position given A in the second.
The rst number is the sum of the relative frequencies of the dinu-
cleotides that have C in the second position, i.e.,
PC PAC PTC PGC PCC ; 3
where PAC , etc., are the relative dinucleotide frequencies of AC, etc.
observed in the gene. This way of calculating what is essentially the
mononucleotide probability for C ensures that the numbers are cor-
rectly normalised. Also, we take into consideration the fact that the
rst base of a codon occurs at the second position of the dinucleotide
that begins at the end of the previous codon. The second number is
calculated as
PAjC PCA =PCA PCT PCG PCC : 4
Similarly the third number is
PAjA PAA =PAA PAT PAG PAC : 5
Again, the amino acid probabilities were calculated as the sum of the
probabilities of the respective codons, normalised to allow for the stop
codons.
 D. Bharanidharan et al. / Biochemical and Biophysical Research Communications 315 (2004) 10971103
Calculation of the expected relative dinucleotide frequencies. The
ratios of the observed relative dinucleotide frequency to the one
expected from the mononucleotide relative frequencies were calculated
as follows. For a dinucleotide XY, this ratio is given as
RXY PXY =PX PY ; 6
where PX denotes the probability of occurrence of the nucleotide X and
PXY is the probability of occurrence of the dinucleotide XY in the gene.
This ratio has also been referred to as genomic signature [20].
Statistical characterisation of composition. The distance between the
observed and predicted composition of amino acid for a given amino
acid aa was calculated as:
s
1X n cients and the distance between the observed and pre-
Daa Paa;i obs
Paa;i pred2 ; 7
n i1
where Paa;i obs and Paa;i pred are the frequencies of the observed
and predicted amino acid compositions, respectively, and the sum-
mation is over all the genomes (n) included in the calculations. The
statistical signicance of this distance was calculated using a two-tailed
paired t test.
Results and discussion
Fig. 1 shows the average composition of each amino
acid in all 115 genomes. Cys, Trp, His, and Met have the
least occurrences. Moreover, the low percentage is
constant in all genomes, as indicated by the low value of
the standard deviation. Clearly, the formation of disul-
phide bridges by Cys is responsible for its scarcity.
Again, the low percentage of Met may be because it is
principally an initiation amino acid [16]. However,
similar reasons are not available to explain the low
percentage of His and Trp. The hydrophobic amino
acids Leu, Ala, and Ile have the highest average com-
position, as well as a large range. Lys, Asn, and Arg also
have a large range of compositions.
Fig. 2 shows observed and predicted values for the
115 bacterial genomes for all twenty amino acids. For
clarity, the predicted values are plotted as lines. The rst
four gures are for GARP residues that have G + C rich
codons, while the last six are for FYMINK residues that
have A + T rich codons. The rest are classied as neutral
[11]. The overall behaviour seen in the gures has been
described earlier [13]. The observed frequencies of
Fig. 1. Average composition of each amino acid in all 115 bacterial
genomes.
1099
GARP amino acids increase with G + C content of the
genome, while those of FYMINK amino acids decrease.
For a majority of the amino acids, the observed values
closely follow the predicted ones. Since the predictions
are based on genome composition, this probably attests
to the strong inuence of mutation pressure in designing
the genome. However, selection pressure by its action on
mono and dinucleotide composition must also be con-
sidered [20,26]. Other common features of the data are
evident in Table 1, which gives the correlation coe-
dicted amino acid frequencies. Both G + C rich amino
acids and G + C poor amino acids (except for Met) have
strong correlations between the observations and pre-
dictions, though this is not the case for the neutral
amino acids. For these amino acids, the correlation
coecients vary from )0.17 to 0.74 in relation to the
mononucleotide-based predictions, and from )0.01 to
0.84 for the dinucleotide-based predictions. In general,
the compositions of the neutral amino acids correlate
better with the dinucleotide frequencies than the
mononucleotide frequencies. The dierences between
the observed and predicted composition, as measured by
the distance, are less than 2.9% points for all amino
acids, except Arg. Considering however that there are 20
amino acids, and the frequency of each is very approx-
imately 5%, even these seemingly small dierences may
be highly signicant.
Of the GARP amino acids (Figs. 2AD) Pro and Arg
show a similar pattern of behaviourat low G + C
content (2232%) the observed values are the same as
the predicted values, while at high G + C content (62
72%) they are signicantly dierent. For Pro, the dis-
tance D between observed and predicted composition
for the highest G + C content genomes is 6.44 percent-
age points (P < 10
14 ) for mononucleotide frequency-
based predictions. For dinucleotide frequency-based
predictions D 4:27 (P < 10
10 ). Likewise for Arg,
D 6:8 (P < 10
19 ) for mononucleotide-based predic-
tions, and 7.7 (P < 10
14 ) for dinucleotide-based pre-
dictions. The other two amino acids in this group, Gly
and Ala, follow an almost identically opposite pattern,
with predicted and observed percentages being the same
at high G + C content and signicantly dierent at low
G + C content (D 2:5 and 2.3, P < 10
12 and <10
13
for Gly; D 2:9 and 2.5, P < 10
11 and P < 10
11 for
Ala). These results are best interpreted as a consequence
of selection at the amino acid level, rather than muta-
tion or selection at the gene level. This is borne out by
the fact that the composition of these four amino acids
does not change appreciably between genomes. For
example, in the case of Arg, the observed percentages
range from 2.8% to 8.3% only, while the predicted
values have a much larger rangefrom 5.2% to 15.5%
in the case of mononucleotide frequency-based predic-
tions, and from 4.7% to 16.7% in the case of dinucleotide
1100 D. Bharanidharan et al. / Biochemical and Biophysical Research Communications 315 (2004) 10971103

Fig. 2. The observed and predicted values in 115 bacterial genomes for all 20 amino acids. The gures (AD), GARP residues, have G + C rich
codons, while the gures (OT), FYMINK residues, have A + T rich codons. The rest are neutral.

frequency-based predictions. Similar values are seen Is the generally lower percentage of Arg seen in the
for the other three amino acids that belong to this proteomes a consequence of CG suppression? Our re-
group. sults show that this is not so. The dinucleotide ratio,
 D. Bharanidharan et al. / Biochemical and Biophysical Research Communications 315 (2004) 10971103 1101

Table 1
The correlation coecients (r), distance (D, in percentage), with signicance levels, between the observed and predicted amino acid frequencies, based
on mononucleotide composition and dinucleotide composition
Amino acid Mononucleotide-based Dinucleotide-based
r D P value r D P value
Gly 0.93 1.71 >5% 0.92 1.57 <10
2
Ala 0.95 2.90 <10
6 0.95 2.20 <10
3
Arg 0.90 4.47 <10
5 0.88 4.38 <10
4
Pro 0.90 2.85 <10
2 0.92 1.95 <10
2
Trp 0.72 0.56 <10
5 0.70 0.64 <10
6
Val 0.12 1.20 <10
2 0.48 1.95 <10
5
Ser )0.11 2.90 <10
4 0.15 2.48 <10
3
Thr 0.35 0.87 <10
4 0.70 0.52 >5%
His 0.56 0.74 <10
7 0.63 0.81 <10
5
Asp )0.17 2.03 <10
7 0.57 2.07 <10
12
Glu 0.74 2.75 <10
10 0.84 2.61 <10
10
Leu )0.07 1.60 <10
1 )0.01 1.89 >5%
Gln 0.33 0.95 <10
3 0.43 0.82 <10
2
Cys 0.38 2.03 <10
5 0.09 2.54 <10
7
Phe 0.80 1.01 <10
2 0.88 1.32 >5%
Tyr 0.87 1.29 <10
1 0.86 0.95 >5%
Met 0.12 0.88 <10
3 0.51 0.60 <10
3
Ile 0.94 1.60 <10
1 0.97 1.45 <10
3
Asn 0.93 0.77 >5% 0.94 0.68 >5%
Lys 0.95 1.56 <10
5 0.98 1.01 >5%

Table 2
Ratio of the observed to predicted values of the dinucleotide frequencies
G+C % AA AT AG AC TA TT TG TC GA GT GG GC CA CT CG CC
2227 1.18 1.18 0.83 0.88 0.68 1.26 1.00 1.10 1.08 0.75 0.99 1.06 1.00 1.00 1.01 1.01
2732 1.14 1.14 0.85 0.84 0.67 1.06 1.09 1.06 1.04 0.80 1.00 1.20 1.07 0.94 1.00 1.02
3237 1.11 1.00 0.97 0.81 0.68 1.07 1.08 1.02 1.09 0.82 1.03 1.25 0.96 1.07 0.83 1.00
3742 1.18 1.00 0.88 0.84 0.72 1.18 1.08 0.94 0.98 0.84 0.92 1.29 1.00 0.98 0.94 0.96
4247 1.13 1.10 0.83 0.93 0.65 1.07 1.16 1.00 1.06 0.85 0.94 1.18 1.09 0.95 0.92 0.92
4752 1.13 1.03 0.81 0.92 0.62 1.03 1.19 1.00 1.01 0.91 0.87 1.28 1.10 0.96 0.99 0.84
5257 1.16 1.01 0.82 0.95 0.68 1.04 1.18 1.05 1.00 0.94 0.86 1.12 1.03 1.02 0.95 0.87
5762 1.20 1.03 0.80 0.88 0.74 1.18 1.17 0.90 0.91 0.88 0.96 1.22 1.00 0.99 1.01 0.90
6267 1.16 0.99 0.87 0.89 0.67 1.11 1.18 0.93 1.00 0.86 0.93 1.24 1.12 1.00 1.00 0.96
6772 1.14 1.21 0.79 0.90 0.72 1.00 1.24 0.83 0.94 0.85 0.87 1.18 1.10 0.83 1.02 0.89
Average 1.16 1.04 0.86 0.88 0.69 1.10 1.13 0.98 1.01 0.85 0.94 1.22 1.04 0.98 0.95 0.95
The G + C content was separated into ten bins at intervals of 5%, from 22% to 72%. The values were averaged for each bin.

Table 2, gives the extent of CG suppression in the
coding regions of genomes obtained as a ratio of the
observed to predicted values, which were derived from
the theoretical sequences on the basis of the mononu-
cleotide frequencies. The average CG ratio in all the
genomes is 0.95. The value ranges from 0.50 to 1.33.
Table 2 also gives the dinucleotide ratios in dierent bins
of G + C content. Clearly, there is no correlation be-
tween CG suppression (or elevation) and G + C content
on the one hand, and CG suppression and the sup-
pression of Arg, on the other. Moreover, from the co-
don usage tables for Arg for all bacterial genomes [27]
we see that codons containing CG are not discriminated
against. Thus, it is not the case that the Arg residues that
we do observe in the proteome are all coded for by CG
decient codons, and that reason for the low values of
Arg is that the number of such codons for this residue is
only a small fraction of the six that code for it. To
summarize this discussion, Arg occurs at lower than
expected rates in the bacterial proteomes, and this sup-
pression is not a consequence of CG suppression.
The case of Pro is somewhat dierent. Only in ge-
nomes of high G + C content is the frequency markedly
lower than the expected value. However, as in the case
of Arg, this may be more because of the selection
pressure towards a more or less constant percentage of
the amino acid. In any case, the dinucleotide ratio for
CC (all four codons for Pro start with this dinucleotide)
is close to 1.0 in all the genomes (Table 2), including
those with high G + C content, and there is no rela-
tionship between this dinucleotide frequency and Pro
composition. Of the other two amino acids in this
1102 D. Bharanidharan et al. / Biochemical and Biophysical Research Communications 315 (2004) 10971103
group, Gly behaves like Pro and has a fairly constant
percentage in all the genomes, no matter what the G + C
content. It is notable that the two amino acids at the
opposite ends of the spectrum of conformational exi-
bility both have such similar behaviour. That is, both
are nearly constant, and both follow the predicted pat-
tern, except Pro in high G + C genomes, and Gly in low
G + C genomes. Ala has a large variation in composition
between the genomes, ranging from 3.6% to 13.7%. The
predicted values for this amino acid also have a large
range, and the predicted patterns of variation follow the
observed pattern closely.
We next discuss the amino acids that are neutral
with respect to the G + C content of their respective
codons. This is the largest of the three classes and con-
tains the following ten amino acids: Cys, Ser, Val, Asp,
Glu, Trp, His, Gln, Thr, and Leu. The pattern of vari-
ation in the amino acid composition is of three types.
Cys and Ser occur with lower frequencies than expected
in all bacterial genomes. For Cys the distance D 2:3
and 2.5 (with signicance levels P < 10
9 and 10
7 ) for
predictions based on mononucleotide and dinucleotide
frequencies, respectively. For Ser, D 2:9 and 2.5
(P < 10
4 and 10
4 ) for the two predictions, respec-
tively. Cys, with two codons, has a strong eect on the
three-dimensional structure of the protein, and if it were
to occur with a higher proportion, it could lead to
misfolding through formation of non-native disulphide
bridges. Ser has six codons, four of which begin with the
dinucleotide TC, and its pattern of variation is remi-
niscent of Arg, which also has six codons. At low G + C
content the frequency is almost identical to that pre-
dicted. At high G + C content, the dierence is the
largest. Like the relationship between Arg and the di-
nucleotide CG, the under-representation of Ser is not a
consequence of TC suppression. Table 2 shows that, in
fact, there is no suppression of TC in any of the G + C
bins. Again like Arg, and indeed like many of the other
amino acids, there is an apparent need for a constant
proportion of this amino acid, no matter what the G + C
content of the genome. The second pattern of variation
is seen in Val, Asp, and Glu. These three residues occur
with signicantly higher frequency than predicted in all
genomes (D 1:2 and 1.9, P < 10
2 and 10
5 for Val;
D 2:0 and 2.1, P < 10
7 and 1012 for Asp; and D 2:8
and 2.6, P < 10
10 and <10
10 for Glu). The third pat-
tern of variation is seen in Trp, His, Gln, and Thr. The
frequencies of these amino acids are not dierent from
the predicted values (average D < 1:0 and average
P < 10
4 for both mononucleotide- and dinucleotide-
based predictions) except Leu, which shows an elevation
in high G + C content genomes (D 3:5 and 3.7,
P < 10
9 and 10
13 ).
The nal class of amino acids are the FYMINK
group with G + C poor codons. As seen in Figs. 2OT,
the amino acid compositions in this group have a close
and inverse relationship with the G + C content of the
genome. At low G + C content, the frequency is high,
while at high G + C content it is low. The composition of
Ile is signicantly dierent from predicted values at high
G + C content (D 2:6 and 2.3, P < 10
11 and
P < 10
15 ). At low G + C content Tyr shows dierent
values (D 2:5 and 1.2, P < 10
10 and P < 10
4 ). Lys
and Asn occur at almost exactly the same percentages as
predicted, and so does Phe, though to a lesser extent.
In conclusion, the above results may be classied in
two dierent ways. First, we consider the correlation
between observed and predicted values. In the G + C
poor and the G + C rich amino acids the two values are
highly correlated in all genomes considered here.
Among the neutral amino acids, the values for Trp and
Glu are highly correlated. Thr and His show correlation
only with the dinucleotide-based predictions, and Asp to
a lesser extent. Others show no correlation. Second, we
consider whether the predicted values are close to the
observed values. Here there are three possibilities: the
observed values could be higher than those predicted
(elevation); they could be lower (suppression) or they
could be approximately the same. Val, Asp, and Glu
belong to the rst category, Pro, Arg, Cys, and Ser to
the second and others to the third.
These results show that, to a large extent, nucleotide
frequencies decide the amino acid composition. How-
ever, the amino acid composition of the proteome is
not decided entirely by nucleotide frequencies, and
show the eect of selection pressure, with some, such
as Pro, Arg, Cys, and Ser, occurring at consistently
less than predicted values, and some, such as Val, Asp,
and Glu, occurring always at greater than predicted
values. The amino acid composition of the proteome is
thus decided partly by mutation pressure (mononu-
cleotide frequencies) and partly selection pressure (di-
nucleotide frequencies) on the genome, and partly by
selection pressure on the proteome. This statement
may be interpreted in two ways. One, the observed
percentage of occurrence of each amino acid is the
resultant of a combination of random (mutation) and
directed (selection) forces. It then follows that while
the proteome requires a certain denite amount of
each amino acid, random variations of this amount are
tolerated. The second interpretation assumes that the
set of twenty amino acids is degenerate, and require-
ments for a particular physical or chemical property in
the protein may be satised by more than one residue.
Therefore, changes in the genome towards higher or
lower G + C content are followed by changes favouring
one or another set of amino acids in the proteome.
Our own results, as well as other, earlier work
[3,10,13,19], in general favour the second interpreta-
tion. These patterns in the amino acid compositions
may thus be one facet of the wider degeneracy seen in
biological systems [28].
 D. Bharanidharan et al. / Biochemical and Biophysical Research Communications 315 (2004) 10971103
Acknowledgments
We acknowledge the nancial assistance from the following or-
ganisations of the Government of India: CSIR under the NMITLI
project, UGC under the SAP programme, and DST under the FIST
programme.
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