The Roles and Evolutionary Patterns of Intronless Genes in Deuterostomes

Abstract

Genes without introns are a characteristic feature of prokaryotes, but there are still a number of intronless genes in eukaryotes. To study these eukaryotic genes that have prokaryotic architecture could help to understand the evolutionary patterns of related genes and genomes. Our analyses revealed a number of intronless genes that reside in 6 deuterostomes (sea urchin, sea squirt, zebrafish, chicken, platypus, and human). We also determined the conservation for each intronless gene in archaea, bacteria, fungi, plants, metazoans, and other eukaryotes. Proportions of intronless genes that are inherited from the common ancestor of archaea, bacteria, and eukaryotes in these species were consistent with their phylogenetic positions, with more proportions of ancient intronless genes residing in more primitive species. In these species, intronless genes belong to different cellular roles and gene ontology (GO) categories, and some of these functions are very basic. Part of intronless genes is derived from other intronless genes or multiexon genes in each species. In conclusion, we showed that a varying number and proportion of intronless genes reside in these 6 deuterostomes, and some of them function importantly. These genes are good candidates for subsequent functional and evolutionary analyses specifically.

Full text

Hindawi Publishing Corporation
Comparative and Functional Genomics
Volume 2011, Article ID 680673, 8pages
doi:10.1155/2011/680673
Research Article
The Roles and Evolutionary Patterns of Intronless
Genes in Deuterostomes
Ming Zou,1, 2 Baocheng Guo,3, 4 and Shunping He1
1The key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology,
Chinese Academy of Sciences, Wuhan 430072, China
2Institute of Hydrobiology, Graduate University of the Chinese Academy of Sciences, Beijing 100039, China
3Institute of Evolutionary Biology and Environmental Studies, University of Zurich, 8057 Zurich, Switzerland
4The Swiss Institute of Bioinformatics, Quartier Sorge-Batiment Genopode, 1015 Lausanne, Switzerland
Correspondence should be addressed to Shunping He, heshunping@gmail.com
Received 23 July 2010; Revised 13 April 2011; Accepted 22 June 2011
Academic Editor: J. Peter W. Young
Copyright © 2011 Ming Zou et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genes without introns are a characteristic feature of prokaryotes, but there are still a number of intronless genes in eukaryotes.
To study these eukaryotic genes that have prokaryotic architecture could help to understand the evolutionary patterns of related
genes and genomes. Our analyses revealed a number of intronless genes that reside in 6 deuterostomes (sea urchin, sea squirt,
zebrafish, chicken, platypus, and human). We also determined the conservation for each intronless gene in archaea, bacteria,
fungi, plants, metazoans, and other eukaryotes. Proportions of intronless genes that are inherited from the common ancestor
of archaea, bacteria, and eukaryotes in these species were consistent with their phylogenetic positions, with more proportions
of ancient intronless genes residing in more primitive species. In these species, intronless genes belong to different cellular roles
and gene ontology (GO) categories, and some of these functions are very basic. Part of intronless genes is derived from other
intronless genes or multiexon genes in each species. In conclusion, we showed that a varying number and proportion of intronless
genes reside in these 6 deuterostomes, and some of them function importantly. These genes are good candidates for subsequent
functional and evolutionary analyses specifically.
1. Introduction
Most eukaryotic genes are interrupted by one or more
noncoding sequences called introns, and intronless genes are
a characteristic feature of prokaryotes. However, researches
on intronless genes in eukaryotes have been reported over the
past few decades [1–4]. Many human genes, like G protein-
coupled receptor genes, are intronless [5]andthehuman
genome report identified 901 predicted intronless genes [6].
Recently, Tay et al. found that many single-copy primate-
specific human transcriptional units are single exon [7].
Moreover, Yang et al. found that species-specific genes in
Arabidopsis,Oryza, and Populus are enriched with intronless
genes [8]. A retrogene, which is formed by homologous
recombination between the genomic copy of a gene and an
cDNA [9], is also considered to be intronless, and it has been
reported that many retrogenes exist in eukaryotic genomes
[10–12]. Intronless genes in eukaryotes, because of their
prokaryotic architecture, provide interesting datasets for
comparative genomics and evolutionary studies. Studying
these genes can help to understand the evolutionary patterns
of related genes and genomes. As a result, systematical
researches on intronless genes in many species from mam-
mals to plants have been reported [13–18]. Several databases
of these single exon genes, such as SEGE [19] and Genome
SEGE [20], have been set up and are of important use
for evolutionary and functional studies. However, former
evolutionary researches on intronless genes have usually been
limited to 1 to 2 species and studies within a phylogenetic
framework are rare. With the development of sequencing
technology, more and more complete genomes have been
sequenced and annotated, which makes comprehensive com-
parative analysis on intronless genes possible. The present
study was designed to identify and analyse intronless genes in

2Comparative and Functional Genomics
6 deuterostomes, sea urchin (Strongylocentrotus purpura-
tus), sea squirt (Ciona intestinalis), zebrafish (Danio rerio),
chicken (Gallus gallus), platypus (Ornithorhynchus anat-
inus), and human (Homo sapiens), which were selected
because of their pivotal phylogenetic positions. We compared
the functions and conservation of these genes between and
within species in an attempt to gain some evolutionary
meaningful insights.
2. Materials and Methods
2.1. Data Source of Intronless Genes. The annotated genomes
(GenBank Flat File Format) of sea urchin, sea squirt, zebra-
fish, chicken, platypus, and human were downloaded from
the NCBI ftp server (ftp://ftp.ncbi.nih.gov/genomes/,10
Jun 2009). Using a customized Perl script, we extracted
protein sequences for all the intron and intronless genes
from each annotated genome. During our processing, a gene
was classified as intron-containing if the “CDS” line in the
FEATURES contains a “join”; otherwise, it was classified as
an intronless gene. Proteins that encoded by mitochondrial
genomes were removed. To avoid any ambiguity, proteins
encoded by genes which have the symbol “<”or“>”in
their annotation (“<” indicates partial on the 5end and “>”
indicates partial on the 3end) were also discarded.
2.2. Functional Assignment and Category. ProtFun is an on-
line procedure designed to produce ab initio predictions of
protein functions from sequences and combines 14 different
sequence-based functional prediction methods. ProtFun
queries a large number of other feature prediction servers to
obtain information on various posttranslation and localisa-
tion aspects of the protein to predict protein function, rather
than relying on sequence similarity compared with other
protein function prediction procedures [21,22]. Therefore,
functional assignments of intronless genes in our study were
done with the webserver ProtFun (http://www.cbs.dtu.dk/
services/ProtFun/) and sequences were clustered according to
their cellular roles and gene ontology (GO) categories.
2.3. Distribution, Conservation, and Paralogue Identification
of Intronless Genes. Genes (both intronless and intron-
containing genes) in archaea, bacteria, fungi, plants, meta-
zoans, and other eukaryotes homologous with our intronless
genes (BLAST score more than 100), were determined on
the basis of sequence similarity using BLink (BLAST Link),
which is a tool that displays the precomputed results of
BLAST searches that have been completed for every protein
sequence in the Entrez proteins data domain [24]andis
available at NCBI.
CD-HIT is a program for clustering the entries in a
large protein database according to sequence identity (with a
high threshold of identity). CD-HIT can remove redundant
sequences and generate a database of only the representatives
[25]. To determine the conservative intronless genes among
the 6 deuterostome species in this study, we clustered all of
our intronless genes using CD-HIT. In order to determine
the relationships among these intronless genes, we clustered
them and identified nonredundant intronless genes in each
genome. We also clustered intron-containing genes to pro-
duce nonredundant multiexon genes in each genome. We
clustered these nonredundant intronless genes with nonre-
dundant multiexon genes in the same genome and produced
a list of corresponding intronless and intron-containing
genes to determine the relationships between intronless and
intron-containing genes. All these data handling were done
with CD-HIT.
3. Results and Discussion
3.1. Intronless Genes in Deuterostomes. Sea urchin, sea squirt,
zebrafish, chicken, platypus, and human were selected to
represent the major groups of deuterostomes and the intron-
less genes in their genomes were identified. Gi number and
protein sequence for each intronless gene in each species
were obtained from processing their annotated genomes. As
a result, there are abundant intronless genes in each of the
6 deuterostome genomes. The numbers of intronless genes
in each species is given in Tab le 1 and details are given in
supplementary material online at doi:10.1155/2011/680673.
Among the selected species, human has the maximum
number of intronless genes (6229) and platypus has the
least (930). We can see the maximum one is nearly seven
times the number of the least one. However, the difference
among numbers of intronless genes in sea urchin (2482),
zebrafish (2169), chicken (1659), and sea squirt (1448) is not
significant and these numbers should increase and be more
accurate when their well-annotated genomes are available.
Since a few previous studies reported a bit lower numbers
of the number of intronless gene [16,20], we compared
protein numbers (encoded by intron and intronless genes)
from Ensemble (Tab l e 1) with ours, and found the former
was always larger. Compared to their numbers, proportions
that intronless genes are accounting for total genes do not
differ significantly, and the maximum one is about twice the
number of the least one (Tab le 1 ). In fact, former researches
reported that 11109, 5846, and 5085 intronless genes reside
in rice, Arabidopsis and mouse genomes, accounting for
19.9%, 21.7%, and 18.9% genes correspondingly [13]. Given
that the total gene numbers and annotation qualities between
species are different, these data may indicate that although
the number of intronless genes varied significantly between
species, the proportions that they account for total genes
are nearly constant. However, the number and percentages
of intronless genes do not correlate with their genome sizes
(P>0.6, |r|<0.3, Spearman’s test). The human genome has
the largest number of intronless genes, which might be due to
the following reasons. Firstly, human has the most complete
expression data, which could result in more annotated genes
compared with other species during the genome annotation
process. Secondly, the human genome has many more
retrogenes compared with other species [26,27]. Thirdly,
duplications of intronless genes are common in the human
genome (see later). Plenty of intronless genes exist in the
6 deuterostomes indicating they may play important roles
during deuterostome evolution. Earlier, Jain et al. found that

Comparative and Functional Genomics 3
Tab le 1: The C-values and statistics for genes (intron and intronless) in each species.
Species Sea-Urchin Sea-Squirt Zebrafish Chicken Platypus Human
C-value (pg) 0.89 0.20 1.75 1.25 3.06 3.5
N— 19858 40585 22194 26836 88237
I2482 (8.6) 1448 (11.2) 2169 (8.6) 1659 (10.2) 930 (7.9) 6229 (16.7)
NR∗676 1263 856 1029 516 2290
NRI∗8792 8502 10620 9502 7840 12823
R∗∗ 621 (92) 110 (9) 274 (32) 156 (15) 86 (17) 1321 (58)
C∗∗ 212 (31) 191 (15) 269 (31) 186 (18) 133 (26) 665 (29)
C: values are obtained from http://www.genomesize.com/.
N: number of proteins encoded by intron and intronless genes, obtained from Ensemble (http://www.ensembl.org/index.html).
I: number of intronless genes in each genome, numbers in parentheses are percentages of intronless genes account for total genes.
NR: number of nonredundant intronless gene clusters.
NRI: number of nonredundant intron gene clusters.
R: number of nonredundant clusters that represent more than one intronless gene.
C: number of clusters that represent both a nonredundant intronless and a nonredundant intron-containing gene.
∗Clustered using CD-Hit (identity =0.3).
∗∗Numbers in parentheses are percentages they account for all nonredundant intronless gene clusters.
intronless genes have a strong bias towards encoding shorter
proteins [13]. Here we testified that the average length of
intronless genes is significantly shorter than multiexon genes
in all the selected species (P<0.001, Mann-Whitney Test).
The average length for intronless genes in sea urchin, sea
squirt, zebrafish, chicken, platypus, and human is 341.75 bp,
389.34 bp, 378.78 bp, 259.53 bp, 294.43 bp, 241.26 bp, and for
intron genes is 530.59 bp, 540.58 bp, 553.04 bp, 541.31 bp,
528.11 bp, and 503.31 bp, respectively.
Among the selected species, chromosomes were well as-
sembled in human, chicken, and zebrafish. To study the dis-
tribution of intronless genes in each selected genome, we
counted the numbers of intronless genes on each of their
chromosomes (Figure 1). Spearman’s test showed that the
number of intronless genes is significantly correlated with
the length of their chromosomes in human (P<0.001, r=
0.721) and chicken (P<0.001, r=0.712). The correlation
may be also significant in zebrafish (P=0.119, r=
0.320) given that nonparametric tests have less “power” to
detect a significant difference. Therefore, we proved that the
distributions of intronless genes in human and chicken (and
maybe in zebrafish) are stochastic, just like previous studies
in mouse, rice, and Arabidopsis [13,18]. However, several
clusters of intronless genes exist in certain chromosomes and
some of these clusters have been reported. For example, the
olfactory receptor gene clusters on human chromosome 17
and odorant receptor genes in the zebrafish genome [28,29].
3.2. Functional Assignment of Intronless Genes. It has been
shown that the distribution of intronless human genes across
molecular function categories is nonrandom [17]. In order
to study the molecular function categories of intronless
genes in the 6 selected species, their cellular roles and GO
categories were predicted using ProtFun (available via web-
server http://www.cbs.dtu.dk/services/ProtFun/). Figure 2
shows the distribution of intronless genes among each
cellular role in 6 species. As in plants, intronless genes that
functionally belong to translation and energy metabolism
are the commonest in most species, followed by the cell
envelope and amino acid biosynthesis [13]. Furthermore, in
these 6 deuterostomes, transport and binding, followed by
regulatory functions and central intermediary metabolism,
are also well represented compared with other function
categories. The percentage of intronless genes with the same
cellular role among the total intronless genes varies sig-
nificantly between species (Figure 2). For example, 7% of
intronless genes are transported and binding in sea squirt
is significantly fewer than in other species. The number of
cellular roles, such as amino acid biosynthesis and central
intermediary metabolism, are quite similar in sea urchin,
sea squirt, zebrafish, and human. However, this is not the
case for chicken or platypus. GO categories can be assigned
to more than 70% of intronless genes except in sea squirt
(which is more than 60%), and the distribution of genes
according to each GO category is shown in Figure 3.As
in plants, proteins associated with the GO category growth
factor, transcription regulation, transport, immune response
and structural proteins are overrepresented in these species
[13]. Furthermore, proteins associated with the GO category
transcription, which might be different between plants and
animals, are well represented in deuterostomes. The percent-
age of total intronless genes that proteins with a certain GO
category, such as growth factor and transporter proteins,
varied significantly among these species. According to their
cellular roles and GO categories, the functional category
distribution of intronless genes in each selected genome
is very similar to those reported for rice and Arabidopsis
[13]. This result might indicate that biological mechanisms
related to intronless genes are common in the biological
kingdom. On the basis of earlier work and this analysis,
we concluded that most plant and deuterostome intronless
genes have the same characteristics, but deuterostomes still
have some lineage-specific and species-specific functional
intronless genes.

4Comparative and Functional Genomics
Human
Chromosome number
12
10
8
6
4
2
0
700
600
500
400
300
200
100
0
xy
2468
10 12 14 16 18 20 22
Number of genes
Genes (%)
(a)
Chick
Chromosome number
2 4 6 8 10 12 14 16 18 20 22
20
15
10
5
0
300
250
200
150
100
50
0
24 26 28wz
Number of genes
Genes (%)
(b)
Chromosome number
Zebrafish
7
6
5
4
3
2
1
0
200
150
100
50
0
24681012141618202224
Number of genes
Genes (%)
Genes (%)
Number of genes
(c)
Figure 1: The numbers of intronless genes on each chromosome in
human, chicken, and zebrafish. Both numbers and percentages are
shown.
3.3. Taxonomic Distribution. To study the evolutionary pat-
terns of intronless genes in major taxonomic groups, we used
BLink, a tool that displays the precomputed results of BLAST
searches for every protein sequence from the entrez proteins
data domain [24], to determine the evolutionarily conserved
proteins among different taxonomic groups (archaea, bac-
teria, fungi, metazoans, plants, and other eukaryotes). The
results of intronless gene clustering on the basis of homology
with each taxonomic group are given in Ta b le 2 ,andthis
will change as more genome sequences become available. We
divided these genes into 7 types of combination according
to their conservation among archaea (A), bacteria (B), and
eukaryote (E) and the distributions are shown in Figure 4.
Majority of intronless genes in each species that have homo-
logues only in eukaryotes (E) suggested that most intronless
0
5
10
15
20
25
30
35
AAB BOC CE CP CIM EM FAM PAP RF RAT T TAB
Cellular role
Genes (%)
su sq z
cph
Figure 2: Distribution of intronless genes among different cellular
roles in each species. Su: sea urchin; sq, sea squirt; z: zebrafish;
c:chicken;p:platypus;h:human.AAB:aminoacidbiosynthesis;
BOC: biosynthesis of cofactors; CE: cell envelope; CP: Cellular pro-
cesses; CIM: Central intermediary metabolism; EM: energy meta-
bolism; FAM: fatty acid metabolism; PAP: purines and pyrimidines;
RF: regulatory functions; RAT: replication and transcription; T:
translation; TAB: transport and binding.
0
5
10
15
20
25
30
ST R H SP T IC VGIC CC TR TRR SR IR GF MIT
GO category
su sq z
cph
Genes (%)
Figure 3: Distribution of intronless genes among different kinds of
gene ontology (GO) categories in each species. su: sea urchin; sq:
sea squirt; z: zebrafish; c: chicken; p: platypus; h: human. ST: signal
transducer; R: receptor; H: hormone; SP: structural protein; T:
transporter; IC: ion channel; VGIC: voltage-gated ion channel; CC:
cation channel; TR: transcription; TRR: transcription regulation;
SR: stress response; IR: immune response; GF: growth factor; MIT:
metal ions transport.
genes emerged after the eukaryotes diverged from prokary-
otes. Another important category of intronless genes is ABE,
in which intronless genes are conserved in all major biolog-
ical kingdoms, and these genes are considered to be func-
tionally important and evolved slowly [30]. Intronless genes
belonging to ABE account for 39% of the total intronless
genes in sea squirt and 30% in sea urchin, which together

Comparative and Functional Genomics 5
0
10
20
30
40
50
60
70
A B E AB AE BE ABE ORFans
Domain combination
su sq z
cph
Genes (%)
Figure 4: Distribution of intronless genes specific to different tax-
onomic group combinations for each species. su: sea urchin; sq: sea
squirt;z:zebrafish;c:chicken;p:platypus;h:human.A:archaea;
B: bacteria; E: eukaryote; AB: archaea and bacteria; AE: archaea and
eukaryote; BE: bacteria and eukaryote; ABE: archaea, bacteria and
eukaryote; ORFans: homologs not found in other organisms.
form the first class. The second class contains zebrafish
(23%), chicken (22%), and the third class includes human
(16%) and platypus (14%). Given their phylogenetic posi-
tions, the first class is more primitive than the second
class, which is more primitive than the third class. These
data show that higher percentage of intronless genes in
primitive species are inherited from the common ancestor
of archaea, bacteria, and eukaryotes than in higher species.
More than 20% of intronless genes are conserved in bacteria
and eukaryotes (BE) in each species, but less than 5% are
conserved in archaea and eukaryotes (AE). This could be
because archaea have lost more homologues with eukaryotes
than bacteria, or because bacteria have obtained more.
Moreover, the percentage of genes conserved in bacteria
and eukaryotes that account for total intronless genes is
significantly higher in zebrafish and chicken than that in
other species, suggesting that these 2 species have a greater
percentage of intronless genes inherited from the common
ancestor of bacteria and eukaryotes. No gene is conserved in
archaea or/and bacteria except one human gene in bacteria
and this might be an example of lateral gene transfer (LGT)
from bacteria to human.
More than 30% of intronless genes are eukaryote specific
in all these species, especially in platypus (61.6%). To inves-
tigate their distributions in eukaryotic groups, we divided
these proteins according to their homogeneity in fungi (F),
metazoans (M), other eukaryotes (O), and plants (P) and
formed 15 types of combination (Figure 5). Generally, ma-
jority of genes have homologues in the combination MO
(metazoans and other eukaryotes) in each species except in
sea squirt, in which only 13% of eukaryote-specific intronless
genes are of this kind. Less than 10% of genes are metazoan-
specific (M) in many species, but inchicken and human there
are 26% and 29%, respectively. Genes conserved in fungi,
metazoans, other eukaryotes, and plants (FMOP), including
histones and ribosomal proteins, were thought to be very
0
10
20
30
40
50
60
F
M
O
P
FM
FO
FP
MO
MP
OP
FMO
FMP
FOP
MOP
FMOP
Genes (%)
su sq z
cph
Domain combination
Figure 5: Distribution of eukaryote-specific intronless genes spe-
cific to different eukaryotic taxonomic group combinations for
each species. su: sea urchin; sq: sea squirt; z: zebrafish; c: chicken; p:
platypus;h:human.F:fungi;M:metazoans;O:othereukaryotes;P:
plants; FM: fungi and metazoans; FO: fungi and other eukaryotes;
FP: fungi and plants; MO: metazoans and other eukaryotes; MP:
metazoans and plants; OP: other eukaryotes and plants; FMO:
fungi, metazoans and other eukaryotes; FMP: fungi, metazoans and
plants; FOP: fungi, other eukaryotes and plants; MOP: metazoan,
other eukaryotes and plants FMOP: fungi, metazoans, other eu-
karyotes and plants.
conservative because they are essential for the survival of all
eukaryotes [30]. The number of FMOP genes is very similar
in sea urchin (314), sea squirt (263) and zebrafish (228) but
the percentage of total eukaryote-specific intronless genes
is much greater in sea squirt (54.7%) than that in sea
urchin (27.8%) and zebrafish (26.9%). Except those cases
mentioned above, very few genes are conserved in other
taxonomic combinations. Moreover, some genes in some
species have homologues in fungi, plants or other eukaryotes
but not in metazoans. These genes might be examples of
lateral gene transfer (LGT) between eukaryotes, which has
been demonstrated recently [31,32]. Since fungi and plants
diverged from metazoan ahead of other eukaryotes, the
distribution pattern of eukaryote-specific intronless genes in
these species can be explained by that much more homologs
have been lost in fungi and plants plus lots of others
have been obtained after their divergence. However, lots of
essential genes (FMOP) were still preserved. Therefore, the
distribution pattern of eukaryote-specific intronless genes
and the gain and loss patterns in this work are in accord with
earlier reports [13,15,33].
The predicted cellular role of each kind of combination
is shown in the supplementary material. Amino acid biosyn-
thesis, cell envelope, energy metabolism, translation, trans-
port and binding are usually well represented. Furthermore,
the distribution of basic functional categories, such as amino
acid biosynthesis, energy metabolism, and translation, are
overrepresented in intronless genes conserved in all major
biological kingdoms (ABE) or all eukaryotic groups (FMOP)
compared to others.

6Comparative and Functional Genomics
Tab le 2: Number of intronless genes with homologousgenes in other taxonomic groups.
Taxonomic group Sea urchin Sea squirt Zebrafish Chicken Platypus Human
Archaea 764 625 524 385 143 1184
Bacteria 1303 873 1161 920 345 2524
Fungi 1492 1149 1067 817 413 2513
Plants 1480 1209 1070 809 441 2617
Metazoans 2447 1403 2027 1575 918 4807
Other eukaryotes 2337 1343 1897 1388 867 4055
ORFans 35 39 136 80 7 1416
ORFans [23]: homologues not found in other species.
AAB BOC CE CP CIM EM FAM PAP RF RAT T TAB
Cellular role
0
10
20
30
40
50
60
70
80
90
su sq z
cph
Genes (%)
Figure 6: Distribution of ORFans according to their functional
categories in each species. The description of functional categories
and species is the same as that given for Figure 2.
0
5
10
15
20
25
30
35
AAB BOC CE CP CIM EM FAM PAP RF RAT T TAB
Cellular role
Genes (%)
Figure 7: Functional distribution of conserved intronless genes in
vertebrates. The description of functional categories is same as that
given in Figure 2.
3.4. ORFans. The protein sequences that have no homologue
in other species are termed ORFans [23]. These proteins
could be responsible for some species-specific characteristics,
and most of these proteins might have evolved faster than
others [34]; in fact, they are part of the most interesting
genome content. Thus, it is important to experimentally
characterize these proteins or use more sensitive bioinfor-
matic approaches to understand their roles and functions
[15]. We found very few ORFans in these species except in
human (Tab le 2 ), about 22.7% of whose intronless genes are
ORFans, and this might be due to their complexities because
they are viviparous and mammalian. Most of these proteins
are annotated as hypothetical; however, majority of ORFans
in all species, except platypus, have mRNA or EST supports
when we checked their annotations. Thus, most of these
ORFans might not be misannotated. Figure 6 shows the pre-
dicted cellular role distribution of ORFans in each species. It
is interesting to note that translation and energy metabolism
are the most frequently represented cellular roles in these
species. The pattern is similar to earlier reports of plants
and human [13,15], suggesting that most species-specific
intronless genes in plants and animals have the same func-
tions and even the components of basic cellular machinery
might evolve to perform species-specific functions in all these
species [13,15]. Moreover, we found the cellular role of cell
envelope is well represented in sea urchin ORFans and more
than half of the intronless genes that have the cellular role of
fatty acid metabolism are ORFans.
3.5. Conserved Intronless Genes in Deuterostomes. To e x a m i n e
the conservation of intronless genes in deuterostomes, we
clustered them together for these 6 species using CD-Hit
(identity =0.3). Only 6 nonredundant sequences (NR) were
shared by these species, and they might perform pivotal
functions in deuterostomes. The predicted cellular roles were
translation for 4 NRs, regulatory function for one NR, and
energy metabolism for one NR. The GO categories of these
NRs were associated with transcription regulation, growth
factor, and transport. When we compared the shared NRs
between any two species, we found that sea urchin and sea
squirt have less than 100 shared NRs with other species, but
the number was more than 200 between any two vertebrate
species (data not shown), suggesting that significantly more
intronless genes are shared by vertebrates than those shared
by deuterostomes. As expected, we found 125 NRs shared
by vertebrates, and Figure 7 shows the distribution of the
predicted functions of these NRs. Most of these proteins
are involved in basic cellular processes, such as transport
and binding, cell envelope, and translation, and these genes
could be one of the important reasons for the emergence of
vertebrates.
3.6. Paralogues of Intronless Genes. Intronless genes in eukar-
yotic genomes have many origins other than inheritance

Comparative and Functional Genomics 7
from ancient prokaryotes, such as duplication (whole ge-
nome duplication or tandem duplication) of existing intron-
less genes and retroposition of intron-containing genes
(retroduplicated genes). Also, there is evidence that ancient
intronless genes were the origin of multiexon genes [35,36].
To investigate these latter patterns, we clustered intronless
genes and nonredundant intronless genes with multiexon
genes using CD-Hit (identity =0.3), and the results were
shown in Ta b le 1 . It shows that about 92% of sea urchin
nonredundant clusters have more than 1 intronless gene and
the value is 58% in human, 32% in zebrafish, and only 9%
in sea squirt. These data suggest that most intronless genes
may originate from other intronless genes in sea urchin,
human, and zebrafish, but much fewer intronless genes
havethesameorigininotherspecies.Ta bl e 1 also shows
the frequency of correspondence between nonredundant
intronless genes and nonredundant intron-containing genes.
About 30% of nonredundant intronless gene clusters have
corresponding nonredundant intron-containing genes in
each species, but in sea squirt and chicken, the proportion
is only 15% and 18%, respectively. This might be due to
the activity of LINE retrotransposable elements in their
genomes. Active LINE retrotransposons that can reversibly
transcribe polyadenylated mRNAs are thought to be the
main reason for the emergence of retrogenes [37,38]. More
than 20% of the human genome is composed of LINE
retrotransposable elements [39], and many studies have
suggested a high rate of retroposition in human [26,40,41],
which might result in the emergence of intronless genes.
In chicken, only about 8% of the genome is comprised of
the CR1 (chicken repeat 1) [42] and this kind of LINE-1 is
not thought to reversibly transcribe polyadenylated mRNAs
[42]. Majority of intronless genes that have intronless or
intron-containing homologs are associated with cellular roles
transport and binding, cell envelope, and translation. It
has long been believed that duplicated genes (including
retroduplicated genes) provide material for the evolution
of genes with new functions [43], but there is evidence
that retrogenes function as their parent genes during the
spermatogenesis X chromosome inactivation of meiosis in
mammals [12] and in the fruit fly [10]. Thus, the selective
advantage of retention of these duplicated intronless genes
might be that these genes can evolve new functions or
help to buffer crucial functions similar to earlier reports on
duplicated genes in angiosperms [44].
4. Conclusion
Both this and earlier studies indicate that the evolutionary
patterns of intronless genes among deuterostomes, as well
as between deuterostomes and plants, have many common
characteristics and might be appropriate for all major
eukaryote kingdoms. However, there are still some lineage-
specific and species-specific characteristics on the evolution
of intronless genes, and this might be one of the reasons for
the existence of biodiversity in this world. As more genome
sequences are sequenced and more exhaustive and accurate
genes are annotated, the evolutionary patterns of intronless
genes will become clearer, providing insights into under-
standing the evolutionary mechanisms underlying gene or
genome evolution in eukaryotes.
Acknowledgments
The authors are thankful to four anonymous reviewers and
M. Yu for their critical reading of this manuscript and helpful
comments and suggestions that greatly improved the paper.
This work was supported by a Grant from the Major State
Basic Research Development Program of China (973 Pro-
gram, no. 2007CB411601).
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