# Tandemly Arrayed Genes in Vertebrate Genomes

Authors: Deng Pan; Liqing Zhang
Journal: Comparative and Functional Genomics
 (2008)

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## Abstract

Tandemly arrayed genes (TAGs) are duplicated genes that are linked as neighbors on a chromosome, many of which have important
physiological and biochemical functions. Here we performed a survey
of these genes in 11 available vertebrate genomes. TAGs account for
an average of about 14% of all genes in these vertebrate genomes, and
about 25% of all duplications. The majority of TAGs (72–94%) have
parallel transcription orientation (i.e., they are encoded on the same strand) in contrast to the genome, which has about 50% of its genes
in parallel transcription orientation. The majority of tandem arrays
have only two members. In all species, the proportion of genes that
belong to TAGs tends to be higher in large gene families than in small
ones; together with our recent finding that tandem duplication played
a more important role than retroposition in large families, this fact
suggests that among all types of duplication mechanisms, tandem duplication
is the predominant mechanism of duplication, especially in
large families. Finally, several species have a higher proportion of large
tandem arrays that are species-specific than random expectation.


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## Body

## 1. Introduction

Although the
importance of duplicated genes in providing raw materials for genetic
innovation has been recognized since the 1930s and is highlighted in Ohno's
bookEvolution by Gene Duplication [1], it is only recently that the
availability of numerous genomic sequences has made it possible to
quantitatively estimate how many genes in a genome are generated by gene
duplication. For instance, it has been estimated that about 38% of the genes in
the human genome and 49% of the Caenorhabditis elegans genome arose from
gene duplication [2, 3]. It is almost certain that current estimates of the
extent of gene duplications are low, as many duplicated genes may have diverged
to such a great extent that their common origin can no longer be recognized.Known mechanisms of gene duplication include unequal
crossover (or equivalently, tandem duplication), retroposition, and segmental
(or genome) duplication [4]. Unequal crossover consists of chromosomal
mispairing followed by the exchange of DNA between nonhomologous regions and
resulting in either gene duplication or gene deletion [5]. Retroposition
refers to reverse transcription of the mRNA transcript of a gene into
double-stranded DNA followed by insertion of the double-stranded DNA into a
location typically distant from the original gene. Genome duplication in
vertebrates is not as frequent as that in plants. According to the two-round
genome duplication hypothesis, the last possible genome duplication in
vertebrates occurred more than 400 million years ago [6]. Recent segmental
duplications cover only about 2% of the mouse genome [7] and 4% of the human
genome [8] and usually do not contain genes [9]. Recently, some general
studies of gene duplications have been undertaken (e.g., [10]), as well as
specific computational identification and characterization of retrotransposed
duplicated genes with respect to their location and dynamics in species such as
human and mouse [11, 12]. There have also been studies of duplicated genes
generated through unequal crossover (tandem duplication) in C. elegans [13], Arabidopsis thaliana [14], Oryza sativa [15], and several
mammals [16].Our current study focuses on tandemly arrayed genes
(TAGs) in available vertebrate genomes. Tandem duplication has been shown to
act as the driving evolutionary force in the origin and maintenance of gene
families [17] and has been a common mechanism of genetic adaptation to environmental
challenges in organisms such as bacteria [18], mosquitoes [19], plants [20], and
mammals [21]. TAGs constitute a large component of several eukaryotic genomes.
For example, at least 10% of the genes in the genomes of C. elegans, Arabidopsis
thaliana, and human are TAGs [13, 14, 16]. TAGs can either promote
genomic diversity to enhance disease resistance, satisfy the requirement for a
large amount of a gene product, or contribute to the fine-tuning of
developmental stages and physiological functions [1, 22].In this work, we performed a genome-wide survey of the
TAGs in 11 completed or nearly completed vertebrate genomes. We provided some
general statistics regarding the number of genes in these genomes that belong
to TAGs, the contribution of TAGs to the total duplications, TAG size (i.e.,
how many genes are in an array) distributions, gene transcription orientations
in TAGs, and the contribution of tandem duplication in the make-up of gene
families of different sizes. We also identified species-specific TAGs and
compared their distribution among species.


## 2. Results

The summary
statistics are presented in Table1. There are a total of 285 801 putative genes in
the 11 genomes. Figure 1 (adapted mostly from [23]; the divergence
time between zebrafish and tetraodon is from [24]) shows the phylogeny of these
species. On average, each genome has 25 982 genes,
with human (31 185) and chicken
(19 399) having the most and least number of genes,
respectively. The number of genes that have been assigned to a specific
chromosome location in the 11 species reduces to 255 293.
More than 90% of the genes have been assigned to a known location in the genome
assembly for human, chimp, mouse, rat, macaca, dog, opossum, and zebrafish,
whereas only 69%, 82%, and 55% have been assigned for cattle, chicken, and
tetraodon, respectively. The numbers of gene families range from 2297 (chicken)
to 4127 (zebrafish), and the numbers of genes contained in these families range
from 7199 (chicken) to 20 187 (zebrafish) among all species. Thus, gene
families on average contain about 3 to 5 members.Table 1
Numbers of nuclear genes in genomes of
species utilized.*TAGs are defined as having zero or one spacer gene.
SpeciesGenesAnnotated genesGene familiesAnnotated genes in familiesTAGs*%TAGs* in duplicated genes%TAGs* in the genomeHuman3118531126361714473339423.5%10.9%Chimp2551024522326212376268621.7%11.0%Macaca2742925990354314439337123.3%13.0%Mouse2796427736364516091498431.0%18.0%Rat2723327194351016446471228.7%17.3%Cattle259771789526169146177919.5%9.9%Dog2280022257316011480206718.0%9.3%Opossum2128819598310112195343828.2%17.5%Chicken193991596622977199143319.9%9.0%Zebrafish2850627457412720187472923.4%17.2%Tetraodon285101555226549702333234.3%21.4%Figure 1
Phylogeny of the eleven species in this
study.


### 2.1. Spacers in TAGs

TAGs are
usually defined as genes that are duplicated tandemly on chromosomes. Spacers
are genes that are not homologous to the members of TAGs (see
Section5 for details). Allowing different numbers
of spacers between two members of an array will result in different numbers
of TAGs. Figure 2 shows TAG statistics with respect to different numbers
of spacers. There are three general patterns. First, for all species, the
number of tandem arrays increases with the number of spacers allowed in the
array, although the extent of increase varies among species (Figure 2(a)). The zebrafish shows the highest extent of increase in the
number of arrays with increase of the number of spacers (P<.01 for all of the one-tailed t-tests between zebrafish and other species).
Similarly, the number of genes included in the tandem arrays also increases
when more spacers are allowed in the TAGs (Figure 2(b)). Second, for
most species, both the number of tandem arrays and the number of genes in the
arrays show the sharpest increase when going from spacer 0 to 1, consistent
with studies in Arabidopsis thaliana and rice [14, 25]. Third, the
similarities in these two quantities (the number of tandem arrays and the
number of genes in the arrays) among species reflect to certain extent their
evolutionary distances (Figure 1). For instance, mouse and rat show a
very similar pattern in both the number of arrays and the number of genes; so
do human and macaca, and dog and cattle. The zebrafish appears to be the most
distinct of the remaining species, having the highest numbers in both arrays
and genes for almost all TAG definitions (P<.01 for all pairwise t-test between zebrafish and the other
species), perhaps because the zebrafish has undergone recent genome duplications
so that the number of tandemly arrayed genes are much larger than for other
species. An exception is seen in the chimp where, despite its being the most
closely related to the human, there is a much greater divergence in the two
quantities from human than in macaca from human. The quality of the chimp
genome assembly has been known to be poor, which might explain the strange
pattern that we observe here.Distribution of tandem arrays and TAGs as a
function of maximum number of spacers allowed.
(a)
Number of tandem arrays(b)
Number of TAGs(c)
% of TAGs in the genomeThe percentages of TAG genes range from about 8% to
19% among all species when no spacers are allowed in TAGs, from about 10% to
26% when allowing 10 spacers. Therefore, TAGs contribute to a large proportion
of genes in vertebrate genomes (Figure2(c)). As previous and current
genome-wide studies of TAGs suggest that allowing 1 spacer between array
members is a good compromise between stringency and gene coverage, we report
for the rest of the study only the results on TAGs that have at most 1 spacer.
Note that according to our definition, allowing at most 1 spacer means that
every pair of the neighboring genes in a TAG array has at most 1 spacer;
therefore, the array can have more than 1 spacer in total.


### 2.2. Contribution to Gene Duplication

Tandem
duplication has been commonly cited in the literature as one of three major
mechanisms of gene duplication [4]. However, a quantitative evaluation of its
contribution to duplications in the vertebrate genomes has not been available
until our recent report [16]. Here, we also examined the percentage of
duplicated genes that are in tandem arrays in these 11 genomes. Results are
shown in Table 1. TAGs not only make up nearly 20% (9%–21%) of the
genes, but also account for up to one third (18%–34%) of all duplications in
these genomes.


### 2.3. Size of Tandem Array

Table2
shows the distribution of tandem array sizes (i.e., the number of genes in a
tandem array) and the percentages of TAG genes in each size category. Among all
species, about 60% to 83% of the tandem arrays are of size two, that is, having
only 2 members in the arrays. The proportions of tandem arrays of larger sizes
decrease rapidly after size two. Mouse (30%), rat (34%), and opossum (38%) have
the least proportions of two-member arrays, in contrast to 41%–73% for all
remaining species. Mouse, rat, and opossum tend to have more larger arrays. In
fact, the average number of genes per array ranges from 3.4 to 4.0 in mouse,
rat, and opossum, from 2.4 and 3.2 in the remaining species.Table 2
The percentages of tandem arrays (PTA) and
the corresponding percentages of TAGs (PTG) in each array size.
Size of TAGSpeciesStatistics2345678910>10HumanPTA65.413.66.64.43.41.01.01.00.62.8PTG40.612.68.26.86.42.32.62.91.815.8ChimpPTA65.316.96.63.23.01.10.90.70.81.6PTG43.617.08.85.46.02.62.42.02.69.6MacacaPTA70.015.25.63.21.31.30.80.50.41.7PTG49.816.38.05.62.83.32.11.61.58.9MousePTA59.714.26.93.73.32.51.61.81.05.2PTG30.110.87.04.75.14.53.24.22.627.9RatPTA62.015.27.14.22.81.71.10.90.94.2PTG34.112.57.85.74.63.32.42.32.524.7CattlePTA77.114.33.62.90.60.40.10.60.10.3PTG63.017.55.85.91.31.20.42.00.62.2DogPTA77.213.03.51.01.31.30.40.60.31.4PTG57.714.55.21.92.93.41.22.21.010.1OpossumPTA64.615.16.03.42.81.61.10.50.34.6PTG38.213.47.15.14.93.32.61.30.923.4ChickenPTA81.811.33.41.50.80.50.20.00.30.2PTG69.614.45.93.12.11.50.60.01.41.4ZebrafishPTA78.011.05.01.81.40.60.40.30.21.3PTG59.612.67.73.53.21.51.21.10.88.8TetraodonPTA82.812.23.11.00.20.10.10.30.00.1PTG72.916.15.52.30.50.20.51.10.00.9


### 2.4. TAG Orientations

Table3 shows the statistics of three types of gene transcription
orientations (parallel →→ or ←←,
convergent →←,
and divergent ←→) for both genomes and TAGs. The proportions
of neighboring genes with three different transcription orientations in the
genome are very similar among all species, with parallel transcription
orientation being the major type (varying within a narrow range of about
50%–57%) and equal proportions of convergent and divergent transcription
orientations (both about 22%–25%). In contrast, for all species, the majority
of gene pairs in TAGs have a parallel transcription orientation, ranging from
about 72% to 94%, much higher than those in the genomes (P<.01, t-test). The proportions of convergent and
divergent transcription orientations in TAGs are similar, ranging from 3% to
14% among species. Statistical tests show that the distribution of the three
types of transcription orientations in TAGs is significantly different from
that of all genes in the genome (the chi-square Goodness-of-Fit test: P-value <1E-36 for all species).Table 3
Occurrence of parallel, convergent, and
divergent orientations among gene pairs. Both
absolute numbers and percentages are shown.
ParallelConvergentDivergentGenomeTAGGenomeTAGGenomeTAGHuman15843137976252467634266(50.9%)(72.9%)(24.5%)(13.0%)(24.5%)(14.1%)Chimp12095106261991916203209(49.4%)(72.6%)(25.3%)(13.1%)(25.3%)(14.3%)Macaca13273125263452306351238(51.1%)(72.8%)(24.4%)(13.4%)(24.5%)(13.8%)Mouse14307240167053776703365(51.6%)(76.4%)(24.2%)(12.0%)(24.2%)(11.6%)Rat13967212766043536602375(51.4%)(74.5%)(24.3%)(12.4%)(24.3%)(13.1%)Cattle883360545181164514117(49.4%)(72.2%)(25.3%)(13.8%)(25.3%)(14.0%)Dog1111680255491385553141(50.0%)(74.2%)(25.0%)(12.8%)(25.0%)(13.0%)Opossum9861164148642344864225(50.3%)(78.1%)(24.8%)(11.1%)(24.8%)(10.7%)Chicken8006581396360396762(50.2%)(82.6%)(24.9%)(8.5%)(24.9%)(8.8%)Zebrafish15228186561022616102299(55.5%)(76.9%)(22.2%)(10.8%)(22.2%)(12.3%)Tetraodon88061563336352336255(56.7%)(93.6%)(21.7%)(3.1%)(21.6%)(3.3%)


### 2.5. TAGs in Gene Families

Table4 shows the proportions of duplicated genes that belong to TAGs in gene families
of different sizes. There is a clear trend that, as family size gets larger,
the proportion of TAGs in the families also increases. For instance, in gene
families of size two (i.e., families that have two members), only around 10% of
gene members belong to TAGs (except for tetraodon),
whereas in families of sizes >10,
35%–60% of the members belong to TAGs. Figure 3 shows the relationship
between family sizes and mean percentages of TAGs (averaged over all species).
Tests of correlation show that the percentages of TAGs in gene families are
positively correlated with gene family sizes (Pearson's correlation coefficient
r varies from .78 to .94 among species, all P-values <.008). The correlation remains significant
even after removing the family size >10 that includes all families with >10 genes for all species (all P-values <.05).Table 4
Percentages of TAGs in gene families of
different sizes. Absolute numbers are omitted for clarity.
Family sizeSpecies2345678910>10CorrelationHuman11.038.9514.5620.9528.8517.2528.3020.7431.3346.75.84Chimp8.659.6815.8625.2725.0021.6021.5729.0830.8046.45.90Macaca11.6411.7720.3423.7326.8821.4323.7827.1934.0739.86.92Mouse11.0212.3915.6719.0229.0423.4232.3333.5743.5759.42.94Rat10.3613.0519.4223.7124.5219.2335.5819.2236.6748.46.84Cattle9.6012.6016.9523.8228.3019.1228.0632.0322.5037.19.84Dog9.0910.8013.7621.0517.4914.9620.7016.6730.0039.17.84Opossum9.9913.8921.2520.6724.9321.1232.2540.8631.2060.11.88Chicken13.4115.6124.8820.6328.0029.6424.3423.6128.3334.65.82Zebrafish11.2012.3616.3515.2818.7819.0326.1319.0732.1338.27.90Tetraodon27.5027.2831.3137.1837.8241.4546.9433.3338.8247.67.78Figure 3
Contribution of TAGs to families of different
sizes.We also examined the homologous tandem arrays (the
TAGs that belong to the same Ensembl gene family) across all the species.
Due to the complex homologous relationship between
the members of TAGs within and across species (see Section3), we did not perform the
standard phylogenetic analysis. Instead, to explore the relationship between
TAGs across species, we clustered the species based on distribution of the
number of TAGs in the same families across these species by the K-means
clustering method [26]. Specifically, each row of the input matrix for K-means
clustering contains a vector with the numbers in the vector corresponding to
the number of TAG occurrence in each of the gene families in a particular
species. Therefore, we used the K-means clustering to take account of the
information of all families in order to group the species that show similar TAG
profiles. Our purpose is to test whether the clustering based on TAGs is
congruent with the species tree (Figure 1). We set the number of
clusters K from 2 to 4.
When K=2,
the resulting two groups are {human, chimp, macaca, mouse, rat, opossum} and
{cattle, dog, chicken, zebrafish, tetraodon}; when K=3,
the resulting three groups are {human, chimp, macaca, opossum}, {mouse, rat}, and
{cattle, dog, chicken, zebrafish, tetraodon}; and when K=4,
the resulting four groups are {human, chimp, macaca, opossum}, {mouse, rat},
{cattle, dog, chicken, tetraodon}, and {zebrafish}. Compared with the species tree,
it turns out that primate species (human, chimp, macaca) and murine species
(mouse, rat) are always clustered correctly, but cattle and dog are more likely
to be clustered with nonmammals.


### 2.6. Species Specific Tandem Arrays

Studies have shown
that species-specific duplications can play an important role in
species-specific traits or life styles that enable species to adapt to certain
environments (e.g., [27–29]). We studied species-specific tandem
arrays (SSTAs), which are defined as the tandem arrays that are present in only
one species while there may be no homologues or
homologues are not tandemly arrayed in all the other species. Table 5
shows the summary of SSTAs in all species. There are about 10% SSTAs in mammals
and more than 20% in nonmammals. The higher proportion of SSTAs in nonmammals
may be mainly due to their much higher divergence from the most recent common
ancestor in the species tree (Figure 1). We also used Gene Ontology
(GO) annotations to see whether there are any GO categories that are highly
enriched in the SSTAs. We found no apparent
preference of specific GO functions even between closely related species, such as mouse and rat.
But as not all SSTA genes have GO information, further evaluation is needed.Table 5
Statistics of species-specific tandem arrays
(SSTAs). The percentages in the parenthesis are shown as follows:thea percentage of SSTAs to the total number of tandem arrays and theb percentage of size-two SSTAs to the total number of SSTAs.
SpeciesNumber ofSSTAsaNumber of size-twoSSTAsbHypergeometric testP-valueHuman75 (7.12%)49 (65.33%)4.62e-01Chimp14 (1.56%)14 (100%)0Macaca85 (7.08%)78 (91.76%)1.00e-07Mouse111 (8.83%)63 (56.76%)7.10e-01Rat92 (7.09%)68 (73.91%)4.48e-03Cattle79 (10.88%)65 (82.28%)9.49e-02Dog46 (5.96%)45 (97.83%)4.48e-06Opossum95 (9.36%)66 (69.47%)1.24e-01Chicken139 (22.79%)118 (84.89%)1.14e-01Zebrafish684 (37.85%)366 (53.51%)1Tetraodon584 (39.81%)457 (78.25%)1As SSTAs are more likely to be recently born than are
the non-SSTA arrays that are shared by multiple species, we expect that under
neutral evolution, the sizes of SSTAs (i.e., the number of genes in an array)
should be on average smaller than the sizes of non-SSTA arrays. As most of the
SSTAs are of size-two (Table5), we expect that the proportion of SSTAs
that are of size two should be higher than the proportion of non-SSTA arrays
that are of size two. Only in chimp, macaca, rat, and dog is the proportion of
size-two SSTAs significantly higher than that of size-two non-SSTAs, which
means that the sizes of SSTAs in most of the species are not significantly
smaller than the sizes of non-SSTA arrays.


## 2.1. Spacers in TAGs

TAGs are
usually defined as genes that are duplicated tandemly on chromosomes. Spacers
are genes that are not homologous to the members of TAGs (see
Section5 for details). Allowing different numbers
of spacers between two members of an array will result in different numbers
of TAGs. Figure 2 shows TAG statistics with respect to different numbers
of spacers. There are three general patterns. First, for all species, the
number of tandem arrays increases with the number of spacers allowed in the
array, although the extent of increase varies among species (Figure 2(a)). The zebrafish shows the highest extent of increase in the
number of arrays with increase of the number of spacers (P<.01 for all of the one-tailed t-tests between zebrafish and other species).
Similarly, the number of genes included in the tandem arrays also increases
when more spacers are allowed in the TAGs (Figure 2(b)). Second, for
most species, both the number of tandem arrays and the number of genes in the
arrays show the sharpest increase when going from spacer 0 to 1, consistent
with studies in Arabidopsis thaliana and rice [14, 25]. Third, the
similarities in these two quantities (the number of tandem arrays and the
number of genes in the arrays) among species reflect to certain extent their
evolutionary distances (Figure 1). For instance, mouse and rat show a
very similar pattern in both the number of arrays and the number of genes; so
do human and macaca, and dog and cattle. The zebrafish appears to be the most
distinct of the remaining species, having the highest numbers in both arrays
and genes for almost all TAG definitions (P<.01 for all pairwise t-test between zebrafish and the other
species), perhaps because the zebrafish has undergone recent genome duplications
so that the number of tandemly arrayed genes are much larger than for other
species. An exception is seen in the chimp where, despite its being the most
closely related to the human, there is a much greater divergence in the two
quantities from human than in macaca from human. The quality of the chimp
genome assembly has been known to be poor, which might explain the strange
pattern that we observe here.Distribution of tandem arrays and TAGs as a
function of maximum number of spacers allowed.
(a)
Number of tandem arrays(b)
Number of TAGs(c)
% of TAGs in the genomeThe percentages of TAG genes range from about 8% to
19% among all species when no spacers are allowed in TAGs, from about 10% to
26% when allowing 10 spacers. Therefore, TAGs contribute to a large proportion
of genes in vertebrate genomes (Figure2(c)). As previous and current
genome-wide studies of TAGs suggest that allowing 1 spacer between array
members is a good compromise between stringency and gene coverage, we report
for the rest of the study only the results on TAGs that have at most 1 spacer.
Note that according to our definition, allowing at most 1 spacer means that
every pair of the neighboring genes in a TAG array has at most 1 spacer;
therefore, the array can have more than 1 spacer in total.


## 2.2. Contribution to Gene Duplication

Tandem
duplication has been commonly cited in the literature as one of three major
mechanisms of gene duplication [4]. However, a quantitative evaluation of its
contribution to duplications in the vertebrate genomes has not been available
until our recent report [16]. Here, we also examined the percentage of
duplicated genes that are in tandem arrays in these 11 genomes. Results are
shown in Table 1. TAGs not only make up nearly 20% (9%–21%) of the
genes, but also account for up to one third (18%–34%) of all duplications in
these genomes.


## 2.3. Size of Tandem Array

Table2
shows the distribution of tandem array sizes (i.e., the number of genes in a
tandem array) and the percentages of TAG genes in each size category. Among all
species, about 60% to 83% of the tandem arrays are of size two, that is, having
only 2 members in the arrays. The proportions of tandem arrays of larger sizes
decrease rapidly after size two. Mouse (30%), rat (34%), and opossum (38%) have
the least proportions of two-member arrays, in contrast to 41%–73% for all
remaining species. Mouse, rat, and opossum tend to have more larger arrays. In
fact, the average number of genes per array ranges from 3.4 to 4.0 in mouse,
rat, and opossum, from 2.4 and 3.2 in the remaining species.Table 2
The percentages of tandem arrays (PTA) and
the corresponding percentages of TAGs (PTG) in each array size.
Size of TAGSpeciesStatistics2345678910>10HumanPTA65.413.66.64.43.41.01.01.00.62.8PTG40.612.68.26.86.42.32.62.91.815.8ChimpPTA65.316.96.63.23.01.10.90.70.81.6PTG43.617.08.85.46.02.62.42.02.69.6MacacaPTA70.015.25.63.21.31.30.80.50.41.7PTG49.816.38.05.62.83.32.11.61.58.9MousePTA59.714.26.93.73.32.51.61.81.05.2PTG30.110.87.04.75.14.53.24.22.627.9RatPTA62.015.27.14.22.81.71.10.90.94.2PTG34.112.57.85.74.63.32.42.32.524.7CattlePTA77.114.33.62.90.60.40.10.60.10.3PTG63.017.55.85.91.31.20.42.00.62.2DogPTA77.213.03.51.01.31.30.40.60.31.4PTG57.714.55.21.92.93.41.22.21.010.1OpossumPTA64.615.16.03.42.81.61.10.50.34.6PTG38.213.47.15.14.93.32.61.30.923.4ChickenPTA81.811.33.41.50.80.50.20.00.30.2PTG69.614.45.93.12.11.50.60.01.41.4ZebrafishPTA78.011.05.01.81.40.60.40.30.21.3PTG59.612.67.73.53.21.51.21.10.88.8TetraodonPTA82.812.23.11.00.20.10.10.30.00.1PTG72.916.15.52.30.50.20.51.10.00.9


## 2.4. TAG Orientations

Table3 shows the statistics of three types of gene transcription
orientations (parallel →→ or ←←,
convergent →←,
and divergent ←→) for both genomes and TAGs. The proportions
of neighboring genes with three different transcription orientations in the
genome are very similar among all species, with parallel transcription
orientation being the major type (varying within a narrow range of about
50%–57%) and equal proportions of convergent and divergent transcription
orientations (both about 22%–25%). In contrast, for all species, the majority
of gene pairs in TAGs have a parallel transcription orientation, ranging from
about 72% to 94%, much higher than those in the genomes (P<.01, t-test). The proportions of convergent and
divergent transcription orientations in TAGs are similar, ranging from 3% to
14% among species. Statistical tests show that the distribution of the three
types of transcription orientations in TAGs is significantly different from
that of all genes in the genome (the chi-square Goodness-of-Fit test: P-value <1E-36 for all species).Table 3
Occurrence of parallel, convergent, and
divergent orientations among gene pairs. Both
absolute numbers and percentages are shown.
ParallelConvergentDivergentGenomeTAGGenomeTAGGenomeTAGHuman15843137976252467634266(50.9%)(72.9%)(24.5%)(13.0%)(24.5%)(14.1%)Chimp12095106261991916203209(49.4%)(72.6%)(25.3%)(13.1%)(25.3%)(14.3%)Macaca13273125263452306351238(51.1%)(72.8%)(24.4%)(13.4%)(24.5%)(13.8%)Mouse14307240167053776703365(51.6%)(76.4%)(24.2%)(12.0%)(24.2%)(11.6%)Rat13967212766043536602375(51.4%)(74.5%)(24.3%)(12.4%)(24.3%)(13.1%)Cattle883360545181164514117(49.4%)(72.2%)(25.3%)(13.8%)(25.3%)(14.0%)Dog1111680255491385553141(50.0%)(74.2%)(25.0%)(12.8%)(25.0%)(13.0%)Opossum9861164148642344864225(50.3%)(78.1%)(24.8%)(11.1%)(24.8%)(10.7%)Chicken8006581396360396762(50.2%)(82.6%)(24.9%)(8.5%)(24.9%)(8.8%)Zebrafish15228186561022616102299(55.5%)(76.9%)(22.2%)(10.8%)(22.2%)(12.3%)Tetraodon88061563336352336255(56.7%)(93.6%)(21.7%)(3.1%)(21.6%)(3.3%)


## 2.5. TAGs in Gene Families

Table4 shows the proportions of duplicated genes that belong to TAGs in gene families
of different sizes. There is a clear trend that, as family size gets larger,
the proportion of TAGs in the families also increases. For instance, in gene
families of size two (i.e., families that have two members), only around 10% of
gene members belong to TAGs (except for tetraodon),
whereas in families of sizes >10,
35%–60% of the members belong to TAGs. Figure 3 shows the relationship
between family sizes and mean percentages of TAGs (averaged over all species).
Tests of correlation show that the percentages of TAGs in gene families are
positively correlated with gene family sizes (Pearson's correlation coefficient
r varies from .78 to .94 among species, all P-values <.008). The correlation remains significant
even after removing the family size >10 that includes all families with >10 genes for all species (all P-values <.05).Table 4
Percentages of TAGs in gene families of
different sizes. Absolute numbers are omitted for clarity.
Family sizeSpecies2345678910>10CorrelationHuman11.038.9514.5620.9528.8517.2528.3020.7431.3346.75.84Chimp8.659.6815.8625.2725.0021.6021.5729.0830.8046.45.90Macaca11.6411.7720.3423.7326.8821.4323.7827.1934.0739.86.92Mouse11.0212.3915.6719.0229.0423.4232.3333.5743.5759.42.94Rat10.3613.0519.4223.7124.5219.2335.5819.2236.6748.46.84Cattle9.6012.6016.9523.8228.3019.1228.0632.0322.5037.19.84Dog9.0910.8013.7621.0517.4914.9620.7016.6730.0039.17.84Opossum9.9913.8921.2520.6724.9321.1232.2540.8631.2060.11.88Chicken13.4115.6124.8820.6328.0029.6424.3423.6128.3334.65.82Zebrafish11.2012.3616.3515.2818.7819.0326.1319.0732.1338.27.90Tetraodon27.5027.2831.3137.1837.8241.4546.9433.3338.8247.67.78Figure 3
Contribution of TAGs to families of different
sizes.We also examined the homologous tandem arrays (the
TAGs that belong to the same Ensembl gene family) across all the species.
Due to the complex homologous relationship between
the members of TAGs within and across species (see Section3), we did not perform the
standard phylogenetic analysis. Instead, to explore the relationship between
TAGs across species, we clustered the species based on distribution of the
number of TAGs in the same families across these species by the K-means
clustering method [26]. Specifically, each row of the input matrix for K-means
clustering contains a vector with the numbers in the vector corresponding to
the number of TAG occurrence in each of the gene families in a particular
species. Therefore, we used the K-means clustering to take account of the
information of all families in order to group the species that show similar TAG
profiles. Our purpose is to test whether the clustering based on TAGs is
congruent with the species tree (Figure 1). We set the number of
clusters K from 2 to 4.
When K=2,
the resulting two groups are {human, chimp, macaca, mouse, rat, opossum} and
{cattle, dog, chicken, zebrafish, tetraodon}; when K=3,
the resulting three groups are {human, chimp, macaca, opossum}, {mouse, rat}, and
{cattle, dog, chicken, zebrafish, tetraodon}; and when K=4,
the resulting four groups are {human, chimp, macaca, opossum}, {mouse, rat},
{cattle, dog, chicken, tetraodon}, and {zebrafish}. Compared with the species tree,
it turns out that primate species (human, chimp, macaca) and murine species
(mouse, rat) are always clustered correctly, but cattle and dog are more likely
to be clustered with nonmammals.


## 2.6. Species Specific Tandem Arrays

Studies have shown
that species-specific duplications can play an important role in
species-specific traits or life styles that enable species to adapt to certain
environments (e.g., [27–29]). We studied species-specific tandem
arrays (SSTAs), which are defined as the tandem arrays that are present in only
one species while there may be no homologues or
homologues are not tandemly arrayed in all the other species. Table 5
shows the summary of SSTAs in all species. There are about 10% SSTAs in mammals
and more than 20% in nonmammals. The higher proportion of SSTAs in nonmammals
may be mainly due to their much higher divergence from the most recent common
ancestor in the species tree (Figure 1). We also used Gene Ontology
(GO) annotations to see whether there are any GO categories that are highly
enriched in the SSTAs. We found no apparent
preference of specific GO functions even between closely related species, such as mouse and rat.
But as not all SSTA genes have GO information, further evaluation is needed.Table 5
Statistics of species-specific tandem arrays
(SSTAs). The percentages in the parenthesis are shown as follows:thea percentage of SSTAs to the total number of tandem arrays and theb percentage of size-two SSTAs to the total number of SSTAs.
SpeciesNumber ofSSTAsaNumber of size-twoSSTAsbHypergeometric testP-valueHuman75 (7.12%)49 (65.33%)4.62e-01Chimp14 (1.56%)14 (100%)0Macaca85 (7.08%)78 (91.76%)1.00e-07Mouse111 (8.83%)63 (56.76%)7.10e-01Rat92 (7.09%)68 (73.91%)4.48e-03Cattle79 (10.88%)65 (82.28%)9.49e-02Dog46 (5.96%)45 (97.83%)4.48e-06Opossum95 (9.36%)66 (69.47%)1.24e-01Chicken139 (22.79%)118 (84.89%)1.14e-01Zebrafish684 (37.85%)366 (53.51%)1Tetraodon584 (39.81%)457 (78.25%)1As SSTAs are more likely to be recently born than are
the non-SSTA arrays that are shared by multiple species, we expect that under
neutral evolution, the sizes of SSTAs (i.e., the number of genes in an array)
should be on average smaller than the sizes of non-SSTA arrays. As most of the
SSTAs are of size-two (Table5), we expect that the proportion of SSTAs
that are of size two should be higher than the proportion of non-SSTA arrays
that are of size two. Only in chimp, macaca, rat, and dog is the proportion of
size-two SSTAs significantly higher than that of size-two non-SSTAs, which
means that the sizes of SSTAs in most of the species are not significantly
smaller than the sizes of non-SSTA arrays.


## 3. Discussion

### 3.1. Contribution of Tandem Duplication

Here we
performed a genome-wide survey of TAGs in 11 assembled vertebrate genomes. In
summary, when using a stringent criterion for TAG identification (e.g.,
allowing at the most 1 spacer between array members), we observed a consistent
pattern of tandem duplication contributing to the number of genes in the
genomes and to genome wide duplications: on average, about 14% of the genes in
vertebrate genomes are TAGs, and about 25% of all duplicated genes are tandemly
arrayed.These numbers most likely underestimate the extent of
tandem duplication in these genomes. Our recent study shows that more than 25%
to 40% of the recent gene duplications are generated by tandem duplications in
human and mouse [30]. Therefore, it is likely that many old tandem arrays
became invisible during evolution owing to various genome rearrangements.
Meanwhile, one may wonder whether duplicated genes arising from duplication
mechanisms other than tandem duplication could get scrambled during evolution
and happen to be arranged as TAGs. However, as shown in our previous study,
this possibility should have minimal effect on the TAG statistics because the
probability that duplicated genes appear as TAGs by chance is very low, about 1
to 2 orders of magnitude lower than the actual extent of tandem duplication
[16].


### 3.2. TAG Transcription Orientation

It has been
shown that∼80% and ∼88% of tandem arrays are in parallel
transcription orientation in Arabidopsis thaliana and rice, respectively
[25]. How this compares to the genome patterns in these species has not yet
been studied. The vertebrate genomes show amazingly consistent patterns in the
proportion of gene pairs in parallel, convergent, and divergent transcription
orientations with a ratio of ∼2:1:1.
In contrast, TAGs have much higher proportions of parallel orientation, ranging
from about 72% to as high as 94%. Therefore, in both plants and animals,
parallel orientation is the dominant type of transcription orientation in TAGs.So why is there disproportionately less convergent and
divergent transcription orientation in TAGs than in the genome? One explanation
is that tandem duplications occur at a higher rate on the same strand than on
different strands. Little is known about what determines the rates of tandem duplication
on the same strand or different strands. Therefore, how much differential rates
of tandem duplication between same-strand and different-strands contribute to
the observed dominance of parallel orientation across all the studied species
remains an open question. Another possible explanation is related to long
inverted repeats (LIRs). It has been shown that LIRs can substantially increase
genome instability. For example, in the mouse, LIRs in germ lines can lead to
elevated genome rearrangements due to increased levels of illegitimate
recombination, gene conversion, and deletion mediated by LIRs [31, 32]. In the
human, several genetic diseases have been reported to be caused by illegitimate
recombination and deletion induced by LIRs [33]. In the case of TAGs, tandem
duplicated genes on opposite strands (in convergent or divergent orientation)
are essentially LIRs, and their initial high sequence identity might increase
the level of illegitimate recombination and various genome rearrangements. The
increased genome instability might have a disastrous effect on the individuals
that carry tandem duplication; strong negative selection against the
duplication will reduce the fixation probability of tandem duplication in the
population. This may at least in part explain why we observe a much lower
proportion of TAGs with convergent and divergent
orientations than in the genome. Meanwhile, the
fact that there are still some TAGs with convergent or divergent orientation
can also be explained by LIR-mediated changes. It has been observed that
illegitimate recombination events induced by LIRs sometimes result in
asymmetric deletion that eliminates the central symmetry of LIRs [31, 32]. When
the deletion does not have a negative effect on the function of the genes, for
example, when the deletion happens to be located in introns, the elimination of
the symmetry in the LIRs can actually prohibit further illegitimate
rearrangements and reduce the levels of gene conversion. Consequently, the
LIRs, that is, tandem duplicated genes on opposite strands, no longer pose a
threat to genome stability and thus can be fixed in the population [5, 31].
More research needs to be done to determine the causes of the higher proportion
of TAGs in parallel orientation than the genome average.


### 3.3. Tandem Array Sizes

All plant and
animal genomes that we have studied so far show that the majority of tandem
arrays contain only two members. It is likely that large arrays are destroyed
by various genome rearrangements and become smaller arrays over time, which
might be the case for most of the tandem arrays. For the large TAG arrays such
as the 18S and 28S ribosomal RNA genes in the vertebrates [22], mechanisms such
as continued concerted evolution (including unequal crossover and gene
conversion) and natural selection need to act on the arrays to prevent
array-size decay.The fluctuation of array sizes has been observed in
natural populations of many species such as humans [34] and flies [35].
Empirical evidence also suggests that the fluctuation can produce visible
phenotypic effects and sometimes can be detrimental. For example, in Drosophila
melanogaster, 18S and 28S rRNA genes contain 150 to 250 tandemly arranged
repeats in wild-type flies [35, 36] and individuals carrying a lower copy
number than the wild-type have so-called bobbed mutations, characterized
phenotypically by having small bristles, abdominal etching, and developmental
delay [37, 38]. These studies show that the size of tandem arrays is important
in the normal function of organisms and the fluctuation of array sizes might be
selected against.At the same time, a variety of mechanisms can reduce
or prevent size change in a tandem array. For example, insertion of irrelevant
genes (i.e., genes with no homology to the array members) into the array may
effectively reduce the frequency of unequal crossovers. The divergence of array
members can also reduce the frequency of unequal crossover. Therefore,
observation on array sizes across multiple animal and plant genomes reflects
not only a snapshot of current genomes, but also most likely a stable state of
TAGs as a result of joint processes of selection, drift, and mutation on the
arrays.


### 3.4. Tandem Duplication and Family Size

The positive
correlation between the extent of tandem duplication and the sizes of gene
families (Figure3 and Table 3) indicates that the contribution
of TAGs to gene families of different sizes weighs more in large gene families
than smaller ones. It may also be possible that large gene families can have
higher percentage of genes belonging to TAGs than small gene families simply by
chance. We performed a permutation test to see whether chance alone can produce
such a strong association between family sizes and TAG percentages. We simulated
10 000 pseudogenomes, each of which has the same
number of gene families and distribution of family sizes as the studied
genomes. We randomly assigned the chromosome location of all the genes in the
pseudogenomes and determined the percentage of TAGs for all the family sizes.
We then examined the correlation between the percentage of TAGs and family
sizes and found that indeed they are correlated. However, the percentage of
TAGs in the simulation is much lower than our observation in all sizes. For
example, in human, we observed about 34% of TAGs in the gene families of size
10, while only about 4% in our simulated distribution. In fact, the simulated
percentages of TAGs in all the gene family sizes are about 10 times lower than
the actual observations. Thus, it is clear that even though chance does
contribute to the positive correlation between the extent of tandem duplication
and the sizes of gene families, it is not a determining factor.Consistent with the current observation, our recent study
shows that tandem duplication generated more duplicated genes than
retroposition did in large families in both humans and mice [30]. Many genes in
large families such as olfactory receptor genes and zinc finger protein genes
are generated by tandem duplication. The question is why tandem duplication has
played a more important role in large families than in small families.To answer the question, we need to compare the
differences among various mechanisms of gene duplication. There are three major
mechanisms of gene duplication: genome duplication, retroposition, and tandem
duplication [4]. Among the three, genome duplication happens the least
frequently in animals. Moreover, it doubles the copy number of all genes and
thus should have a similar contribution to different-sized families. In
contrast, tandem duplication is more frequent and more specific as it
duplicates only specific genes instead of every gene in the genome. It may be
difficult for a gene to change from single copy to duplicated states because
sequence homology around the gene, required by unequal crossover to generate
tandem duplication, is not always present. However, once a tandem duplication
occurs, it is easy for unequal crossover to quickly expand the array due to the
availability of sequence homology. Thus, tandem duplication has the advantage
of being fast and easy in generating a large number of genes and providing
opportunities for the divergence and refinement of gene function among
duplicated members.It has been shown that retroposition seems to be more
active in highly expressed genes in germline cells [39, 40]. However, members
in large gene families are not necessarily highly expressed. Our recent study
suggests that the expression level seems to be more important than gene family
size in determining what genes get retroposed [41]. Moreover, due to the nature
of retroposition, the retroposed copy does not have ancestral regulatory
regions and its survival is thus dependant upon the probability of being able
to capture a regulatory region. The large amount of retroposed pseudogenes in
the human and mouse genomes suggests that the probability of survival of the
retroposed copy is very small. In contrast, many fewer pseudogenes are
generated by tandem duplication [42], suggesting that the survival rate of
tandem duplication is much higher than the retroposed genes. Therefore, as
unequal crossover is the most efficient mechanism to generate and maintain gene
copies among the three major gene duplication mechanisms, it may explain why
TAGs are more frequent in large families than in small ones.


### 3.5. TAG Homology and SSTAs

Identifying the
homologous relationships (orthologous and paralogous) for TAGs across species
is a challenging task. Frequent gene conversion within tandem arrays [43] and
gene losses and gains of different array members in different species make
genome-wide orthologue assignments computationally intractable [44]. One good
example that shows the difficulty of homology assignments in TAGs can be seen
in the HOX genes. Numerous studies of these genes have shown that there is a
tremendous amount of variation in the number of HOX clusters in different
species. Moreover, there are losses and gains of different members in different
species and frequent gene conversion or concerted evolution in some members
(e.g., [45–48]). Computationally, it is nearly impossible to
identify a correct homology relationship for these genes across multiple
species. There have been computational attempts to infer an evolutionary history
of tandem repeated sequences in multiple species (e.g., [49–51]).
However, it is clear that correct inference of evolutionary history relies on
correct homology assignment, which remains a computationally challenging
problem.To circumvent the homology assignment problem, we
studied two aspects regarding the evolution of TAGs in the 11 species that do
not require identification of exact homology relationships among TAGs. The
first aspect is to examine the evolutionary closeness of the 11 species in
terms of TAG quantities in different gene families. There are two TAG
quantities that one can describe for a particular gene family. One is the total
number of arrays in the family and the other is the total number of
tandemly-arrayed genes in the family. It is expected that the two quantitative
descriptions should show similar evolutionary closeness to what the species
tree reflects. OurK-means clustering result
suggests that the two quantities, to a large extent, are able to reflect the
phylogenetic relationship of the species. The exception is the grouping of dog
and cattle, which is always clustered with the nonmammals. A possible
explanation is that many genes have not yet been annotated in these two
species, especially those mammalian-specific TAGs, which makes them appear
closer to nonmammals. Alternatively, it may also mean that some ancestral
mammalian TAGs are broken up in dog and cattle.The second aspect is related to species-specific
tandem arrays (SSTAs). Apparently, the definition of SSTAs determines that SSTA
statistics are sensitive to the number, the kind, and the annotation quality of
the species that are sampled. For example, it is expected that the more species
included in a sample, the less likely an array will be an SSTA. Meanwhile, the
number of SSTAs in a certain species is directly influenced by the species'
distance to its closest related species in the sample. For instance, the number
of SSTAs in human in the human-mouse-rat sample will certainly be higher than
the number of SSTAs in human in the sample that also includes chimp. Moreover,
if the annotation qualities of the two species are different, for instance in
the case of human and chimp, there would be more SSTAs in the better annotated
species human than in the less well-annotated species chimp.Despite these caveats, study of SSTAs, or more
generally, species-specific duplication, can potentially provide insight into
the adaptive evolution of species-specific traits and life styles. For example,
one of the human SSTAs, the sperm protein associated with the nucleus on the X
chromosomeSPANX gene family, containing two tandem arrays with a total
of 6 genes, has been reported to have gone through rapid evolution and
amplification in hominids [52]. Our analyses show that the proportion of tandem
arrays with more than two members in SSTAs is significantly higher than that in
non-SSTAs in some of the studied mammals, which suggests that nonneutral forces
may maintain relatively recent-born arrays. However, caution must be taken to
interpret the results as the species sampling is not homogeneous and some of
the SSTAs might not be truly species-specific due to the deep divergence.


## 3.1. Contribution of Tandem Duplication

Here we
performed a genome-wide survey of TAGs in 11 assembled vertebrate genomes. In
summary, when using a stringent criterion for TAG identification (e.g.,
allowing at the most 1 spacer between array members), we observed a consistent
pattern of tandem duplication contributing to the number of genes in the
genomes and to genome wide duplications: on average, about 14% of the genes in
vertebrate genomes are TAGs, and about 25% of all duplicated genes are tandemly
arrayed.These numbers most likely underestimate the extent of
tandem duplication in these genomes. Our recent study shows that more than 25%
to 40% of the recent gene duplications are generated by tandem duplications in
human and mouse [30]. Therefore, it is likely that many old tandem arrays
became invisible during evolution owing to various genome rearrangements.
Meanwhile, one may wonder whether duplicated genes arising from duplication
mechanisms other than tandem duplication could get scrambled during evolution
and happen to be arranged as TAGs. However, as shown in our previous study,
this possibility should have minimal effect on the TAG statistics because the
probability that duplicated genes appear as TAGs by chance is very low, about 1
to 2 orders of magnitude lower than the actual extent of tandem duplication
[16].


## 3.2. TAG Transcription Orientation

It has been
shown that∼80% and ∼88% of tandem arrays are in parallel
transcription orientation in Arabidopsis thaliana and rice, respectively
[25]. How this compares to the genome patterns in these species has not yet
been studied. The vertebrate genomes show amazingly consistent patterns in the
proportion of gene pairs in parallel, convergent, and divergent transcription
orientations with a ratio of ∼2:1:1.
In contrast, TAGs have much higher proportions of parallel orientation, ranging
from about 72% to as high as 94%. Therefore, in both plants and animals,
parallel orientation is the dominant type of transcription orientation in TAGs.So why is there disproportionately less convergent and
divergent transcription orientation in TAGs than in the genome? One explanation
is that tandem duplications occur at a higher rate on the same strand than on
different strands. Little is known about what determines the rates of tandem duplication
on the same strand or different strands. Therefore, how much differential rates
of tandem duplication between same-strand and different-strands contribute to
the observed dominance of parallel orientation across all the studied species
remains an open question. Another possible explanation is related to long
inverted repeats (LIRs). It has been shown that LIRs can substantially increase
genome instability. For example, in the mouse, LIRs in germ lines can lead to
elevated genome rearrangements due to increased levels of illegitimate
recombination, gene conversion, and deletion mediated by LIRs [31, 32]. In the
human, several genetic diseases have been reported to be caused by illegitimate
recombination and deletion induced by LIRs [33]. In the case of TAGs, tandem
duplicated genes on opposite strands (in convergent or divergent orientation)
are essentially LIRs, and their initial high sequence identity might increase
the level of illegitimate recombination and various genome rearrangements. The
increased genome instability might have a disastrous effect on the individuals
that carry tandem duplication; strong negative selection against the
duplication will reduce the fixation probability of tandem duplication in the
population. This may at least in part explain why we observe a much lower
proportion of TAGs with convergent and divergent
orientations than in the genome. Meanwhile, the
fact that there are still some TAGs with convergent or divergent orientation
can also be explained by LIR-mediated changes. It has been observed that
illegitimate recombination events induced by LIRs sometimes result in
asymmetric deletion that eliminates the central symmetry of LIRs [31, 32]. When
the deletion does not have a negative effect on the function of the genes, for
example, when the deletion happens to be located in introns, the elimination of
the symmetry in the LIRs can actually prohibit further illegitimate
rearrangements and reduce the levels of gene conversion. Consequently, the
LIRs, that is, tandem duplicated genes on opposite strands, no longer pose a
threat to genome stability and thus can be fixed in the population [5, 31].
More research needs to be done to determine the causes of the higher proportion
of TAGs in parallel orientation than the genome average.


## 3.3. Tandem Array Sizes

All plant and
animal genomes that we have studied so far show that the majority of tandem
arrays contain only two members. It is likely that large arrays are destroyed
by various genome rearrangements and become smaller arrays over time, which
might be the case for most of the tandem arrays. For the large TAG arrays such
as the 18S and 28S ribosomal RNA genes in the vertebrates [22], mechanisms such
as continued concerted evolution (including unequal crossover and gene
conversion) and natural selection need to act on the arrays to prevent
array-size decay.The fluctuation of array sizes has been observed in
natural populations of many species such as humans [34] and flies [35].
Empirical evidence also suggests that the fluctuation can produce visible
phenotypic effects and sometimes can be detrimental. For example, in Drosophila
melanogaster, 18S and 28S rRNA genes contain 150 to 250 tandemly arranged
repeats in wild-type flies [35, 36] and individuals carrying a lower copy
number than the wild-type have so-called bobbed mutations, characterized
phenotypically by having small bristles, abdominal etching, and developmental
delay [37, 38]. These studies show that the size of tandem arrays is important
in the normal function of organisms and the fluctuation of array sizes might be
selected against.At the same time, a variety of mechanisms can reduce
or prevent size change in a tandem array. For example, insertion of irrelevant
genes (i.e., genes with no homology to the array members) into the array may
effectively reduce the frequency of unequal crossovers. The divergence of array
members can also reduce the frequency of unequal crossover. Therefore,
observation on array sizes across multiple animal and plant genomes reflects
not only a snapshot of current genomes, but also most likely a stable state of
TAGs as a result of joint processes of selection, drift, and mutation on the
arrays.


## 3.4. Tandem Duplication and Family Size

The positive
correlation between the extent of tandem duplication and the sizes of gene
families (Figure3 and Table 3) indicates that the contribution
of TAGs to gene families of different sizes weighs more in large gene families
than smaller ones. It may also be possible that large gene families can have
higher percentage of genes belonging to TAGs than small gene families simply by
chance. We performed a permutation test to see whether chance alone can produce
such a strong association between family sizes and TAG percentages. We simulated
10 000 pseudogenomes, each of which has the same
number of gene families and distribution of family sizes as the studied
genomes. We randomly assigned the chromosome location of all the genes in the
pseudogenomes and determined the percentage of TAGs for all the family sizes.
We then examined the correlation between the percentage of TAGs and family
sizes and found that indeed they are correlated. However, the percentage of
TAGs in the simulation is much lower than our observation in all sizes. For
example, in human, we observed about 34% of TAGs in the gene families of size
10, while only about 4% in our simulated distribution. In fact, the simulated
percentages of TAGs in all the gene family sizes are about 10 times lower than
the actual observations. Thus, it is clear that even though chance does
contribute to the positive correlation between the extent of tandem duplication
and the sizes of gene families, it is not a determining factor.Consistent with the current observation, our recent study
shows that tandem duplication generated more duplicated genes than
retroposition did in large families in both humans and mice [30]. Many genes in
large families such as olfactory receptor genes and zinc finger protein genes
are generated by tandem duplication. The question is why tandem duplication has
played a more important role in large families than in small families.To answer the question, we need to compare the
differences among various mechanisms of gene duplication. There are three major
mechanisms of gene duplication: genome duplication, retroposition, and tandem
duplication [4]. Among the three, genome duplication happens the least
frequently in animals. Moreover, it doubles the copy number of all genes and
thus should have a similar contribution to different-sized families. In
contrast, tandem duplication is more frequent and more specific as it
duplicates only specific genes instead of every gene in the genome. It may be
difficult for a gene to change from single copy to duplicated states because
sequence homology around the gene, required by unequal crossover to generate
tandem duplication, is not always present. However, once a tandem duplication
occurs, it is easy for unequal crossover to quickly expand the array due to the
availability of sequence homology. Thus, tandem duplication has the advantage
of being fast and easy in generating a large number of genes and providing
opportunities for the divergence and refinement of gene function among
duplicated members.It has been shown that retroposition seems to be more
active in highly expressed genes in germline cells [39, 40]. However, members
in large gene families are not necessarily highly expressed. Our recent study
suggests that the expression level seems to be more important than gene family
size in determining what genes get retroposed [41]. Moreover, due to the nature
of retroposition, the retroposed copy does not have ancestral regulatory
regions and its survival is thus dependant upon the probability of being able
to capture a regulatory region. The large amount of retroposed pseudogenes in
the human and mouse genomes suggests that the probability of survival of the
retroposed copy is very small. In contrast, many fewer pseudogenes are
generated by tandem duplication [42], suggesting that the survival rate of
tandem duplication is much higher than the retroposed genes. Therefore, as
unequal crossover is the most efficient mechanism to generate and maintain gene
copies among the three major gene duplication mechanisms, it may explain why
TAGs are more frequent in large families than in small ones.


## 3.5. TAG Homology and SSTAs

Identifying the
homologous relationships (orthologous and paralogous) for TAGs across species
is a challenging task. Frequent gene conversion within tandem arrays [43] and
gene losses and gains of different array members in different species make
genome-wide orthologue assignments computationally intractable [44]. One good
example that shows the difficulty of homology assignments in TAGs can be seen
in the HOX genes. Numerous studies of these genes have shown that there is a
tremendous amount of variation in the number of HOX clusters in different
species. Moreover, there are losses and gains of different members in different
species and frequent gene conversion or concerted evolution in some members
(e.g., [45–48]). Computationally, it is nearly impossible to
identify a correct homology relationship for these genes across multiple
species. There have been computational attempts to infer an evolutionary history
of tandem repeated sequences in multiple species (e.g., [49–51]).
However, it is clear that correct inference of evolutionary history relies on
correct homology assignment, which remains a computationally challenging
problem.To circumvent the homology assignment problem, we
studied two aspects regarding the evolution of TAGs in the 11 species that do
not require identification of exact homology relationships among TAGs. The
first aspect is to examine the evolutionary closeness of the 11 species in
terms of TAG quantities in different gene families. There are two TAG
quantities that one can describe for a particular gene family. One is the total
number of arrays in the family and the other is the total number of
tandemly-arrayed genes in the family. It is expected that the two quantitative
descriptions should show similar evolutionary closeness to what the species
tree reflects. OurK-means clustering result
suggests that the two quantities, to a large extent, are able to reflect the
phylogenetic relationship of the species. The exception is the grouping of dog
and cattle, which is always clustered with the nonmammals. A possible
explanation is that many genes have not yet been annotated in these two
species, especially those mammalian-specific TAGs, which makes them appear
closer to nonmammals. Alternatively, it may also mean that some ancestral
mammalian TAGs are broken up in dog and cattle.The second aspect is related to species-specific
tandem arrays (SSTAs). Apparently, the definition of SSTAs determines that SSTA
statistics are sensitive to the number, the kind, and the annotation quality of
the species that are sampled. For example, it is expected that the more species
included in a sample, the less likely an array will be an SSTA. Meanwhile, the
number of SSTAs in a certain species is directly influenced by the species'
distance to its closest related species in the sample. For instance, the number
of SSTAs in human in the human-mouse-rat sample will certainly be higher than
the number of SSTAs in human in the sample that also includes chimp. Moreover,
if the annotation qualities of the two species are different, for instance in
the case of human and chimp, there would be more SSTAs in the better annotated
species human than in the less well-annotated species chimp.Despite these caveats, study of SSTAs, or more
generally, species-specific duplication, can potentially provide insight into
the adaptive evolution of species-specific traits and life styles. For example,
one of the human SSTAs, the sperm protein associated with the nucleus on the X
chromosomeSPANX gene family, containing two tandem arrays with a total
of 6 genes, has been reported to have gone through rapid evolution and
amplification in hominids [52]. Our analyses show that the proportion of tandem
arrays with more than two members in SSTAs is significantly higher than that in
non-SSTAs in some of the studied mammals, which suggests that nonneutral forces
may maintain relatively recent-born arrays. However, caution must be taken to
interpret the results as the species sampling is not homogeneous and some of
the SSTAs might not be truly species-specific due to the deep divergence.


## 4. Conclusions

We have
provided a quantitative account of TAGs and their contribution to duplications
in vertebrate genomes. This is a first step towards understanding the evolution
of these genes. As it has been increasingly realized that how genes are
arranged on chromosomes plays an important role in determining gene function,
TAGs stand out for their unusual spatial arrangement. Future research can be
directed towards further understanding the intricate differences of tandem
duplication from other types of duplication and the impact on the ultimate fate
of duplicated genes.


## 5. Materials and Methods

There are
altogether 11 vertebrate genomes assembled and available in Ensembl Version 41
(http://www.ensembl.org/). Therefore, we focused on these 11 species including
human (Homo sapiens), chimp (Pan troglodytes), mouse (Mus
musculus), rat (Rattus Norvegicus), macaca (Macaca mulatta),
cattle (Bos taurus), dog (Canis familiaris), opossum (Monodelphis
domestica), chicken (Gallus gallus), zebrafish (Danio rerio),
and tetraodon (Tetraodon nigroviridis). Previously, we studied TAGs in
the genomes of human, mouse, and rat [16]. However, as the annotation quality
has been continually improved over time and this paper is intended to be a
comprehensive overview of TAGs in all available vertebrate genomes, we
reanalyzed these species using the latest version 41 as well.Annotation of genes for all 11 species was obtained
using Ensembl Biomart (http://www.ensembl.org/). The total number of genes is shown
in Table 1. Genes annotated as unknown and mitochondrial were removed and
only those with known chromosome location were kept, as we needed the
information to determine TAGs. We also required that each gene should be equal
to or longer than 300 nucleotides. Family information was also obtained using
Ensembl Biomart. In Ensembl, gene families are clustered using Markov
clustering algorithms (MCL) based on sequence similarities (see
http://www.ensembl.org/ for details). All data were stored in MySQL database for
subsequent analysis.TAGs are usually defined as genes that are duplicated
tandemly on chromosomes. Members of tandem arrays may be separated by other
unrelated genes (called spacers). During evolution, various genome
rearrangements, such as transposition and insertion of genes that are unrelated
to array members (i.e., not through duplication), can disrupt the spatial
arrangement of the TAGs. Allowing different numbers of spacers in between two
members of an array will result in a different number of TAGs. For example,
consider an array with the spatial arrangement ofA1-B-A2-A3-C-A4-A5,
where all As are duplicated genes, and B and C are spacers. When allowing 0 spacers, we will
have 2 tandem arrays with each having 2 members (A2 and A3; A4 and A5); allowing 1 spacer, we will have 1 array
with 5 members (A1, A2, A3, A4,
and A5).To obtain TAGs, we sorted all the genes of each
species chromosome by chromosome and indexed them in ascending order based on
their physical locations. Letd denote the absolute difference of the indices
between two genes on the same chromosome. d−1 is equal to the number of spacers between
these two genes S.
When S=0,
it is a perfect TAG gene pair with no spacers. For certain S,
we marked those gene pairs with d≤S+1 and clustered them using a single-linkage
algorithm, which ensures that within each tandem array, there exists at least
one TAG link between any two array members. A TAG link is the relationship of
two genes that can be seen as a TAG pair under the certain number of spacers
allowed. We screened TAGs under each TAG definition (spacers 0–10) for every
species.



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*Source: 545269-2008-09-21.xml*