Molecular Biology, Vol. 35, No. 2, 2001, pp. 157–167. Translated from Molekulyarnaya Biologiya, Vol. 35, No. 2, 2001, pp. 196–207.
Original Russian Text Copyright © 2001 by Arkhipova.
UDC 575.11:595.773.4
Transposable Elements in the Animal Kingdom
I. R. Arkhipova
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA;
E-mail: [email protected]
Received August 23, 2000
Abstract—Transposable elements (TEs) are commonly thought to be of universal occurrence in eukaryotes.
Analysis of complete higher eukaryotic genomes confirms the TE status as substantial genome components and
provides insights into their role in shaping the genome structure of extant eukaryotes. This review addresses
several recently investigated problems in transposon biology, including the potential roles of promoter organization in transposon function and evolution, the ubiquity of TEs in numerous phyla of the animal kingdom, and
the possible connection between transposon content and mode of reproduction.
Key words: DNA transposons, retrotransposons, animal phyla, sexual/asexual reproduction
INTRODUCTION
As the genome sequencing era is rapidly advancing
our understanding of what the eukaryotic genomes are
in fact composed, it’s time to take a new look at the
role which transposable elements (TEs) have played
in shaping the present structure of these genomes over
millions of years of evolution. In this review, I will
address a number of recently investigated problems in
the field of transposon research. The emphasis will be
placed on TEs in the animal kingdom, in accordance
with primary research interests of the author and also
because reviews on TEs in other kingdoms were published relatively recently [1–3]. However, a comprehensive review is not the main purpose of this paper;
rather, it is concerned with several subjects which
were of particular interest to the author in recent years
and are especially attractive for presenting speculations rarely affordable within the frameworks of a
research paper.
For convenience, I will consider transposons and
host cells as separate interacting entities, with transposons being foreign DNA and the host cell providing
them with the appropriate environment. However, in
many cases transposons became so tightly associated
with host genomes and intertwined with host systems
of transcription, translation etc. that it becomes difficult to make such distinctions. Moreover, it is plausible that transposons have become such an integral part
of higher eukaryotic genomes that it would be almost
impossible to imagine evolutionary consequences
resulting from the loss of the transposon component.
One of the main unanswered questions is how
transposons got established so profoundly in genomes
of higher eukaryotes. In prokaryotes, they do not normally form stable associations with the host genomes.
In lower eukaryotes with streamlined genomes, they
proliferate in relatively small numbers, mostly by
finding themselves “safe havens” for insertion in
which they do not cause much damage to their hosts
[4]. With increase in the genome size, however, an
increase in transposon content also comes about.
What are the properties of higher eukaryotic organisms and their TEs that allow such permanent associations? Some of these properties are discussed below,
while others still await further consideration.
TRANSCRIPTION AND PROMOTERS
One of the first aspects of transposon–cell interaction concerns its ability to function as a transcriptional
unit. For the two major classes of eukaryotic transposons, class I or retrotransposons and class II or
DNA transposons, transcription would serve slightly
different purposes. In DNA transposons, it is primarily needed to generate mRNA serving as a template
for synthesis of transposase, in most cases the only
enzyme needed for transposition. Hence, their level of
transcription need not be as high as for retrotransposons, which in addition to the enzymes such as
reverse transcriptase (RTase) also produce large
amounts of structural proteins involved in RNP formation, and generate enough RNA to serve as a template for reverse transcription. Consequently, there is
often a pronounced difference between levels of transcription for DNA transposons and retrotransposons.
Transcript levels of the former can be relatively low or
even not reach detectable levels at all, and fusion to
inducible promoters is typically used to achieve
higher levels of expression [5–8]. In fact, DNA transposon-based constructs are the genetic tools of choice
over retrovirus-based constructs, which require prior
0026-8933/01/3502-0157$25.00 © 2001 MAIK “Nauka /Interperiodica”
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ARKHIPOVA
removal of enhancers, in large-scale enhancer-trap
screens based on the ability of weak promoters to
acquire expression patterns determined by adjacent
enhancers upon insertion in their vicinity [9–13].
Retrotransposon transcripts, on the contrary, are
relatively abundant at the stages and in tissues when
and where they are produced. This is correlated with
the absence or presence of various stage- and tissuespecific enhancer elements, which are far more diversified and individualized in different retrotransposon
families than their coding regions. The variety of
expression patterns covers virtually all developmental
stages and tissues (reviewed in [14–16]), implying
that the process of acquisition of such control elements could be more or less random. However, transposition should take place in the germ line in order to
establish the transposon in the host genome and transmit it to the progeny. Retrotransposons cannot transpose without an RNA template, and their transcripts/reverse transcripts need to be present in the
germ line in order to give rise to heritable transposition events. It is therefore not surprising that a large
number of retrotransposons has been demonstrated to
possess enhancer elements providing expression in
the germ line and/or to generate transcripts detectable
in germline cells [17–28].
Let us summarize the strategies available for transposons as transcriptional units with respect to transcription control elements:
Class I or retrotransposons: RNA is not only a template for protein synthesis but also is used as genetic
material during transposition, therefore transcription
of the entire unit should be ensured by either avoiding
loss of non-transcribed control regions or compensating for it.
Non-LTR retrotransposons could achieve this by:
(i) having completely internal promoter/enhancer
elements at the 5' end, with an RNA start site located
upstream at the 5' boundary of the transposon, with no
dependence on adjacent transcriptional control elements, although possibly subject to influence by such
elements;
(ii) same but at the 3' end, thus requiring a tandem
head-to-tail arrangement;
(iii) repeating the promoter sequence several times
at the 5' end;
(iv) having no promoter elements of their own, but
inserting site-specifically into a sequence which provides a promoter for readthrough transcription;
(v) relying on a “master copy” which had by
chance landed in a proximity of a cellular promoter
and since then is giving rise to all subsequent copies.
LTR-containing retrotransposons:
(vi) promoter (and terminator) is located in the
LTR and regenerated after each retrotransposition
cycle, so that loss of the 5'-nontranscribed promoter
sequences after reverse transcription is avoided.
A uniform LTR structure (U3-R-U5) includes location of the transcription start site upstream of the transcription termination site, so that the short region in
between gives rise to a terminal redundancy (R) at the
ends of the transcript [29].
Strategy (i) appears to be the most common one used
by a large number of non-LTR retrotransposons studied (e.g., jockey, F, I, Doc, L1Hs, TRAS1) [30–36]. It is
especially widespread in Drosophila, perhaps because
downstream RNA polymerase II promoter elements
work efficiently in this species and occur in a very
large number of cell TATA-less promoters [37, 38].
(ii) is an ingenious invention used by HeT-A, the
telomere-associated retrotransposon of Drosophila
melanogaster, in which an element transposing to the
end of the chromosome attaches the 3'-terminally
located promoter to its downstream neighbor, being
therefore quite unselfish [39]. (iii) so far has been
described in rodent L1 elements [40, 41]. (iv) was
hypothesized for the site-specific ribosomal insertion
elements [42]. Finally, strategy (v) is usually invoked
whenever all the other options are ruled out (e.g., [36]).
Promoters of LTR-retrotransposons, while possessing the uniform retrovirus-like structure, usually
conform to the standards used by other promoters in
the host cell, since they also exploit the same machinery. Thus, the LTRs of retroviruses typically contain
TATA boxes [43], while many Drosophila LTR retrotransposons possess TATA-less promoters with
well-pronounced initiator sequences and downstream
promoter elements [44, 45]. Moreover, even such nonstandard combinations as overlapping RNA polII and
polIII promoters have been described, both for LTR
and non-LTR retrotransposons [46, 47], strengthening
the belief that their acquisition was largely a matter of
chance. Transcriptional enhancers and other control
elements typically reside in the U3 region of the LTR,
although they may also be localized in the 5'-and 3'untranslated regions (reviewed in [14, 15]), offering
targeting opportunities for control of transposition at
the transcriptional level.
Class II elements, or DNA transposons, in principle do not need to carry efficient promoter/enhancer
elements and may possess very weak promoters (see
above); in fact, a basal level of transcription would do
almost as well. A basal promoter could be located
anywhere between the 5'-ITR and ORF, and a precise
RNA start would not be necessary, because RNA is
not a genetic material (even readthrough transcription
might occasionally suffice). This offers very limited
opportunities to control transposition at the level of
transcription, compared to retrotransposons, and posttranscriptional control mechanisms appear to be
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TRANSPOSABLE ELEMENTS IN THE ANIMAL KINGDOM
favored, including splicing, RNA interference, transposase subunit interaction, etc. [6, 48–53].
TANDEM PROMOTERS AND HYPOTHESES
FOR EVOLUTIONARY ORIGINS
OF LTR-RETROTRANSPOSONS
The tandem type of transcriptional unit organization, such as the one discovered in the HeT-A retrotransposon [39], deserves special attention. It might
represent a mechanistically possible pathway of transition from non-LTR to LTR retrotransposons and
could have resulted, at some distant point in evolutionary history, in acquisition of the LTR structure
previously lacking in non-LTR retrotransposons. In
this respect, it is worth mentioning that HeT-A might
not be the only element using this strategy: the Penelope transposon of D. virilis [53a] appears to possess
a promoter organization which is in fact very similar
to that of HeT-A (M.B. Evgen’ev, personal communication).
It has been noted that the arrangement of two transcriptional units in tandem bears resemblance to the
LTR structure: both promoter and terminator
sequences are present in duplicate on either side of the
coding region, and the resulting RNA has a small terminal redundancy similar to the transcripts of retroviruses and LTR-retrotransposons [39]. However, the
actual transition requires acquisition of a different
pathway of reverse transcription.
Non-LTR retrotransposons are undoubtedly the
most diverse and arguably the most ancient group of
retroelements, with significant sequence homology to
group II introns and telomerase reverse transcriptases
[54–57]. They have been around long before the
fusion of the RTase and integrase domains, characteristic of LTR-retrotransposons, took place early in evolutionary history [58], perhaps having occurred independently in the progenitors of copia-like and gypsylike retrotransposons which have a reciprocal arrangement of these domains. If we assume that these
fusions indeed occurred independently, it is of interest
to trace common features that might have been
acquired, making different superfamilies of LTR-retrotransposons so structurally similar. The features that
are important for an evolutionary transition from nonLTR to LTR-retrotransposons are acquisition of internal priming (tRNA-dependent, self-priming, etc.), the
ability of RTase to perform template jumps, and participation of more than one RNA molecule in the act
of reverse transcription.
An imaginary intermediate step would be a LINElike element with a tendency to form a tandem headto-tail arrangement of identical transcriptional units.
If the propensity of LINE elements for “3' transduction” [59] could simultaneously lead to capture of a
cellular promoter at the 3' end, its activity might
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159
supersede the previously used 5'-terminal promoter,
which tends to be lost during incomplete reverse transcription, and result in production of a terminally
redundant transcript (Fig. 1). If the RTase previously
possessed affinity to the sequence or secondary structure at the 3' end of a nonredundant transcript, as
seems to be the case for some LINE-like RTases [60],
a terminally redundant transcript, when used by RTase
as a template, could have RTase bound to either 3' or
5' R region with equal probability. At this stage, acquisition of an internal priming mode should be postulated. Namely, the enzymatic machinery bound at the
5' end could find a way to introduce a cut into the secondary structure formed by the 5' end (Tf1-like selfpriming mechanism [61]), or to recruit one of the cell
tRNAs, especially if an RTase displays a certain affinity for this tRNA (similar to the RTase of a Neurospora mitochondrial plasmid that recognizes a tRNAlike structure at the 3' end of the RNA template [62]).
Note that the LTR formation occurs automatically,
once the priming is moved to an internal location and
the ability to switch templates allows the full-length
cDNA synthesis to complete. The process of reverse
transcription is eventually relocated from the nucleus
to the cytoplasmic nucleoprotein particles containing
another molecule of the RNA template to facilitate
extension of newly formed strong-stop cDNAs. The
RNA binding capacity, which is typically provided by
the ORF1 located upstream from the pol gene in both
LTR- and non-LTR retroelements, should be an
important factor in a transition to sequestering cDNA
synthesis within RNP particles.
HORIZONTAL TRANSFER AND ITS POSSIBLE
PREREQUISITES
To relocate into another host species, a TE must
undergo an act of horizontal transfer: introduce a copy
of itself, previously residing in a TE-containing host
species, into the genomic DNA of another, naive species (reviewed in [63, 64]). To succeed, it needs to get
established in the host germ line for further vertical
transmission to the progeny, and to proliferate in individual genomes and ultimately throughout the entire
population. All this, however, should be accomplished
without doing a lot of harm to the host, as this will
also result in death of a TE.
If a transposon starts over as a foreign DNA invading the host cell, the first challenge it faces is to evade
any restriction system to which it might be vulnerable.
Then, it needs to establish a long-term association
with the host by becoming compatible with the host
systems involved in transcription, translation, posttranscriptional and post-translational processes. There
are several options available to transposons to establish themselves in a new environment:
160
ARKHIPOVA
PT
(A)
PT
(A)
A
RNA
R
R
AAA
B
RT
RT
R
R
AAA
C
RT
R
RT 3'
AAA
R
Fig. 1. Possible steps in evolution of LTR structure from
tandem arrays of retroelements. P and T, promoter and terminator sequences arranged to generate terminal redundancy R at both ends of the polyadenylated (AAA) RNA
transcript. Step A represents transcription; B, translation;
and C, acquisition of internal priming (in this case tRNAmediated) versus target priming by reverse transcriptase
(RT), leading to evolution of retroviral pathway of reverse
transcription.
(i) If a transposon comes from a species that is not
too distantly related to the new host, there is a good
chance that its control elements, such as promoters/enhancers, might still be compatible with the new
host species, with no need to acquire novel regulatory
elements. Introgression-type mechanisms might then
be responsible for exchange of genetic material,
including TEs, between related species. This would
imply that horizontal transfers are more likely to
occur between species belonging to the same genus
than between species belonging to different phyla.
Indeed, the best-documented cases of horizontal
transfer are known for host species belonging to the
same genus Drosophila [65, 66].
(ii) There is limited chance that regulatory elements, especially transcriptional regulators, would be
compatible between very distantly related species.
Therefore, such distant horizontal transfers should be
quite infrequent. To circumvent the non-functionality
of control elements, the easiest way would be to
acquire the resident ones by de novo incorporation
into transposon structure, rather than to modify the
pre-existing sequences. For this event to occur, a retrotransposon must land in the vicinity of a functional
promoter/enhancer, and readthrough transcription
from the cell promoter or 3'-transduction could lead to
formation of a hybrid message containing the necessary regulatory regions. After that, RTase would be
able to convert such transcript into cDNA, either in
the chromosome or in a nucleoprotein particle, assuming that the synthesized RTase would still preserve its
intrinsic ability to initiate reverse transcription of the
corresponding RNA. Indeed, most of the conservation
is usually observed within open reading frames of a
TE and not in the adjacent untranslated regions which
exhibit a lot of variability, and insertions of retroelements into promoter/enhancer sequences are a rule
rather than an exception (reviewed in [15]).
For LINE-like elements, the RTase and, in most
cases, the associated endonuclease activity would be
sufficient to perform subsequent integration via target-primed reverse transcription [67]. LTR-containing
elements, in addition, would need to incorporate the
cis-acting packaging and priming signals compatible
with their enzymatic machinery. Ability to associate
more than one molecule during reverse transcription
would increase the chances of such incorporation by
recombinational processes at the RNA level, mediated
by intermolecular jumps of RTase within a nucleoprotein particle.
In addition to the above-mentioned problem of
interchangeability of regulatory elements between
different host species, a similar problem would also
emerge if intermediate hosts serving as shuttle vectors
are used to transfer TEs between host species [68]. It
is conceivable that such complex entities as retrotransposons would require vectors compatible with
eukaryotic hosts, such as eukaryotic viruses or parasites. In contrast, the simplest transposition units such
as mariner-like transposons essentially consist of a
single intron-lacking transposase gene embedded
between two short inverted terminal repeats, with very
small untranslated regions and little room for regulatory elements such as enhancers, insulators, etc. These
simplest units could be temporarily maintained in
prokaryotic vectors, thereby expanding the range of
available host organisms and facilitating horizontal
transfers. However, experimental observations capturing such acts are yet to be made.
It is therefore not surprising that for DNA transposons horizontal transfer appears to be the predominant mode of transmission [51, 69]. This, however,
can easily be reversed if the transposition process,
which is essentially host factor-free for mariner-like
transposons [70–72], becomes dependent on hostencoded proteins (e.g., [73]). LTR retrotransposons
seem to undergo horizontal transfers only sporadically [66, 74, 75], although the gypsy group elements
containing an env-like gene have a potential to be
transmitted as extracellular virus-like particles, without resorting to any vectors [76, 77]. Finally, non-LTR
retrotransposons apparently move horizontally very
rarely if ever, probably in part because they do not
have a relatively stable extrachromosomal DNA intermediate in their transposition cycle ([78], but see [79]).
ARE TRANSPOSONS UBIQUITOUS
IN THE ANIMAL KINGDOM?
The universality of TE occurrence and the mode of
their transmission may be assessed to a certain extent
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TRANSPOSABLE ELEMENTS IN THE ANIMAL KINGDOM
by analyzing the distribution of TEs on a wide scale in
a large number of phylogenetically diverse organisms.
Previously, screens of such nature were conducted for
copia-like retrotransposons in plants [80, 81] and
revealed the ubiquity of copia-like elements throughout the plant kingdom and the predominance of the
vertical mode of transmission, with rare occasional
cases of suspected horizontal transfers. However,
copia-like retrotransposons in the animal kingdom are
found only sporadically, in a few isolated species, and
the most prominent superfamilies of TEs are LINElike and gypsy-like retrotransposons, as well as DNA
transposons of the mariner/Tc1 superfamily.
The way that has proven effective to rapidly screen
for transposons on a large scale is to employ a single
step of PCR amplification with degenerate primers
spanning the most conserved domains of RTases or
transposases. This approach has successfully been
used to amplify those transposon sequences which
contain at least two six-amino-acid blocks of similarity to a given query transposon-encoded protein
[80−83]. A problem with this approach lies in the relative shortness of the blocks of conserved amino acids
in many RTases and transposases, making the detection of distantly related transposons uncertain. RTases
contain at least seven characteristic “signature”
domains distributed over more than a kilobase with
variable spacings (Fig. 2). However, each domain displays only two or three amino acids that are well conserved across the entire superfamily [54, 84]. In DNA
transposons, several analogous short conserved
blocks of homology exist in most transposases [85].
To compensate for the lack of extensive conservation in reverse transcriptases, a two-step PCR amplification procedure with nested primers was designed
[86]. The procedure takes advantage of the most conserved reverse transcriptase domains, designated A,
B, C, and E (Fig. 2a). The first set of highly degenerate
primers is targeted to domains A and E, and the firstround amplification products, substantially enriched
in retrotransposon-related sequences, are subjected to
a second round of amplification using highly degenerate primers against the most conserved residues in the
internally located superfamily-specific domains B and
C. This procedure typically yields bands of sizes diagnostic for the corresponding superfamily. The identity
of such bands can be confirmed by cloning and
sequencing. A similar strategy can be employed for
amplification of DNA transposases, using primer
pools directed against conserved residues in domains
depicted in Fig. 2b.
Retrotransposons are commonly believed to be of
universal occurrence in eukaryotes, even though only
a few major phyla have actually been examined. We
tested the validity of this belief by screening representatives of 24 animal phyla for two retrotransposon
superfamilies. PCR assays for LINE-like RTase
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161
sequences yielded one or more prominent bands of
120−170 bp, the size range of the B–C interval in
known LINE-like RTase clades (table). Comparisons
of amino acid sequences in the B–C interval place
most sequences in the known LINE-like clades R4,
L1, RTE, R1, Jockey, or CR1, all of which are
believed to be of ancient origin [78]. Groups of LINElike RTase sequences apparently representing new
clades were found in an acanthocephalan, a flatworm,
and a diplomonad protozoan. Within a given clade,
more closely related species typically contain more
similar RTase sequences, although it is not always
possible to determine phylogenetic relationships
because the segments in question are fairly short and
in many cases quite divergent. It is notable, however,
that the region between the second-step primers
exhibits significant clade-specific conservation of
amino acid sequences, which only becomes evident in
a large-scale comparison between many members of
the clade.
Similarly, bands of 120–130 bp, diagnostic for
gypsy-like RTases, were found in two-step amplifications of DNA from 35 species, representing 21 of the
23 phyla tested (table). Amplicons from the species
further investigated by sequencing could be assigned
to known clades of gypsy-like RTases [87, 88], such as
gypsy, Osvaldo, sushi, and ZAM.
These data demonstrate that LINE-like elements
are virtually universal and gypsy-like elements are
ubiquitous throughout the animal kingdom, being easily detectable by the two-step PCR procedure in the
overwhelming majority of the phyla tested.
The genome sequence data [89, 90] for model
higher eukaryotic organisms such as D. melanogaster,
C. elegans, and humans indicate that recognizable
TEs, and especially retrotransposons, represent a substantial fraction of the total genomic DNA (10–20%,
not to mention plant genomes such as maize where
they represent 50–85% of the genome). Comparison
of band intensities in DNA from model organisms and
from representatives of the major phyla of the animal
kingdom indicates that in most of them retrotransposons also form a substantial component of genomic
DNA. The multicopy nature of these sequences is also
confirmed by the fact that none of the cloned copies
from any LINE-like family in any of the species studied had identical nucleotide sequences. They were
always present as multiple divergent copies and
apparently inhabited these genomes for a long period
of time. The gypsy-like elements are generally present
in lower copy number than LINE-like elements, since
it takes additional 20 cycles of amplification to detect
the corresponding bands at the second PCR step.
It is also evident that no closely related retrotransposon sequences are present in distantly related species, indicating that horizontal transfers of retroelements occur very rarely. DNA transposons of the mar-
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ARKHIPOVA
(a)
LINE
1
2
A
B
C
D
E
Gypsy
1
2
A
B
C
D
E
(b)
DE
D
(34)
DE
D
(35)
D
Mariner
Tc
E
Fig. 2. Domain structure of reverse transcriptases (a) and transposases (b) indicating location of nested PCR primers. Conserved
domains are denoted by filled boxes. Domains A–E [84] correspond to RTase domains 3–7 [54], respectively. One or two feathers
on the arrows indicate first- and second-step amplification primers, respectively. Filled bars represent second-step amplification
products specific for each superfamily.
iner/Tc1 superfamily, in agreement with previous
studies [51, 69, 83, 91], exhibit patchy distribution,
and it is not uncommon to find closely related
sequences in very distant species, indicating that the
horizontal mode of transmission for these elements is
predominant.
TRANSPOSON CONTENT AND MODE
OF REPRODUCTION
It has long been noted that sexual reproduction
allows mobile elements, even if deleterious, to spread
in populations, and the loss of sex was predicted to
eventually result in populations free of such elements
[92]. Therefore, it was of particular interest to test this
expectation on a very special taxonomic group—rotifers of the class Bdelloidea, comprising 4 families,
18 genera, and some 360 species. This is the largest
metazoan taxon in which males, hermaphrodites, and
meiosis are unknown, and the only taxon in which
ancient asexuality was supported by molecular
genetic evidence [93]. The molecular data obtained
were in agreement with the expectation that after millions of years of evolution without sexual reproduction or genetic exchange individual genomes of bdelloid rotifers would no longer contain closely similar
haplotypes and instead would contain highly divergent descendants of former alleles.
The PCR-based assays described above were performed on five species of bdelloid rotifers representing three of the four known families of class Bdel-
loidea, and, for comparison, on five species of rotifers
belonging to classes in which sexual reproduction is
either constitutive (Acanthocephala) or facultative
(Monogononta). All five species of non-bdelloid rotifers, as expected, tested positive for LINE-like
RTases, and three of them also tested positive for
gypsy-like RTases. In contrast, no diagnostic LINElike or gypsy-like bands were visible under the same
amplification conditions in any of the five species of
bdelloid rotifers tested. Although the complete
absence of these retrotransposons cannot be demonstrated without knowing the entire genomic sequence,
it is evident that very few if any copies are present in
the bdelloid genomes we examined.
Transposable elements, even if deleterious, are
able to spread through a sexually reproducing population if a given copy can be transmitted to more than
half the progeny [92, 94]. If sexual reproduction
ceases, spread within a population is limited to rare
horizontal transmission events. After a sufficiently
long time, if the population has not become extinct,
random mutation and selection for lineages with
reduced insertional load will eventually result in a
population free of deleterious transposons. In bdelloid
rotifers, which appear to have abandoned sexual
reproduction many millions of years ago [93], any
such elements should have been lost or have diverged
so greatly as to become undetectable by PCR.
Although the apparent lack of retrotransposons
may be interpreted as a consequence of long-term
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TRANSPOSABLE ELEMENTS IN THE ANIMAL KINGDOM
163
Tests for LINE-like and gypsy-like RTases and mariner/Tc1 transposases in diverse eukaryotic species
Types
Sarcomastigophora
Porifera
Cnidaria (L, M)
Ctenophora
Platyhelminthes (M)
Acanthocephala
Rotifera (Monogononta)
Rotifera (Bdelloidea)
Gastrotricha
Nemertea
Priapulida
Sipuncula
Annelida
Echiura
Mollusca (L)
Brachiopoda
Bryozoa
Phoronida
Nematoda (L, G, M, T)
Onychophora
Arthropoda (L, G, M, T)
Tardigrada
Chaetognatha
Echinodermata (G)
Hemichordata
Chordata (L, G, M, T)
Species
Giardia lamblia
Halichondria bowerbanki
Spongilla sp.
Hydra littoralis
Aurelia aurita
Condylactus sp.
Dugesia tigrina
Moniliformis moniliformis
Brachionus plicatilis
Brachionus calyciflorus
Sinantherina socialis
Monostyla sp.
Philodina roseola
Philodina rapida
Habrotrocha constricta
Adineta vaga
Macrotrachela quadricornifera
Lepidodermella sp.
Lineus sp.
Priapulus caudatus
Themiste alutacea
Glycera sp.
Lissomyema mellita
Chione cancellata
Glottidea pyramidata
Amathia convoluta
Phoronis architecta
Caenorhabditis elegans
Euperipatoides rowelli
Drosophila melanogaster
Drosophila pseudoobscura
Drosophila virilis
Lasius niger
Formica polyctenum
Aphis sp.
Milnesium sp.
Sagitta sp.
Echinometra mathaei
Strongylocentrotus purpuratus
Saccoglossus kowalevskii
Branchiostoma floridae
Danio rerio
Onchorhynchus keta
Xenopus laevis
Mus musculus
Bos taurus
Mariner/Tc1
+
+
+S
+
–
+S
–
+
–
–
–
+
+
+S
+S
+S
+
+
–
–
–
+
+S
–
+
+
+
+
–
+
+
+
+
+
+S
–
–
–
+
–
–
–
–
–
+
+
–
+
+
+S
+
+
+
LINE
+S
+
+
+
+
+
+S
+S
+S
+
+S
+S
–
–
–
–
–
+S
+S
+S
+S
+S
+S
+S
+S
+S
+
+S
+S
+S
+
+
+S
+
+S
+
+S
+
+
+S
+S
+
+
+
+
+
Gypsy
+S
+S
–
+
+S
+
+S
+
–
–
–
–
–
–
+
+
+
+
+S
+
+
+
+
+
+S
+S
+S
+S
+
+S
+
+
+
+S
+
+
+
+S
+
+
+S
+
Note: Presence or absence of diagnostic PCR bands is indicated by + or -, respectively. S, verified by sequencing; blank, not done. Superfamilies
previously reported to be present in representatives of a phylum are indicated in parentheses: L, LINE; G, gypsy; M, mariner; T, Tc1.
MOLECULAR BIOLOGY
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ARKHIPOVA
Escherichia coli
Giardia lamblia
Halichondria bowerbanki
Hydra littoralis
Dugesia tigrina
Lineus sp.
Priapulus caudatus
Themiste alutacea
Glottidea pyramidata
Amathia convoluta
Phoronis architecta
Glycera sp.
Lissomyema mellita
Chione cancellata
Euperipatoides rowelli
Drosophila melanogaster
Lepidodermella sp.
Monostyla sp.
Habrotrocha constricta
Caenorhabditis elegans
Sagitta sp.
Saccoglossus kowalevski
Branchiostoma floridae
100 bp ladder
Fig. 3. Ubiquity of LINE-like RTases as demonstrated by
nested PCR.
asexuality, it is also possible that it has allowed bdelloid rotifers to avoid the early extinction that usually
follows the loss of sex in other taxa. This could be the
case if sexual reproduction plays a significant role in
limiting the load of deleterious insertions, by mechanisms involving recombination [95–97], or by other
mechanisms dependent upon sexual reproduction or
meiosis. Indeed, a major advantage of sex may be in
limiting the deleterious insertional load (see [15],
chapter 8). If so, the loss of sex would allow the load
to increase, driving the population to extinction. The
bdelloid lineage may be unusual in having escaped
this fate by somehow becoming free of active retrotransposons, either before or not long after abandoning sex.
It will be of particular interest to obtain sequences
of large stretches of genomic DNA from bdelloid rotifers in an attempt to identify remnants of transposons
that were apparently present in the genomes of bdelloid ancestors, which are presumed to be sexual diploids. While retrotransposons are not expected to constitute a major component of bdelloid genomes, it is
plausible that identification and sequence comparisons of such inactive transposon relics might provide
some clues as to the timing and possible mechanisms
accounting for their loss.
Although deleterious transposons are not expected
to be retained in ancient asexual lineages, those which
have been co-opted to perform certain functions in the
host have the potential to be preserved by natural
selection. It is difficult to guess what kind of function,
if any, might they perform. So far, the best known
example of useful transposons are telomere-associated retrotransposons in Drosophila. Upon loss of
telomerase gene, they have apparently taken over the
function of telomeric repeats normally synthesized by
telomerase to protect chromosome ends from DNA
loss during replication. This is not too surprising
because telomerase is a specialized reverse transcriptase and the underlying principle of telomere
maintenance is restoration of lost DNA by RNAdependent DNA synthesis [57, 98].
While RTases diagnostic for retrotransposons were
not detected in bdelloids, bands diagnostic for mariner-like DNA transposases were readily detectable in
at least three of the bdelloid species tested. Sequence
analysis demonstrated that they belong to the known
lineata and elegans subfamilies of mariner-like transposons [91], and Southern analysis revealed that they
are present in high copy numbers. The degree of
sequence divergence between subfamily members
varies from very low to relatively high, indicating that
some of the copies proliferated relatively recently but
others were present in the genome for a long time (an
alternative explanation would be multiple reinvasions
from an unknown source). The most interesting feature of these transposase sequences was a strong bias
toward synonymous substitution within subfamilies,
indicating that they are or recently were under prolonged selection for function. This is very unusual for
mariner-like transposases: the majority of copies in a
given genome are usually nonfunctional [51, 69],
partly owing to the fact that a transposase transposes
functional and mutated copies with equal efficiency.
While sexually transmitted deleterious transposons are expected to be absent in ancient asexuals,
transposons that undergo frequent horizontal transfer
and are not significantly deleterious may occur in both
sexual and ancient asexual taxa. This may explain
why bdelloids lack retrotransposons, which seldom
move horizontally and typically possess enhancer
and/or suppressor elements that can disrupt the
MOLECULAR BIOLOGY
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TRANSPOSABLE ELEMENTS IN THE ANIMAL KINGDOM
expression of nearby genes, while mariner-like transposons are often transmitted horizontally, lack such
control elements and, as perhaps indicated by the conservation of amino acid sequence in bdelloids, may
not be significantly detrimental to their hosts.
Are there any other organisms in which these correlations could be tested? There are many species and
a few genera for which sexual reproduction is not
known, and even more that reproduce asexually but
resort to sexual reproduction once in a while. The
uniqueness of the bdelloid rotifers is in their ancient
asexuality, which manifests itself in changing their
allelic structure over many millions of years of evolution. So far, bdelloid rotifers top the list of organisms
claimed to represent “ancient asexual scandals” [99],
since in many other asexual groups cryptic sex has
been discovered upon close examination and they
have been removed from the list. The most recent
example is the pathogenic yeast Candida albicans,
which for a very long time was thought to be completely asexual. Upon progress of the Candida
genome sequencing project and discovery of a number of mating-type genes, two groups were able to
induce a sexual cycle in this yeast [100, 101]. Interestingly, Candida also differs substantially in the transposon content from the model S. cerevisiae genome.
While the latter contains a few dozen Ty retrotransposon copies belonging to five families, most of which
are intact and apparently active [102, 103], the Candida genome contains several hundred fragments of
highly rearranged retrotransposons belonging to at
least 34 families [104]. The vast majority of these are
non-functional degenerate elements, and only three
copies still remain intact. This peculiar pattern might
be connected with the overwhelming predominance of
the asexual mode of reproduction in this yeast.
It is also of interest to note that the majority of the
C. elegans retrotransposons is apparently inactive and
have never been reported to cause insertional mutations, while in D. melanogaster they represent the
main cause of spontaneous mutation and most of the
copies appear to be functional [89, 90]. Whether this
is in any way connected with the primarily self-fertilizing mode of C. elegans reproduction and the enormous outcrossing capabilities of fruit flies remains to
be investigated. It has been proposed that there is an
inverse relationship between transposon aggressiveness
and the ratio of asexual to sexual cycles of reproduction
in the host, with selection for benign transposons in
hosts that are mostly selfing or asexual [105].
CONCLUSIONS
Transposable elements continue to bring us new
surprises in the field of genome structure and evolution. Remembering the skepticism with which
Howard Temin’s hypothesis about the origin of retroMOLECULAR BIOLOGY
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165
viruses from cellular movable genetic elements [106]
was initially met, it is only fair that these elements are
no longer being regarded as products of viral degeneration, and the searches for putative progenitors of retroviruses in sequences of vertebrate genomes hold
much promise. Similarly, extensive analyses of TE
phylogenies should reveal a complete picture of their
evolution from simplest to very complex units via
domain acquisition and shuffling, very much in parallel to the way the evolution of protein modules
occurred during transition from primitive to more
complex organisms. The enormous potential of TEs
for genome restructuring is difficult to deny, and
future studies will reveal novel examples of TE participation in the evolution of eukaryotic genomes. At the
same time, the bulk of RTase-containing elements is
apparently dispensable and even deleterious, as indicated by their lack in uniparentally transmitted
organelles and in ancient asexual taxa.
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