The Plant Journal (1998) 13(5), 699–705
SHORT COMMUNICATION
Gypsy-like retrotransposons are widespread in the plant
kingdom
Annu Suoniemi, Jaakko Tanskanen and
Alan H. Schulman*
Institute of Biotechnology, University of Helsinki,
Biocenter 1, PO Box 56, Viikinkaari 9, FIN-00014 Helsinki,
Finland
Summary
Retrotransposons propagate via an RNA intermediate
which is then reverse-transcribed and packaged into viruslike particles. They are either copia- or gypsy-like in coding
domain order and sequence similarity, the gypsy-like
elements sharing their organization with the retroviruses
but lacking retroviral envelope domains. Copia-like retrotransposons, or at least their reverse transcriptase
domains, appear broadly distributed in higher plants, but
gypsy-like elements have been reported only for scattered
species. The authors have exploited the difference in
domain order between these groups to amplify and clone
segments bridging the reverse transcriptase – integrase
region of specifically gypsy-like retrotransposons. Species
representative of the diversity of higher plants yielded
products whose sequences establish that gypsy-like transposons are dispersed throughout the plant genomes. This
class of plant elements has been named romani retrotransposons. The presence of both types ubiquitously in the
fungi, plants and animals support their existence as ancient
distinct lineages and subsequent, vertical radiation.
gypsy-like or copia-like based both on the order of their
protein-coding domains found between the long-terminal
repeats (LTRs) and on their sequence similarities (Xiong
and Eickbush, 1990). A major difference between the copialike and gypsy-like retrotransposons is the placement of
their in domain with respect to the rt domain, with the
gypsy-like elements and retroviruses both arranged LTR–
gag–proteinase–rt–in–LTR, and the copia-like elements
organized LTR–gag–proteinase–in–rt–LTR (gag encoding
the structural protein for the capsid).
Although copia-like retrotransposons, or at least their
reverse transcriptase domains, appear broadly distributed
in higher plants (Flavell et al., 1992; Voytas et al., 1992),
gypsy-like elements have only been reported for scattered
species (Springer and Britten, 1993). Retroviruses, or infectious gypsy-like retrotransposons, have never been
reported for a plant host. In view of this, we set out
to systematically establish the distribution of gypsy-like
retrotransposons in the plants. The difference in domain
order between the two classes of retrotransposons was
exploited in the design of PCR primers to specifically
amplify segments from gypsy-like elements spanning the
rt–in region.
We examined 24 species, including representatives of
the gymnosperms and all classes of angiosperms, and
detected PCR products of appropriate size from all but one.
Our data indicate that these elements are both widespread
and conserved in sequence. We have therefore named the
plant gypsy-like elements romani elements to reflect their
existence as a distinct class of plant retrotransposons.
Introduction
The retrotransposons are genomic elements which, like
the retroviruses, propagate intracellularly through transcription and translation, followed by packaging of the
transcript into particles (Adams et al., 1987). The RNA is
then reverse-transcribed (Boeke and Corces, 1989; Goff,
1990) by the encoded reverse-transcriptase-RNase H (RT)
into the integrative DNA form which is inserted back into
the genome by integrase (IN). Retrotransposons have been
characterized and named according to the Drosophila type
elements (Emori et al., 1985; Marlor et al., 1986), as either
Received 20 June 1997; revised 11 November 1997; accepted 24
November 1997.
*For correspondence (fax 1 358 9708 59570;
e-mail [email protected]).
© 1998 Blackwell Science Ltd
Results
Strategy for identifying gypsy-like elements
An alignment of the polyproteins or predicted translations
for retroviruses and gypsy-like retrotransposons was constructed over the diagnostic RT–IN region and examined
for conserved boxes suitably spaced for the construction
of PCR primers. An RT and IN box were identified (Figure 1)
and spaced ™ 1600 bp apart with respect to the del element
of Lilium (Joseph et al., 1990). The equivalent RT box from
plant copia-like elements (Figure 1) was fairly divergent
and the IN box unconserved with respect to the gypsy-like
elements and retroviruses. Primers were thus designed to
match residues from gypsy-like retrotransposons and were
made degenerate at areas of low conservation. The peptide
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700 Annu Suoniemi et al.
Figure 1. Scheme for PCR amplification of
gypsy-like retrotransposons.
(a) Map of the gypsy (Marlor et al., 1986)
retrotransposon (accession M12927) showing
long-terminal repeats (LTR), untranslated
leader (UTL), the two (gag, pol 1 env)
translated open-reading frames (Lozovskaya
et al., 1995; Pélisson et al., 1994), and the
regions for which PCR primers were
constructed.
(b) Alignment for retrotransposon and
retroviral polyproteins at the PCR primer sites.
Shown below (bold, upper case), are residues
for which the corresponding codons would
hybridize to the PCR primers were there no
mismatches, as well as those residues (bold,
lower case) the codons of which would be
recognized with a single base mismatch.
Arrows indicate PCR primer orientation.
sequence below the IN and RT boxes in Figure 1 shows the
residues corresponding to the sequences of the primers.
Widespread occurrence of romani (gypsy-like) elements
Plants were chosen for survey based on current molecular,
morphological, and anatomical phylogenies (Albert et al.,
1994; Donoghue, 1994; Doyle et al., 1994; Nixon et al., 1994;
Takhtajan, 1980), so that all classes of flowering plants, as
well as the gymnosperms, would be represented
(Figure 2a). Of the 24 species examined, 16 yielded PCR
fragments of approximately the expected 1.6 kb, with the
others giving slightly larger (™ 2.0 kb) or smaller (™ 1.3)
products (Figure 2). Only one species, Polygonum
capitalum, failed to yield a product in this size range, but
instead a fragment of ™ 800 bp. The yield of this shorter
product, detected in other species as well, depended on
PCR conditions. For Cycas circinalis and Anenome
numerosa, use of ‘touchdown’ PCR eliminated or shifted
amplification of the 800 bp band to the 1.6 kb product.
The 1.6 kb bands from nine species were isolated from
gels, cloned and sequenced, most only partially, from both
ends. The complete sequence of one of these, from barley
(Hordeum vulgare L. cv. Kymppi), was determined. The
sequences were deposited in the EMBL Nucleotide
Sequence Database as accessions AJ002611–AJ002628.
Analyzed elements were named romani elements to reflect
their gypsy-like organization (from the Gypsy people’s
name for themselves) and their existence as a distinct and
widespread class in the plants. We have assigned names
as ‘romani-xx#’, where xx is the pair of initials for the
genus and species and # is sequential numbering within
that species. One of 800 bp products, from Anemone nemorosa (romani-An1), was cloned and sequenced completely.
The sequence corresponded to a gypsy-like in without an
rt at the 59 end (Figure 3). We believe that these reaction
products result from priming by the rt primer at a secondary, internal site because these were either completely
replaced or reduced in prevalence in favor of the ™ 1.6 kb
bands when ‘touchdown’ PCR was used. Furthermore, the
rt primer is predicted to anneal at 15/18 nucleotides at its
39 end at the point of truncation, based on the translational
consensus for that region. Finally, it would seem unlikely
that a class of replicationally incompetent (lacking rnase h
and most of rt) elements should be maintained in at least
one-quarter of the species examined, despite these species’
evolutionary divergence, with apparently the same
deletion.
Conservation of IN and RT functional motifs in the
romani elements
An alignment of the predicted primary structures for the
sequenced romani regions (Figure 3) reveals blocks of
residues previously identified as highly conserved (Barber
et al., 1990; Khan et al., 1990; Kulkosky et al., 1992; Springer
and Britten, 1993; Xiong and Eickbush, 1990). The 59 PCR
primer bears the invariant DD of the RT active site (Figure 3,
box a), and the region immediately beyond contains the
highly conserved block SKCEF (box b) which includes
the invariant Lys of RNA-dependent polymerases in the
‘scaffold’ segment (Barber et al., 1990). The RNase H region
was defined by a conserved TDAS motif (box c), identical
in most other gypsy-like elements (Springer and Britten,
1993), bearing a key active-site aspartate (Campbell and
Ray, 1993). In addition, a Glu and Asp residue essential for
RNase H catalysis, as well as an N-3-DXL motif (box d)
spatially close in crystallized RNase H to the catalytic acidic
residues (Campbell and Ray, 1993), are conserved and in
register in the romani alignment. The N-terminal DNAbinding domain of IN (Khan et al., 1990) is revealed in the
romani translations as a conserved H-6-H-29-C-2-C motif
(box e). The enzymatic core domain of IN may begin with
the highly conserved N-terminal GLLQPLPI motif (box f)
visible in the romani alignment; its N terminus is 17
residues upstream of the first catalytic Asp (Figure 3,
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 699–705
Plant gypsy-like retrotransposons 701
Figure 2. Gypsy-like elements in the plant kingdom.
(a) The plant species sampled are arranged in a phylogeny based on rbcL (Albert et al., 1994; Rice, 1995), rDNA (Doyle et al., 1994), and morphological
characters (Nixon et al., 1994) with taxonomy according to Takhtajan (Takhtajan, 1980). Branch lengths are not scaled to phylogenetic distance or divergence
time. Lengths of the fragments amplified by PCR are displayed to the right, with those cloned and sequenced underlined and with minor products in
parentheses. Names for the sequenced elements appear at the far right.
(b) Gel electrophoresis of PCR products (1/10 reaction) for some of the species. Lane labels correspond to species listed in 2 A, with M denoting length markers.
first marked D in hatched bar) whereas the active and
crystallized (Dyda et al., 1994) HIV-1 core domain begins
14 residues upstream of the Asp. The aligned romani
translations contains a D-60-D-35-E geometry (D, D, E in
hatched bar); the D,D-35-E motif is completely conserved
in retroviral and retrotransposon integrases and is essential
for enzymatic activity (Baker and Luo, 1994; van Gent et al.,
1993; Kulkosky et al., 1992).
divergent sequences. The non-plant retrotransposons, with
the exception of Maggy, do branch at the deepest nodes,
and romani-Av1 and -Cc1, respectively, of Abies and Cycas,
are on deep nodes not well resolved by bootstrapping.
The romani elements from the Gramineae generally cluster.
Nevertheless, the elements from the dicotyledonous plants
neither cluster nor share a branching level within the
dendrogram.
Phylogenetic analysis of romani sequences
Discussion
A neighbor-joining phylogenetic estimate based on this
alignment (Figure 4) is only somewhat congruent with the
host organisms’ evolutionary relationships (Figure 2a).
Phylogenetic estimates, based on retroelements, over such
evolutionary distances are fraught with difficulties:
sampling errors where species contain large families of
divergent elements; parologous comparisons stemming
from the emergence of distinct lineages before speciation
events; limitations in the modeling algorithms for very
The gypsy- and copia-like retrotransposons differ in the
domain order of their encoded polyproteins, the gypsylike elements resembling the retroviruses. While members
of one class might conceivably be derived from the other
by sequence rearrangements, sequence comparisons and
phylogenetic analyses support these groups as independent evolutionary lines (Doolittle et al., 1989; Xiong and
Eickbush, 1990). Either or both classes of retroelements
may have entered into the genome of a given evolutionary
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 699–705
702 Annu Suoniemi et al.
Figure 3. Alignment of predicted translations for romani and gypsy-like retrotransposons.
Residues were shaded by GeneDoc© 2.1 (http://www.cris.com/~ketchup/genedoc.shtml) as identical (black shading), chemically similar (grey shading), or
dissimilar, with thresholds, respectively, of 100%, 70% and 40% (counting gaps), applying the amino acid groups FYW, ILVM, RKH, DE, GA, TS, and NQ. The
romani-Hv1 and -An1 sequence was determined completely; the others were sequenced from the ends only, with dotted regions (....) indicating the missing
sequence. Dashes (– -) indicate gaps added to optimize the alignment. Arrows below the termini of the alignment indicate the position of the PCR primers
and primer-derived sequence. Bold letters above the alignment and boxed regions bearing italicized labels indicate key residues conserved in all related
enzymes (see text). The hatched bar denotes the core domain of integrase. Sequences: Gypsy, POL protein of Drosophila melanogaster gypsy, P10401;
Ty3–2, TyB protein of Saccharomyces cerevisiae Ty3–2, S53577; Del, translation of Lilium henryi del1–46, X13886; Maggy, conceptual translation of
retrotransposon from Magnaporthe grisea, the rice blast fungus, L35053; R-At1 and others, romani elements named as in Figure 2, accessions AJ002611–
AJ002628.
line of plants either by horizontal transfer or by vertical
transmission from the ancestral plant group. Recent and
rare horizontal transfer events would restrict the occurrence
of a given retrotransposon type to narrow phylogenetic
groups within the plants. In contrast, the earlier the presence of a retrotransposon type in plant genome evolution,
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 699–705
Plant gypsy-like retrotransposons 703
Figure 4. Neighbor-joining phylogenetic model inferred from the romani–
gypsy-like protein alignment.
Numbers at nodes are bootstrap values for 100 replicates. Labels for
elements are as in Figure 3.
the more widely the expected distribution of that type
would be today. Hence, the distribution pattern of copiaand gypsy-like elements is of interest.
Copia-like retrotransposons, or at least their rt components, have been shown to be ubiquitous in the plant
kingdom (Flavell et al., 1992; Voytas et al., 1992). Nevertheless, the occurrence of gypsy-like retrotransposons has not
previously been systematically examined. A gypsy-like
family, magellan, is widespread in the Zea genus and in
one species of the related Tripsacum genus, but is absent
from all other Tripsacum species (Purugganan and Wessler,
1994). Besides these, only del (Joseph et al., 1990) and
another maize element, Reina (U69258) have been reported
within the Liliopsida (monocots). In the Magnoliopsida
(dicots), the Tna1 element of Nicotiana elata was identified
as gypsy-like on the basis of similarity to the putative in
domain of del (Royo et al., 1996) and gypsy-like rt fragments were detected in the mitochondrial genome of
Arabidopsis thaliana (Knoop et al., 1996). A pine rt segment,
IFG7, clusters with gypsy-like elements (Springer and
Britten, 1993). In sum, only a few gypsy-like sequences or
elements have been identified in the plants.
The conservation of sequence and domain order that
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 699–705
these elements share, however, has enabled us to establish
a method robust enough to isolate diagnostic components,
the rt–in region of specifically gypsy-like retrotransposons,
from across the plant kingdom. Virtually all species
examined contained bands of the size expected for gypsylike retrotransposons. Arabidopsis thaliana, with one of
the smallest genome sizes known for a plant and a very
limited retrotransposon complement – 0.1% of the genome
(Konieczny et al., 1991) compared with at least 7% for
barley (Suoniemi et al., 1996a) – contains both gypsylike and copia-like retroelements. In recognition of their
widespread occurrence and sequence similarity, the gypsylike elements of the plants were named romani. The predicted translations of the rt–in region of the cycad and
gymnosperm romani elements show close resemblance to
the angiosperm romani clones.
The widespread presence of both gypsy-like and copialike retrotransposons in every class of angiosperms, in the
gymnosperms, and in ferns supports the early presence
and subsequent, vertical passage of both groups during
the radiation of the plant kingdom. Furthermore, for the
romani sequences determined, the predicted translations
contain conserved residues established to be critical for
enzymatic activity of both IN and RT. The clones produced
were from genomic DNA rather than cDNA, which would
have selected only transcriptionally active romani elements. The genomic romani elements therefore likely
represent, or were recently derived from, active retrotransposons. Hence, gypsy-like retrotransposons have
been, and likely remain, actively propagated genomic components throughout the plant kingdom. Since both types
of retrotransposons are found as active components of the
fungi and animal genomes, the gypsy and copia lineages
appear to have diverged, remained active, and been passed
vertically since early in eukaryotic evolution.
Experimental procedures
Plant material and DNA preparation
Leaf samples were obtained from specimens in the Helsinki
University Botanical Garden or from chamber-grown plants. The
DNA samples were isolated as described previously (Dellaporta
et al., 1983; Rogers and Bendich, 1985); Pinus taeda DNA was
a kind gift from Claire Kinlaw (USDA Forest Service, Albany,
CA, USA).
PCR amplification and cloning
The PCR primers (IUPAC ambiguity codes) were: forward (rt) 59
(CTGGTTCGGCCCA)GTITAWYKTIGAYGAYRTIYTIRT 39, degeneracy of 256; reverse (in) 59 (CTCGCTCGCCCA)ICKYTCISWYTGICCRTCISTYTGIGG 39, degeneracy of 512. Sequences in
parentheses are extensions for cloning in the PCR-Direct vector
(Clontech). For PCR reactions, 300 ng-1 µg denatured template
DNA was added to 0.1 mM each dNTP, 25 pmol each primer, Taq
704 Annu Suoniemi et al.
buffer (Promega), and H2O in a 100 µl reaction. The mix was
overlaid with paraffin oil and brought to 95°C 3 min before adding
5 U Taq DNA polymerase (Promega). The reaction cycles consisted
of: 6 3 (94°C 30 sec, 42°C 30 sec, ➚ @ 1°C (3 sec)–1, 72°C 3 min);
31 3 (94°C 30 sec, 56°C 30 sec, ➚ @ 1°C (3 sec)–1, 72°C 3 min);
72°C 10 min; 4°C. For some species (Cycas circinalis, Anenome
numerosa), bands of anticipated (™ 1.6 kb) size were obtained
only using ‘touchdown’ PCR: 6 3 (94°C 30 sec, 47°C 30 sec, ➚ @
1°C (3 sec)–1, 72°C 3 min), with the annealing temperature reduced
1°C per cycle, followed by 31 cycles as above. PCR products
were purified from gels (Qiaex, Qiagen) for cloning. Sequencing
reactions on plasmid minipreps were carried out with Sequenase
v2.0 (Amersham) and analyzed under standard conditions either
manually or with an automated system (ALF, Pharmacia).
Alignments and phylogenetic estimates
Alignments of retrotransposon sequences in the database for the
purpose of primer design were made using PileUp of Wisconsin
Package Version 8.1-UNIX (http://www.gcg.com/) (Anonymous,
1994). Nucleotide sequences from the amplified and cloned gypsylike elements were translated into all six reading frames, and the
translations then aligned using Clustalw vers. 1.6 (Thompson
et al., 1994). Alignments were manually corrected. Distances
between sequences were calculated with Protdist of Phylip 3.5c
of Felsenstein (http://utmmg.med.uth.tmc.edu/mmg/genetics/
phylip_info/main.html) using the PAM matrix of Dayhoff. Trees
were produced by the neighbor-joining method (Saitou and Nei,
1987) implemented in Phylip 3.5c; 100 bootstrap replicates were
analyzed. The method is based on all pairwise comparisons in
which positions for which there was no sequence data – the
central regions for many of the sequences – were treated as
missing data (coded as ‘X’) rather than as gaps.
Acknowledgements
We thank AnneMari Narvanto for excellent technical assistance,
Victor Albert (New York Botanical Garden) for helpful discussions
on plant phylogeny and systematics, and Aune Koponen (Botanical
Garden, University of Helsinki) for providing plant material.
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