# Coe1 in Beta vulgaris L. Has a Tnp2-Domain DNA  Transposase Gene within Putative LTRs and Other  Retroelement-Like Features

Authors: David Kuykendall; Jonathan Shao; Kenneth Trimmer
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

We describe discovery inBeta vulgaris L. of Coe1, a DNA transposase gene within putative long terminal repeats (LTRs), and other retrotransposon-like features including both a retroviral-like hypothetical gene and an Rvt2-domain reverse transcriptase pseudogene. The central DNA transposase gene encodes, in eight exons, a predicted 160-KDa protein producing BLAST alignments with En/Spm-type transposons. Except for a stop signal, another ORF encodes a Ty1-copia-like reverse transcriptase with amino acid sequence domain YVDDIIL. Outside apparent LTRs, an 8-mer nucleotide sequence motif CACTATAA, near or within inverted repeat sequences, is hypothetical extreme termini. A genome scan of Arabidopsis thaliana found another example of a Tnp2-domain transposase gene within an apparent LTR-retrotransposon on chromosome 4.


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## Body

## 1. Introduction

Since the discovery of transposable elements (TEs) in corn [1],
DNA sequencing has revealed that genomes of eukaryotic organisms are largely comprised
of evolutionarily significant TEs responsible for creation of considerable genetic
diversity [2]. The movement of transposable
elements is either autonomous or dependent on other elements.  Classified according to mode of transposition,
Class I TEs, or retrotransposons, are retroviral-type elements which may or may
not have long terminal (direct) repeats (LTRs) [3]. Movement of Class I elements necessarily involves
an RNA inter-mediate in what can appropriately be termed “replicative” transposition. Retrotransposons are transcribed into RNA,
and then the reverse transcriptase and integrase make and insert a DNA copy at a
secondary genomic location. Class II TEs
are often called “DNA transposons,” but it is important to note that Class I retrotransposons
are also comprised of DNA except during transposition. Transposases permit Class
II transposable elements to move by a “cut and paste” process, first excising from
one site and then reintegrating at another. DNA replication is not required. “Footprints,”
telltale evidence for a previous DNA transposon insertion, result from imprecise
excision. Class II transposons characteristically
have relatively short inverted repeat sequences near their termini and an excision
site at each end recognized by the transposase.One of the first plant transposons
McClintock described [1], En1, or the
maize suppressor-mutator (Spm), is
the original example of a “CACTA” class, or superfamily, of transposons. CACTA transposons were thought until recently
to be found only in plants, but a similar element was discovered [4] in the
genome of Schistosoma mansoni, the causative agent of schistosomiasis.Evidence that retrotransposons account for
much of the sugar beet (Beta vulgaris L.) genome was first obtained by Schmidt et al. [5],
who described (1) repetitive DNA sequences in Beta vulgaris similar to long interspersed nuclear elements
(LINEs), a type of retrotransposon without LTRs, and (2) other repetitive DNA
sequences that resembled LTR retrotransposons of the Ty1-copia class. Vulmar1,
a mariner-class transposon in Beta
vulgaris, [6], 3 909 bp in length, has 32 bp terminal inverted repeats and
carries a single ORF that encodes a transposase with a characteristic DDE signature
motif in a single exon.Our interest in repetitive DNA developed
from our recent discovery of a number of LTRs and retrotransposon genes as well
as a transposase gene in the region between two clusters of core plant genes on
a 130 Kb sugar beet BAC [7, 8]. One gene
cluster has an NPR1-class disease
resistance-potentiating gene adjacent to another core plant gene whose
predicted product has high similarity to a heat shock factor protein. The other cluster consists of a signal
peptide calmodulin-binding protein kinase gene located near a CK1-class protein
kinase gene. In this communication, we report the discovery
in Beta vulgaris of Coe1, a Class II DNA transposase gene within
putative LTRs and other features that are characteristic of Class I LTR-retrotransposons.
Also, a genome scan of Arabidopsis
thaliana found a similar arrangement of transposon and retro-element genes
on chromosome 4.


## 2. Materials and Methods

The identification of a sugar
beet genome-derived bacterial artificial chromosome (BAC) carrying theNPR1 disease resistance control gene has
been previously described [7] as well as the basic methods used for DNA sequence
analysis. In this study, analysis of the NPR1 BAC was performed using LTR_STRUC (http://www.genetics.uga.edu/retrolab/data/LTR_STRUC.html),
RepFind (http://zlab.bu.edu/repfind/form.html) analysis identified
identical direct repeats. Etandem (http://bioweb.pasteur.fr/seqanal/interfaces/etandem.html)
and Einverted (http://edukon.biologie.uni-konstanz.de/cgi-bin/emboss/einverted)
were used to identify tandem and inverted repeats. EMBOSS [9] (http://emboss.sourceforge.net/)
was also utilized to identify tandem repeats and inverted repeats.  Repeats were also found using NCBI’s BLASTprogram (http://www.ncbi.nlm.nih.gov/BLAST) by
BLAST of a contig against itself using BLASTn.
A sugar beet expressed sequence tag (EST) database (http://genomics.msu.edu/sugarbeet/blast.html)
was employed for nucleotide and protein BLASTs in order to identify possible functional
gene expression. Subsequent
analyses of DNA sequence data were performed using Lasergene ver. 6 (DNASTAR, Madison, Wis). Multiple alignments were performed using MegAlign
from the DNASTAR suite. Phylogenetic tree
analysis was performed using Mega 4 software (http://www.megasoftware.net/).A new multicopy direct tandem repeat (MDTR) within an intronic region within theCoe1 transposase gene was identified by BLAST of the intron against the entire 130 kb
BAC and plotting a diagram of repeat versus DNA base positions with respect to the
BAC. In order to find the starting points of the repeats, a window which was of a constant length less than one
repeat was used in a sliding window technique. The first base used in this
window was the putative starting point of the first repeat in the MDTR, as
determined by the BLAST output. This window was BLASTed against the whole
intron. If BLAST found any repeated DNA using this template, the window was actually still within the repeated segment;
therefore, the starting base of the first repeat was deduced to be further
towards the 5th end. The window was then
moved a few bases in the 5th direction and the amended sequence was subjected
to another BLAST. This process continued
until BLAST no longer identified repeats.GenBank accession EF101866 provides annotation
of the 130 KbNPR1-carrying BAC
derived from thesugar beet genome. Conserved microsynteny of four core plant
genes was observed with other eudicots (Kuykendall et al., submitted).To scan theArabidopsis thaliana genome for a Coe1-like element(s), each chromosome was individually subjected to
LTR_STRUC analysis, and then each putative LTR-retrotransposon element was examined
for both a DNA transposase gene and a retrotransposon-like integrase or reverse
transcriptase gene within its LTRs.


## 3. Results and Discussion

BLAST and LTR_STRUC analyses performed on
an annotated 130 KbNPR1 gene-carrying sugar beet BAC (GenBank accession EF 101866) revealed the
presence of Coe1, which appears to be
a new and unique composite of Class I and Class II transposable elements. Coe1 was chosen as its name to honor Dr. Gerald Coe who originally bred and
developed a new U.S. hybrid sugar beet genotype, US H20 [10]. US H20 was the source of genomic DNA for a sugar beet Bacterial Artificial Chromosome
(BAC) library 8 from which a BAC clone carrying the NPR1 disease resistance control gene was
recently identified [7]. Initially detected by LTR_STRUC analysis as a LTR-retrotransposon, Coe1, defined as 14.5 Kb by two
putative 169 bp LTRs, has both an Rvt2-domain reverse transcriptase pseudogene and another
retroviral-like hypothetical gene. However, a DNA transposase gene was found
within its central region (Figure 1). In
addition to Coe1, LTR_STRUC analysis performed on the 130-Kb NPR1 BAC
identified at least two other LTR-retrotransposons, briefly: (1) a copia or Ty1-like retroelement, BvRTR1, which has a reverse transcriptase with
active site YVDDIIF; and (2) BvRTR2, a gypsy or Ty3-like retroelement
with active site FIDDILI in its conserved Rvt1 domain (unpublished). Precedence in the literature exists for similar yet
considerably smaller repetitive DNA sequences from sugar beet largely
uncharacterized except for genomic distribution.Figure 1
A schematic diagram ofCoe1, a DNA transposon
within an LTR retrotransposon. Inverted repeats are in checkerboard with arrows
indicating direction. Dark green lines depict the 8-mer DNA sequence motif CACTATAA, whereas lighter green lines show
the sequence motif CACTA. Heavily cross-hatched regions depict the putative LTRs. The lightly dotted blue and red
regions show the composite element. Darker shaded red or blue boxes are exons of retroelement ORFs or of a central DNA
transposase gene, respectively. A red-highlighted box with downward (backward)
slanting lines depicts an apparent polymerase binding site (PBS). Lighter red
or blue boxes, between exons, are introns. Repeating units of a light blue to dark blue gradient depicts repeating “MDTR”
units within an intron of the DNA transposase gene (blue). A red box with horizontal lines depicts the apparent
active site (save for a stop signal) of the predicted protein product encoded
by retroelement integrase/reverse transcriptase. A box with upward (forward)
slanting lines depicts a polypurine tract. Scale is in kilobases is shown with 2 Kb increments. Names of putative
genes are shown above the respective ORFs in Coe1.The question then arises of whether the
transposase gene ofCoe1 represents a
Class II transposon inserted into a Class I LTR-retrotransposon. This is probably
how Coe1 originated. In any case, Coe1 has salient features of a Class II
DNA transposon within a Class I retrotransposon (Figure 1) as described below.TheTnp2-domain
transposase gene central to Coe1 consists
of eight exons. The transposasegene of Coe1 has a predicted protein product that is evidently a CACTA
superfamily DNA transposase as deduced from the results of BLAST amino acid
sequence alignments of the predicted protein product with En/Spm-type DNA transposons (Figure 2).Amino acid residue
alignment of a conserved region of the predicted product ofBeta vulgaris Coe1’s DNA transposase
gene with predicted products of DNA transposase genes from various other
plants. Amino acids matching the
consensus sequence are shaded. Numbers indicate cumulative amino acid
positions. Antirrhinum magus Tam1 (X57297), Arabidopsis thaliana (BAB09502), Beta vulgaris (ABM55245), Brassica
rapa (BAA85462), Cleome spinosa (ABD96944), Oryza sativa indica (CAH66091), Oryza
sativa japonica (NP_001062816), Sorghum bicolor (AAM94290), Vitis vinifera (CAN82870), Zea mays En/Spm (AAA66266).
(a)(b)(c)(d)Evidence for  probable expression ofCoe1’s transposase
gene, or at least a similar gene, were
two sugar beet (expressed sequence tags)(ESTs) [11] whose nucleotide sequence aligned
by BLAST with Coe1’s DNA transposase
gene: CF542726 (e=0.0) and BQ595658 (2e−96).TheCoe1 DNA transposase gene is flanked by inverted repeats and a CACTA
sequence motif (Figure 1). In addition to Coe1, the prototypical CACTA superfamily transposon En/Spm of corn, Tam1 of snapdragon [12] and seven other Tnp2-domain transposons, from various plant species, were compared
using MegAlign.  Figure 2 shows the amino
acid sequence alignments obtained with one of two conserved domains.  Cluster
analysis of these data (Figure 3), a neighbor joining analysis tree, indicates
that the Coe1 transposase falls into a
group we designate as I subgroup A, with other plant Tnp2-domain transposases
from Arabidopsis thaliana (BAB09502), Cleome spinosa (ABD969441), and Brassica rapa (BAA85462.1). Another subgroup
of group I, B has En/Spm of Zea mays (AAA66266) and Oryza sativa japonica NP_001062816. In
the amino acid sequence alignments of this particular conserved region (Figure 2),
two other dissimilar groups (II and III) had the remaining four DNA transposases
(Figure 3). The En/Spm-like superfamily
of plant transposons, exemplified by Barbara McClintock’s suppressor/mutator
transposons of corn, has been named CACTA for the sequence motif recognized for
excision. Conservation is well established over the taxonomic divide between
eudicots, and monocots.Figure 3
Phylogenetic tree, constructed by neighbor joining
analysis, of the amino acid alignments shown in Figure2. Genbank accession
numbers of amino acid sequences follow the plant species name.Coe1 has a centrally located transposase gene, flanked downstream by a pair of imperfect 51 bp I14 inverted repeat sequences (94% match) separated by only 10 bp, and
upstream by another repeated sequence, I24,
that  aligns with I14 with about 75% identity over 41 bp.  These
distal I24/I14 inverted repeats are each
flanked by a CACTA sequence motif.Coe1 has
a total of three ORFs: a retroviral-like hypothetical gene ORF1, the Tnp2-domain transposase gene and an
apparent Rvt2-domain reverse
transcriptase pseudogene, ORF2. Coe1 has putative long terminal repeats
characteristic of LTR retrotransposons (Figure 1). These relatively short
169-bp direct repeats share only
96.4% identity. The 3.6% sequence divergence
in the LTRs is consistent with possibility that the retroelement-like features
of Coe1 are no longer active. The 5th end of the Coe1 positive strand has a pair of I13 inverted repeats 173 bp apart with 76% match over 190 bp and an
internal CACTATAA sequence motif. I15 inverted repeats are found downstream
of Coe1 and these inverted repeats, near
another CACTATAA sequence motif, are 52 bp apart with about 74% match over 51 bp.ORF1, the first retroelement-like gene ofCoe1, encodes, in a single exon, a
hypothetical protein for which no significant BLAST alignment is currently found.
The predicted protein product of ORF1 had initially produced a significant
BLAST with a “polynucleotidyl transferase” but that accession has been
withdrawn.ORF2 produced only a relatively weak
nucleotide BLAST alignment (2e−25) to EST BI643401. ORF2 is apparently a
pseudogene since a stop signal occurs in the sequence prior to that part of the
sequence that would otherwise encode the active site of an Rvt2 domain reverse transcriptase.  Although a sequencing error is possible, it is
unlikely; therefore, it is reasonable to deduce from the sequence data that ORF2
of Coe1 is a reverse transcriptase
pseudogene. Disregarding the stop signal, the predicted protein product of the Rvt2-like gene of Coe1 aligned well with other Rvt2-domain
gene products (Figure 4). The Coe1Rvt2 domain reverse transcriptase has a Ty1-copia-like YVDDIIL active site which
is most highly conserved in comparison with that of the Medicago truncatula accession (Figure 4). Among the protein alignments
performed, the hypothetical sugar beet Rvt2-domain
containing gene product also showed higher similarity with retrotransposon-type
reverse transcriptase proteins encoded by genes in two subspecies (indica and japonica) of Oryza sativa than with most others from
a wide taxonomic range (Figure 4).Figure 4
Amino acid residue alignment of a conserved region of the
predicted product ofBeta vulgaris Coe1 ORF2, albeit interrupted with a stop codon, which otherwise would have contained an Rvt2 domain with those of other retroelement
reverse transcriptase genes from different plants. A conserved YVDDIIL active site is highlighted
in green. Beta vulgaris (ABM55246), Citrus
sinensis (CAJ09951), Glycine max (AAO73527),
Ipomoea batatas (AAV88069), Medicago truncatula (ABE85780), Oryza longistaminata (AAB82754), Oryza sativa indica (CAH67061),
Oryza sativa japonica (NP_001067469), Solanum demissum (AAT38758).We recently performed a genome-wide scan or
survey of theArabidopsis thaliana genome looking specifically for
a composite DNA transposon within LTR retrotransposon features similar to Coe1, and a similar single Tnp2-domain transposase gene flanked by putative
LTRs and other retrotransposon-like features was identified, as described below.AnArabidopsis thaliana DNA transposase gene within putative LTRs and between LTR-retrotransposon genes is
depicted in Figure 5. The apparent LTR-retrotransposon is 9 078 bp and has 506 bp
and 471 bp LTR regions with about 90% identity. This element was found on BAC T26N6 from chromosome IV at 19.3 cM
(accession AF07243). The first ORF (At4g04426) appears to be a highly degraded pseudogene of a reverse transcriptase, the central
ORF (At4g04430) is a CACTA-class transposase gene, and the third ORF (At4g04440) has a predicted protein product with an Rvt2 reverse
transcriptase domain similar to Ty1-copia-like retrotransposons. Alignment of a conserved region of the predicted
product of the reverse transcriptase and a neighbor joining tree from cluster analysis (not shown) revealed a number of similar
BLAST hits but none of these was very nearly identical to the predicted product of the Arabidopsis element. The
finding of a Coe1-like element in Arabidopsis supports the concept that Coe1 in
Beta vulgaris is not a unique instance of a Class II transposon within a Class I retrotransposon.Figure 5
A schematic diagram of anArabidopsis Coe1-like composite
Class II transposon within a Class I LTR-retrotransposon. A central gene colored
blue encodes a CACTA-superfamily transposase between retrotransposon genes
colored red, all within retrotransposon
LTRs. Light green lines depict the DNA sequence motif CACTA. Heavily dotted regions show LTRs. The lightly and darkly red and blue dotted box
regions show the composite. A red box
with downward (backward) slanting lines shows a RNA polymerase binding site
(PBS). Darker red or blue boxes show exons.
Dark red retrotransposon ORFs (dark) are interrupted by lighter red
introns. A central DNA transposase gene is dark blue with light blue introns. A
red box with upward (forward) slanting lines is a polypurine tract. Scale is in
kilobases located below has 2 Kb increments.
Names assigned putative genes are above their representations.Regarding the subject ofCoe1 in Beta vulgaris, it is noteworthy that within the transposase gene most
of the DNA sequence of an intron, when BLAST against itself yielded multipl,
relatively small regions of imperfect DNA sequence identity. These small regions
aligned, albeit imprecisely, with each other. These results, when graphed, showed
a distinct pattern (Figure 6) that suggested a whole series of multicopy direct
tandem repeats (MDTRs).Figure 6
Diagram showing repeats within an intron of theCoe1 transposase gene
as initially identified by BLAST. Sets of multicolored lines
represent lengths and position of a direct repeats. Green boxes depict the exons
of the transposase gene. Scale is in base pairs in increments of 1000 base pairs.Repeats of the apparent MDTR found by BLAST
were of different lengths, but the first repeat in each set began at the same
position, and the second repeat in each set ended at the same position. Each
successive set of repeats was shorter by about the same number of bases. The
intron was thus divisible into visually evident repeats (Figure6).Individual nonidentical MDTR repeats are 173 bp. Each repeated DNA sequence, aligned
in MegAlign, gave the following consensus sequence:
aggaacatgaaacccaaaaaagggct cgaaatggcttagtttctatcattttcattggctaagtgtattaaacttgcttagaatcata caaccatgtagtagaaagtttaaatgagtattttaggacttgcatgagccattagaactt gaaataggcatagaggtaggatta.An intron withinCoe1’s transposase gene was thus determined to have 15 complete 173
bp MDTR and a partial repeat at the 3′ end. The sequence of this MDTR-carrying intron
did not yield any MegaBLAST hits against the NCBI database. One or more individual
repeat(s) could have transposed into the intron followed by duplication(s), or possibly
the entire series of multicopy tandem direct repeats transposed into the intron
altogether. The latter possibility may
be supported by the observation that the last repeat is partial, presumably a
deletion.How didCoe1, or an arrangement of transposable genes that resembles Coe1, originate? A DNA transposon could have moved into the middle
of an LTR-retrotransposon. In other words, Coe1’s
central transposase gene, flanked by inverted repeats and CACTA sequence
motifs, could have transposed into an LTR-retrotransposon.The CACTATAA sequence motif, located 16.3 Kb apart, outside the putative LTRs near large inverted repeats, could perhaps be
extreme boundaries ofCoe1 instead of
the two putative LTRs (Figure 1) separated by 14.5 Kb.It is possible that the
intact larger and more complex composite transposon could move
about using DNA transposase. The LTRs are flanked by pairs of inverted repeat sequences
that may be nonautonomous, miniature inverted [repeat] sequence transposable
elements (MITEs) (Figure1). MITEs, sometimes called “Class III transposons,” are
dependent on DNA transposase. The 8-mer sequence motif CACTATAA flanks the Coe1 LTRs near or within relatively
large inverted repeats that are perhaps MITEs.We hypothesize that originally a Class I
LTR-retrotransposon inserted between pairs of MITEs. Then, a Class II DNA transposon moved into the
middle of the LTR-retrotransposon, and voila, a composite of Class II and Class
I elements resemblingCoe1, at least conceptually.
To summarize, Coe1 has a Tnp2-domain transposase gene flanked by
putative LTRs and between two retrotransposon-like genes, all within CACTATAAs
near or within pairs of inverted repeats (Figure 1).One may ask, “what possible selective
advantage would a CACTA DNA transposon within an LTR retrotransposon have over
a simple Class I or Class II element alone?” A combination of Class I and Class
II features may offer little if any selective advantage, and thus such a composite might be unique.
The finding of a similar gene arrangement inArabidopsis provides a second example of a composite Class II
transposon within a Class I retrotransposon.A change in the expression of a gene when placed under the control of another can
confer a selective advantage on the host plant. Increased fitness might also be
characteristic of the host of a versatile element which can hypothetically  transpose in either of two known mechanisms.
Such versatility could facilitate more rapid genetic change due both to transposition
and to subsequent blockage of gene conversion.


## 4. Conclusion

In conclusion, based on results ofin silico analyses, Coe1,
found in the sugar beet genome, can be viewed as an incipient or emerging CACTA
super-family DNA transposon amalgamated within an LTR-retrotransposon. A similar arrangement of a central Tnp2-domain transposase gene within LTRs and between retrotransposon
genes was found in chromosome 4 of Arabidopsis
thaliana by a genome scan.More DNA sequencing of the sugar beet genome,
either of larger stretches or of the complete genome, is likely to be needed in
order to distinguish whetherCoe1 represents an evolutionarily significant gene arrangement or a mere coincidental
merging of transposable genes.In either case, as far we know, the two
examples shown here of a Class II DNA
transposon within a Class I retrotransposn are novel.



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*Source: 360874-2008-06-11.xml*