RESEARCH Open Access
Analysis o CACTA ansposases e eals in on loss
as majo ac o in luencing hei exon/in on
s uc u e in monoco yledonous and
eudico yledonous hos s
Jan P Buchmann
1,4*
, A i Löy ynoja
1
, Thomas Wicke
2
and Alan H Schulman
1,3
Abs ac
Backg ound: CACTA elemen s a e DNA ansposons and a e ound in nume ous o ganisms. Despi e hei low ac i i y,
se e al housand copies can be iden i ied in many genomes. CACTA elemen s anspose using a ‘cu -and-pas e’
mechanism, which is acili a ed by a DDE ansposase. DDE ansposases om CACTA elemen s con ain, despi e hei
conse ed unc ion, di e en exon numbe s among a ious CACTA amilies. While ea lie s udies analyzed he ances al
his o y o he DDE ansposases, no s udies ha e examined exon loss and gain wi h a iew o mechanisms ha could
d i e he changes.
Resul s: We analyzed 64 ansposases om di e en CACTA amilies among monoco yledonous and eudico yledonous
hos species. The anno a ion o he exon/in on bounda ies showed a ange om one o six exons. A obus mul iple
sequence alignmen o he 64 ansposases based on hei p o ein sequences was c ea ed and used o phylogene ic
analysis, which e ealed eigh di e en clades. We obse ed ha he exon numbe s in CACTA ansposases a e no
speci ic o a hos genome. We ound ha ancien CACTA lineages di e ged be o e he di e gence o monoco yledons
and eudico yledons. Mos exon/in on bounda ies we e ound in h ee dis inc egions among all he ansposases,
g ouping 63 conse ed in on/exon bounda ies.
Conclusions: We p opose a model o he ances al CACTA ansposase gene, which consis s o ou exons, ha
p eda es he di e gence o he monoco yledons and eudico yledons. Based on his model, we p opose pa hways o
in on loss o gain o explain he obse ed a ia ion in exon numbe s. While in on loss appea s o ha e p e ailed, a
pu a i e case o in on gain was ne e heless obse ed.
Keywo ds: T ansposases, In on loss, Molecula e olu ion, DNA ansposons, Plan s
Backg ound
CACTA elemen s a e DNA ansposons ound in genomes
ac oss he phylogene ic spec um, om algae [1] o ascu-
la plan s [2-6] o animals [7,8]. The i s CACTA elemen
desc ibed a he molecula le el was En-1 in Zea mays [2];
since hen, hey ha e been well documen ed in he g asses.
Al hough CACTA elemen s usually do no accoun o he
la gegenomesizes ounding asses,CACTA amilies
ne e heless can be highly abundan . In a ew cases,
howe e , including Tpo1 in Lolium pe enne ( yeg ass)
and Caspa in he T i iceae, CACTA elemen s a e
known o ha e con ibu ed conside ably o he expan-
sion o he genome size o hei hos [9-12]. Mo eo e ,
CACTAs can in luence he e olu ion o he hos gen-
ome in o he ways [12]. In Glycine max (soybean),
CACTA elemen s can a ec lowe colo and cap u e
hos genes [13-16]. CACTA elemen s a e some imes as-
socia ed wi h egula o y elemen s o genes, he e o e
possibly in luencing gene exp ession [10,17]. Despi e
hei p e alence and impac , e olu iona y s udies abou
* Co espondence: [email p o ec ed]
1
Ins i u e o Bio echnology, Viikki Biocen e , Uni e si y o Helsinki, PO Box 65,
FIN-00014 Helsinki, Finland
4
P esen add ess: Ma ie Bashi Ins i u e o In ec ious Diseases and
Biosecu i y, Cha les Pe kins Cen e , Uni e si y o Sydney, Sydney NSW 2006,
Aus alia
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2014 Buchmann e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly c edi ed. The C ea i e Commons Public
Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his
a icle, unless o he wise s a ed.
Buchmann e al. Mobile DNA 2014, 5:24
h p://www.mobilednajou nal.com/con en /5/1/24
CACTA elemen s, o DNA ansposons in gene al, a e
sca ce.
The CACTA supe amily belongs o he Class II o
ansposable elemen s, p oli e a ing by a ‘cu and pas e’
mechanism. In con as o Class I elemen s, which ans-
pose ia an RNA in e media e and he e o e copy he
o iginal elemen , CACTAs anspose he o iginal elem-
en i sel . CACTA elemen s cons i u e app oxima ely 2
o 5% o a g ass genome [16,18]. Howe e , only ew ac i e
CACTA elemen s ha e been iden i ied in plan s [2-6,19].
In addi ion, only se en pu a i e ansc ibed ansposases
ha e been iden i ied in he T i iceae [10].
A ull-leng h CACTA elemen consis s o wo e minal
in e ed epea s (TIRs) bo de ing wo open eading
ames(ORFs), one encoding a ansposase and he o he ,
called ORF2, a p o ein o unknown unc ion. The i s
and las 5 bp o he TIRs consis o he highly conse ed
CACTA and TAGTG mo i s, espec i ely, hence he
name o he elemen . The unc ion o he ORF2 p o ein
has been de e mined in speci ic CACTA amilies o sup-
po excision and ansposi ion [20]. Howe e , he ans-
posase is he key ansposi ion enzyme. I binds o he
TIR du ing excision, c ea ing a 3-bp a ge si e duplica-
ion (TSD) [21]. The ca aly ic cen e o he ansposase
is he acidic iad known as he ‘DDD/E’mo i , which is
highly conse ed [22].
The p esence o CACTA elemen s ac oss he phylo-
gene ic spec um and he highly conse ed ca aly ic co e
o hei ansposases indica e an ancien p esence. In e -
es ingly, he numbe o exons in ansposases among
CACTA ansposons di e s e en among he g asses.
T ansposases in ice we e ound ha ha e ou exons
[23], while s udies in maize epo ed up o ele en exons
o CACTA ansposases [2,24]. In he ecen ly se-
quenced g ass B achypodium dis achyon, he exon num-
be o ansposases among CACTA supe amilies anges
om one o h ee. The e o e, he analysis o he exon/
in on con igu a ion o CACTA ansposases o e s an
excellen oppo uni y o s udy he e olu iona y mecha-
nisms o in on gain and loss in DNA ansposons. In
addi ion, analyzing exon numbe a ia ions in such a
highly conse ed and ancien gene as he CACTA ans-
posase can o e a pe spec i e on he ‘in on-ea ly’and
‘in on-la e’models [25,26].
The goal o his s udy was o analyze he di e ences in
exon numbe s in CACTA ansposases in monoco yle-
donous and eudico yledonous plan s and o iden i y an
e olu iona y mechanism o explain hose di e ences.
This was accomplished using phylogene ic and compa a-
i e analyses, which equi ed a solid and obus mul iple
sequence alignmen (MSA). We cons uc ed such an
MSA based on p o ein consensus sequences o 64 ans-
posases om CACTA amilies anno a ed in en mono-
co yledonous and eudico yledonous species.
Ou phylogene ic analysis e ealed ha ancien CACTA
lineages di e ged be o e he di e gence o he monoco yle-
dons and eudico yledons, suppo ing an in on-ea ly model
o CACTA ansposases. The analysis o he MSA iden i-
ied conse ed exon/in on bounda ies and pu a i e in on
gain among he ansposases examined. Combining hese
analyses lead o a model o a pu a i e ancien CACTA
ansposase, in which in on loss was he main mechanism
shaping he exon/in on con igu a ions o cu en anspo-
sases ound in monoco yledonous and eudico yledonous
plan s.
Resul s
We analyzed 64 au onomous CACTA ansposases om
en di e en monoco yledonous and eudico yledonous
species. All analyzed ansposases a e de i ed om con-
sensus sequences om dis inc i e CACTA amilies. Be-
cause amilies o ansposable elemen s (TEs) di e om
each o he based on he 80-80-80 ule, hey we e consid-
e ed o hologous [27]. The e o e, he name o he amily,
o example, Cal in, will indica e he consensus se-
quence o he ansposase and no he consensus o he
whole elemen . We e e o he plan in which a CACTA
amily and i s ansposase we e anno a ed as i s hos .
Excep o ansposases iden i ied in B. dis achyon,we
sea ched he PTREP [28] and Repbase [29] da abases
o CACTA amilies wi h anno a ed ansposases (see
Ma e ials and Me hods). The selec ion was based on
wo c i e ia: i) he anno a ion had o clea ly s a e ‘ anspo-
sase’, ha is anno a ions wi hou ORFs desc ibed as ans-
posases we e omi ed because CACTA elemen s ha e wo
ORFs, he ansposase and ORF2; ii) he p esence o wo
ORFs was expec ed, he eby a oiding selec ion o anno a-
ions ha ing a p edic ed ansposase ha spans mos o a
consensus sequence, such as ATENSPM10 in Repbase,
whe e he consensus is 8,272 bp and he p edic ed
ansposase co e s posi ions 1,201 o 7,766. We se-
lec ed nine ansposases om So ghum bicolo ,eigh
ansposases om Z. mays, i e ansposases om
T i icum aes i um,13 omO yza sa i a,and11 om
B.dis achyon (Addi ional ile 1). This esul ed in a o al
o 46 ansposases om monoco yledonous hos s. Fo
he eudico yledonous da ase , we selec ed all anspo-
sases om eudico yledonous hos s in Repbase i ing
ou c i e ia, o aling in eigh een elemen s: se en ans-
posases om elemen s anno a ed in A abidopsis hali-
ana, i e omF aga ia esca, h ee omVi is ini e a,
and one each om Pe unia hyb ida, Malus domes ica,
and G. max (Addi ional ile 1).
Anno a ion o exon/in on bounda ies on CACTA
ansposases
Fo simplici y, he e m ‘bounda y’will indica e exon/
in on bounda ies in his s udy. Excep o ansposases
Buchmann e al. Mobile DNA 2014, 5:24 Page 2 o 15
h p://www.mobilednajou nal.com/con en /5/1/24
in B. dis achyon, bounda ies we e ex ac ed om he e-
spec i e PTREP and Repbase en ies (Table 1, Ma e ial
and Me hods). The ele en B achypodium dis achyon
ansposases we e de i ed om consensus sequences o
he au onomous amilies in his genome [18]. We manu-
ally anno a ed he ansposases and bounda ies by align-
ing o he mos simila BLASTX hi wi hin he PTREP
da abase. Addi ional alignmen s agains ansc ip ion da-
abases om ice and B. dis achyon did no inc ease he
quali y o he bounda y p edic ions, because ansc ip-
ome da a is sca ce o CACTA ansposases. De no o
gene p edic ion did no e u n signi ican esul s.
Ou inal da ase consis ed o 64 ansposases wi h
86 anno a ed bounda ies on he 40 ansposases ha
con ained mo e han one exon (Table 1). Ou o he 64
anno a ed ansposases, 24 con ained only one exon
and he e o e no bounda ies. On he emaining 40
ansposases, we anno a ed be ween wo and six exons
(Addi ional ile 1). The leng h o he ansposases
anged om 552 amino acids (amino acids; PSL, 1
exon) o 4,785 amino acids (EnSpm4_F es, 4 exons),
and a e aged 1,163 amino acids. The six ansposases
Isido , Ru us, Sand o, Radon, I an, and Isaac we e an-
no a ed on he 3’end o he co esponding CACTA
consensus sequence (Addi ional ile 1).
Gene a ion o a obus mul iple sequence alignmen
using con idence sco es
Ou phylogene ic and compa a i e analyses we e based on
an MSA de i ed om he selec ed 64 consensus anspo-
sase p o ein sequences. Due o he possibly ancien o igin
o ce ain CACTA ansposases and hei gene ally low ac-
i i y, we assumed ha some pa s o sequences migh be
mo e e olu iona ily di e ged han o he s. In addi ion, he
o ma ion o consensus sequences can in oduce weak e-
gions in o an MSA. A obus MSA is he e o e c ucial be-
cause e o s o unce ain ies can in luence he downs eam
analysis. In addi ion, iden i ying weakly aligned egions o
posi ionsinanMSAand hen emo ing hemmayim-
p o e downs eam phylogene ic analysis [30].
GUIDANCE is a me hod o in e un eliable egions in
an MSA and emo e he po en ially e oneous signal
om subsequen analyses ([31]; Ma e ials and Me hods).
The inal MSA was 2,516 esidues long and con ained
i e uns able egions placed be ween posi ions 120 o
186, 196 o 251, 381 o 416, 728 o 766, and in he 3’
end, s a ing om posi ion 1,665 (Addi ional ile 2).
GUIDANCE sco es ange om 0 (low con idence) o 1
(high con idence) and a e calcula ed o single esidues
as well as o whole columns. Because he e is no ec-
ommended con idence sco e o esidues and columns
in an MSA, a ade-o be ween sensi i i y and speci i-
ci y is equi ed. High sensi i i y (low cu o alue) e-
ains as many columns as possible while high speci ici y
(high cu o alue) keeps only columns o e y high
con idence.
The de aul GUIDANCE cu o o 0.93 emo ed 638
columns (app oxima ely 25%) om he alignmen , in-
cluding he badly aligned egions and 34 anno a ed
bounda ies. Howe e , GUIDANCE kep columns wi h
only one esidue, o example, mos o he badly aligned
3’end. To e ain as many bounda ies as possible o he
analysis we applied ou own imming: we emo ed col-
umns con aining only esidues wi h sco es below 0.804
(keeping bounda ies) and columns wi h only one esidue
(no compa able and/o bad aligned). This app oach e-
mo ed 1,398 columns (app oxima ely 44%): he badly
aligned egions bu only 13 anno a ed bounda ies. This
inal MSA was 1,118 esidues long and con ained 73 an-
no a ed bounda ies in 64 ansposases (Figu e 1). Be-
cause he i s bounda y is also he beginning o he i s
in on, in ons we e named in he 5’ o 3’di ec ion and
designa ed as subsc ip s o he name o he ansposase,
o example, he i s in on and bounda y o ansposase
Ba on is desc ibed as Ba on
1
. We mapped conse ed
DDE mo i s [22] on o he MSA, which we e all in posi-
ions wi h high con idence alues (Figu e 1). This MSA
was used o all u he analysis.
Exon numbe s in CACTA ansposases a e no speci ic o a
hos genome
RAxML [32] was used o calcula e he phylogene ic ee
(Figu e 2). A maximum likelihood (ML) ee was gene -
a ed based on 200 dis inc , andomized, maximum pa si-
mony ees and i s obus ness assessed by using 1,000
boo s ap eplica es and by es ing he in luence o se -
e al ou g oups (Addi ional ile 3, Ma e ial and Me hods).
The esul ing ee shows he ela ion be ween indi idual
ansposases bu no hei e olu ion o e ime; ha is
he b anch leng hs do no indica e he ime when ans-
posases di e ged om each o he bu how close hey a e
on he molecula le el (Figu e 2). We iden i ied eigh
clades, designa ed α o θ(Figu e 2). C ucially, he ans-
posases g ouped p ima ily by hei exon numbe s a he
han by hei hos s and he analysis o he clus e s ound
no hos -speci ic exon numbe s o CACTA ansposases
(Figu e 2).
Ancien CACTA lineages di e ged be o e he di e gence
o monoco yledons and eudico yledons
We iden i ied h ee clades in which monoco yledonous
and eudico yledonous ansposases clus e ed oge he .
EnSpm2_Gmax om soybean g ouped in Clade αwi h
ansposases om se e al monoco yledonous hos s, analo-
gous o EnSpm3_F es and EnSpm4_F es om s awbe y
in Clade ζ. Clade δg ouped ansposases om s awbe y,
apple, and se e al g asses. The o he clades con ained
only ansposases om ei he eudico yledonous o
Buchmann e al. Mobile DNA 2014, 5:24 Page 3 o 15
h p://www.mobilednajou nal.com/con en /5/1/24
Table 1 Exon/in on bounda ies o he 34 analyzed CACTA ansposases wi h mo e han one exon.
12 3 4 5
EnSpm12_F es 462 | 564
G
C 718 | 771
I
EnSpm10_F es 826 | 846
Joey 842 | 893
II
Janus 837 | 894
II
F 846 | 894
II
G 847 | 894
II
No man 879 | 921
III
En1 879 | 925
III
Al ed 885 | 925
III
H 838 | 972
EnSpm3_V in 821 | 894
II
856 | 925
III
EnSpm8_Sbic 754 | 783
I
976 | 0
S o m 827 | 782
I
951 | 0
She man 831 | 782
I
954 | 0
J 495 | 521 750 | 885
EnSpm2_Mdom 755 | 782
I
886 | 893
II
Baldu 731 | 782
I
837 | 895
II
I 834 | 782
I
954 | 910
Isido 857 | 894
II
892 | 920
III
Radon 841 | 894
II
877 | 921
III
Ru us 851 | 894
II
887 | 921
III
EnSpm13_V in 821 | 894
II
856 | 925
III
EnSpm5_V in 824 | 894
II
859 | 925
III
Isaac 861 | 894
II
900 | 925
III
Sand o 744 | 782
I
851 | 928
III
Balduin 850 | 895
II
890 | 930
III
DOPPIA 843 | 894
II
890 | 936
III
K 744 | 7,82I 850 | 936
III
Ho ace 712 | 711 981 | 1,054
EnSpm4_F es 812 | 0 992 | 0 1,244 | 0
EnSpm3_F es 681 | 0 770 | 781
I
919 | 0
Seamus 730 | 782
I
833 | 892
II
878 | 925
III
Da io 726 | 711 842 | 839 895 | 890
III
A on 851 | 833 899 | 879
II
1,013 | 1,060
Ko bin 510 | 567
G
718 | 782
I
814 | 894
II
853 | 0
Ches e 520 | 563
G
728 | 777
I
823 | 889
II
858 | 920
III
Ba on 522 | 568
G
730 | 781
I
825 | 893
II
861 | 925
III
EnSpm8_F es 158 | 163 830 | 893
II
975 | 0 1,219 | 0 1,500 | 0
ATENSPM6_A hal 802 | 809 918 | 922
III
978 | 981 1,011 | 1,012 1,141 | 0
The posi ions a e ela i e o he beginning o he ansc ip ion s a and gi en as ollows:
On he p o ein sequence | on he immed mul iple sequence alignmen (MSA). 0 and numbe s in i alic indica e bounda ies wi h GUIDANCE sco es below 0.804
and emo ed in he inal MSA. Supe sc ip s indica e Regions I o III and G clus e , espec i ely (Figu e 1).
Buchmann e al. Mobile DNA 2014, 5:24 Page 4 o 15
h p://www.mobilednajou nal.com/con en /5/1/24
monoco yledonous hos s (Figu e 2). Despi e he long e o-
lu iona y ime sepa a ing monoco yledonous and eudico-
yledonous hos s, he p esence o mixed clades and he
close ela ion o clades wi h only monoco yledonous o
eudico yledonous hos s sugges s ha he CACTA anspo-
sase phylogeny a he han he hos phylogeny is p ima y,
ha is ha he main ansposase b anches di e ged
al eady be o e he di e gence o monoco yledons and
eudico yledons. Indeed, a close look a he phylogene ic
ee e ealed ha ansposases wi hin clades end o ha e
hesamenumbe o exons(Figu e2).
The majo i y o CACTA ansposase bounda ies a e ound
in h ee egions on he MSA
To analyze he e olu ion o exon/in on a angemen s in
CACTA ansposases, we compa ed he bounda ies om
he 33 ansposases con aining 73 in ons ha we e no
emo ed in he imming p ocess (Table 1, Figu e 1).
We iden i ied 3 egions, labeled I o III, in he MSA,
which con ain 63 ou o he 73 bounda ies (Figu e 1).
Ou side hose egions, we iden i ied eigh bounda ies in-
side he DDE mo i , ou bounda ies be ween Regions I
and II, one bounda y be ween Regions II and III and i e
bounda ies downs eam o Region III. Mos bounda ies
a e close o each o he bu no in he same posi ion on
he alignmen . This can be due o small e o s in o-
duced by calcula ing he MSA o consensus sequences.
The e o e, we analyzed he dis ances be ween bounda -
ies o iden i y which we e sha ed among ansposases.
We analyzed he bounda ies by clus e ing hem based
on hei posi ions on he MSA. We se he maximal dis-
ance be ween bounda ies s ill conside ed o be in he
Figu e 1 Mul iple sequence alignmen based on p o ein sequences o he 64 analyzed CACTA ansposases. Colo ed boxes indica e
amino acids, g ay boxes indica e esidues wi h a GUIDANCE sco e below 0.804, and whi e boxes indica e gaps in he mul iple sequence alignmen
(MSA). The plo below he MSA shows GUIDANCE sco es o he co esponding posi ion in he MSA. Columns wi h a sco e below 0.804 a e indica ed
in ligh blue while columns wi h a sco e o 0.804 and abo e in da k blue. Posi ions ela i e o he MSA and co esponding GUIDANCE sco e a e shown
be ween he MSA and he plo . Highly conse ed DDE ansposase mo i s as desc ibed in [22] a e depic ed on op. In he phylogene ic ee, colo s
indica e he hos as shown in he legend. Majo clades a e depic ed α o θ. Exon/in on bounda ies a e depic ed as blue ci cles i hei GUIDANCE
sco e was abo e 0.804 and ed o he wise. The numbe in he bounda y indica es he bounda y numbe on he co esponding ansposase. Regions I
o III a e indica ed by dashed lines and co esponding oman capi als. Posi ions o pu a i e in on gain a e depic ed as desc ibed in he legend.
Buchmann e al. Mobile DNA 2014, 5:24 Page 5 o 15
h p://www.mobilednajou nal.com/con en /5/1/24

same egion o 16 esidues, which is hal he leng h o he
sho es in on anno a ed (33 amino acids in ATENSP-
M_A hal
3
). Bounda ies ha we e close han 16 esidues o
each o he we e g ouped oge he . No bounda ies wi hin a
egion we e u he han 16 esidues apa (Tables 2, 3,
Addi ional iles 4, 5, 6). The dis ances be ween he closes
bounda ies o Regions I and II is 98 esidues (Addi ional
ile 7), bu 30 esidues be ween Region II and III
(Addi ional ile 7). The closes bounda y ups eam o
Region I is 60 esidues away, whe eas he closes bounda y
downs eam o Region III is 36 esidues away. This
clus e ing con i med he p e iously iden i ied egions as
clea ly dis inc . The ou bounda ies EnSpm10_F es
1
,
Da io
2
,A on
1
, and ATENSPM6_A hal
1
be ween Region I
Figu e 2 Majo i y- ule based phylog am o he 64 analyzed CACTA ansposases. The phylogene ic ee is he same as in Figu e 1.
Boo s ap alues ep esen he pe cen age ou o 1,000 boo s ap eplica es. Only boo s aps below 100% a e indica ed. T ansposase hos s a e colo ed
as indica ed in he legend. Numbe s in pa en heses indica e he numbe o exons. Clades a e indica ed by dashed lines and labeled α o θ.
Table 2 Dis ances be ween exon/in on bounda ies wi hin Region I
Baldu
1
Ba on
2
1 Ba on
2
C
1
11 10 C
1
Ches e
2
5 4 6 Ches e
2
EnSpm2_Mdom
1
0 1 11 5 EnSpm2_Mdom
1
EnSpm3_F es
2
1 0 10 4 1 EnSpm3_F es
2
I
1
01115 0 1 I
1
K
1
0 1 11 5 0 1 0 K
1
Ko bin
2
0 1 11 5 0 1 0 0 Ko bin
2
Sand o
1
0 1 11 5 0 1 0 0 0 Sand o
1
Seamus
1
0 1 11 5 0 1 0 0 0 0 Seamus
1
She man
1
0 1 11 5 0 1 0 0 0 0 0 She man
1
S o m
1
0 1 11 5 0 1 0 0 0 0 0 0
Dis ances be ween exon/in on bounda ies in he MSA wi hin Region I (depic ed in Figu e 1). The dis ances a e gi en in esidues in he alignmen .
Buchmann e al. Mobile DNA 2014, 5:24 Page 6 o 15
h p://www.mobilednajou nal.com/con en /5/1/24
and II, as well as I
2
be ween Region II and III could no be
clus e ed in hose Regions. We iden i ied only one add-
i ional clus e con aining ou bounda ies ou side Regions
I o III. I g oups he i s in ons om all membe s o
Clade γand was he e o e named Region G.
Based on hese analyses o dis ances be ween all bounda -
ies, we es ablished ha Regions I o III and G in he MSA
we e clea ly sepa a ed om each o he as well as om all
o he bounda ies. Gi en he dis inc ness o he ou bound-
a y egions, we examined i he bounda ies hemsel es we e
conse ed among he analyzed ansposases.
Bounda ies in Regions I o III a e conse ed among mos
ansposases while Region G ep esen s pu a i e in on gain
Due o he p oximi y o bounda ies in Regions I o III and
hei clea sepa a ion om o he bounda ies, we es ab-
lished ha bounda ies wi hin a egion a e sha ed be ween
he di e en ansposases. The clus e ing o bounda ies
wi hin Regions I o III indica es ha he bounda ies a e
conse ed among he analyzed ansposases. This is sup-
po ed by he phylogene ic ee, in which pu ely mono-
co yledonous o eudico yledonous clades sha e bounda ies
(Figu e 1). Bounda ies in Region I a e on, o close o, he
posi ion o he conse ed E om he DDE mo i , suppo -
ing he claim ha Region I ep esen s conse ed bounda -
ies among he ansposases (Figu e 1). The e o e, we
conside ed he 63 bounda ies in Regions I o III as con-
se ed wi hin each egion. All ansposases in Clade γ
sha e hei i s in ons wi h a maximum dis ance o i e
esidues (Figu e 1, Table 4). This is a unique clus e in he
whole ee, indica ing in on gain since all membe s o
Clade γsha e his in on bu none o i s ances o nodes
and ansposases in o he clades.
Only wo bounda ies om a monoco yledonous hos a e
ound ou side Regions I o III
We iden i ied 17 bounda ies ou side Regions I o III
(Figu e 1). Only J
1
and H1 a e om a monoco yledonous
hos , whe eas he emaining 15 bounda ies we e anno-
a ed in ansposases om eudico yledonous hos s.
Bounda ies I
1
and ATENSPM6
1,2,3
canno be clus e ed
and he e o e we e no u he cha ac e ized. The ans-
posases Ho ace, Da io, and A on ha e h ee sepa a e
bounda ies which a e no a he apa han six esidues:
Ho ace
1
and Da io
1
, Da on
2
and A on
1
, Ho ace
2
and
A on
3
. While his appea s as ano he case o in on gain,
hei ela ion in he phylogene ic ee is no p ope ly e-
sol ed and does no suppo his in e p e a ion.
Ou analysis o he bounda ies iden i ied 63 conse ed
bounda ies and 4 cases o pu a i e in on gain in Region
G. Mos conse ed in ons we e iden i ied in anspo-
sases om monoco yledonous hos s. In con as , all
unique bounda ies excep wo we e iden i ied in eudico-
yledonous hos s. We decided o combine he esul s o
he phylogene ic and bounda y analyses o de elop a
model o unde s and how he obse ed exon/in on con-
igu a ion e ol ed.
De ining consensus exon numbe s o each phylogene ic
clade
A compa ison o he phylogene ic ee and he conse ed
bounda ies e ealed a high consis ency be ween clades
and bounda y posi ions. Based on he majo i y o exons
pe clade, we cons uc ed a loose consensus o ep esen
he exon numbe o ansposases in he co esponding
clade. Fo example, Clade ζg oups oge he se en ans-
posases o which ou , he majo i y, ha e wo exons.
The e o e, a ep esen a i e ansposase om Clade ζhas
wo exons and one consensus bounda y. We used his ap-
p oach o each clade (Figu e 3). Ou app oach esul ed in
ollowing exon numbe s o ep esen a i e ansposases:
one exon o Clade α;Cladesβ,δ,andθ h ee exons each;
Clade η ou exons; Clade γ i e exons. Designa ing con-
sensus exon numbe s o each clade simpli ied u he he
analysis o de elop a model o he loss and gain o
bounda ies in CACTA ansposases.
A model o loss and gain o exon/in on bounda ies in
CACTA ansposases
Because i had he la ges numbe o con i med exons, we
compa ed all consensus bounda ies o Clade γ(Figu e 3).
Clade αhas no anno a ed in ons. The second, hi d, and
ou h in on o Clade γcan be ound h oughou he phylo-
gene ic ee, whe eby he hi d in on o Clade γis he mos
conse ed, ollowed by i s ou h and second in on. The
ou h in on o Clade γis ound among Clades β,θ,ι,and
in Isaac. The hi d in on is missing in he Clades EnSpm8,
δ,andθ, bu o he wise is ound in all clades con aining in-
ons. The second in on o Clade γis p esen in Clades δ,
EnSpm8, and η. This compa ison indica es ha CACTA
ansposases we e as a whole losing a he han gaining in-
ons. Howe e , Clades γand ζha e in ons ha a e no
ound in o he clades (Figu e 3), he i s in on in Clade γ
ep esen ing an in on gain. The unique in ons in Clade ζ
canno be classi ied as losses o gains because he phylogen-
e ic ee does no allow a de ini i e classi ica ion.
We p opose ha he consensus ansposase in Clade γ
ep esen s he mos likely exon/in on con igu a ion o
an ancien ansposase, con aining a leas ou exons
and h ee in ons (Figu e 3). The h ee bounda ies co -
espond o hose iden i ied in Regions I o III in he
MSA (Figu es 1, 3). Using he pu a i e ances o model
ansposase, we can in e he eme gence o he known
ansposases h ough in on loss and gain (Figu e 3).
Discussion
In sum, we analyzed 64 CACTA ansposases om 11
monoco yledonous and eudico yledonous hos s. Ou
Buchmann e al. Mobile DNA 2014, 5:24 Page 7 o 15
h p://www.mobilednajou nal.com/con en /5/1/24
Table 3 Dis ances be ween exon/in on bounda ies wi hin Region III
Al ed
1
Balduin
2
5 Balduin
2
Ba on
4
0 5 Ba on
4
Ches e
4
5 10 5 Ches e
4
En1
1
0 5 0 5 En1
1
EnSpm13_V in
2
0 5 0 5 0 EnSpm13_V in
2
EnSpm3_V in
2
0 5 0 5 0 0 EnSpm3_V in
2
EnSpm5_V in
2
0 5 0 5 0 0 0 EnSpm5_V in
2
Isaac
2
0 5 0 5 0 0 0 0 Isaac
2
Isido
2
5 10 5 0 5 5 5 5 5 Isido
2
No man
1
4 9 4 1 4 4 4 4 4 1 No man
1
Radon
2
4 9 4 1 4 4 4 4 4 1 0 Radon
2
Ru us
2
4 9 4 1 4 4 4 4 4 1 0 0 Ru us
2
Sand o
2
3 2 3 8 3 3 3 3 3 8 7 7 7 Sand o
2
Seamus
3
05 05 00 0 0 054 443
Dis ances be ween exon/in on bounda ies in he MSA wi hin Region III (depic ed in Figu e 1). The dis ances a e gi en in esidues in he alignmen .
Buchmann e al. Mobile DNA 2014, 5:24 Page 8 o 15
h p://www.mobilednajou nal.com/con en /5/1/24
phylogene ic analysis indica es di e gence o ancien
CACTA lineages al eady be o e he di e gence o he
monoco yledons and eudico yledons. The analysis o 73
bounda ies ac oss 33 ansposases wi h mo e han one
exon iden i ied 55 conse ed exon/in on bounda ies
and allowed us o econs uc he exon/in on con igu -
a ion o a CACTA ansposase ep esen ing he ances al
s a e be o e he di e gence o monoco yledonous and
eudico yledonous plan s. The model consis s o a leas
ou exons. We p opose a mechanism o he e olu ion
o he ex an CACTA ansposases in which hey we e
shaped mainly by in on loss, al hough one case o pu a-
i e in on gain was ound.
Po en ial o g ea e egula ion o CACTA elemen s in
eudico yledons
S udies o he PElemen in D osophila and Ac/Ds in
maize ha e shown ha al e na i e splicing can egula e
issue-speci ic ansposi ion o elemen s. Fo example,
he Pelemen e ains i s hi d in on in soma ic cells,
inhibi ing ansposi ion [33,34]. Should his occu wi h
CATCA ansposases as well, ou da a sugges s ha ele-
men s in dico yledonous hos s ha e mo e possibili ies
o egula ion. In e es ingly, mos non-clus e ed bound-
a ies and he pu a i e in on gain clus e we e ound in
ansposases om dico yledonous hos s, whe eas he
majo i y o bounda ies in Regions I o III we e ound in
ansposases om monoco yledonous hos s. The num-
be o ansposable elemen s in eudico yledonous ge-
nomes is gene ally lowe han in monoco yledonous
genomes, consis en wi h a igh e con ol o anspos-
able elemen s in eudico yledonous hos s. The e o e, he
la ge numbe o unique bounda ies ound ou side Re-
gions I o III could be associa ed wi h mo e con ol o
exp ession o CACTA elemen s in eudico yledons han
in monoco yledons.
Di e ences in in on gain and loss among TE
ansposases
P e iously, in on gain and loss in ansposases o DNA
ansposable elemen s was s udied o Ma ine -like ele-
men s in lowe ing plan s [35]. In ha s udy, degene a e
p ime s we e used o ex ac agmen s o DDE anspo-
sases om 54 plan species o phylogene ic analysis.
The esul s we e consis en wi h e ical ansmission
Table 4 Dis ances be ween exon/in on bounda ies
wi hin Clus e G
Ba on
1
Ches e
1
5 Ches e
1
EnSpm12_F es
1
4 1 EnSpm12_F es
1
Ko bin
1
14 3
Dis ances be ween exon/in on bounda ies in he MSA wi hin Clus e G
(depic ed in Figu e 1). The dis ances a e gi en in esidues in he alignmen .
Figu e 3 Model o he loss and gain o in ons in CACTA ansposases. Simpli ied phylogene ic ee based on he consensus exon numbe s
pe clade as desc ibed in he ex . Below he ee he pu a i e ances o ansposase wi h ou exons is depic ed. Exons a e depic ed as g ay
ec angles wi h in ons as colo ed lines. Blue, ed and g een depic in ons conse ed in Regions I o III, G indica es clus e G wi h he pu a i e
in on gain. Conse ed in ons sha e he same colo band. In on loss is depic ed by i s co esponding colo and ci cled −, in on gain by an
enci cled +. G ay balloons indica e how he obse ed con igu a ion a ose om he pu a i e ances o .
Buchmann e al. Mobile DNA 2014, 5:24 Page 9 o 15
h p://www.mobilednajou nal.com/con en /5/1/24