MUSTANG is a novel family of domesticated transposase genes ...

MUSTANG is a novel family of domesticated transposase genes found in diverse 

angiosperms 

Rebecca K. Cowan, Douglas R. Hoen, Daniel J. Schoen, and Thomas E. Bureau* 

McGill University, Biology Department, 1205 Dr. Penfield Avenue, Montreal, Quebec H3A 1B1 

* corresponding author: 1205 Ave Docteur Penfield, Montreal, Quebec, Canada H3A 1B1 

phone: (514) 398-6472; fax: (514) 398-5069; email: thomas.bureau@mcgill.ca 

This manuscript is intended as a Research Article. 

Running head: Selfish DNA domestication in flowering plant genomes 

Key words: Genome; Evolution; Transposons; Plants 

Abbreviations: 

FAR1 = FAR-RED IMPAIRED RESPONSE PROTEIN 1 

MUG = MUSTANG 

MULE = Mutator-like element 

TAM = transposon-associated mudrA 

TDM = transposon-dissociated mudrA 

TIR = terminal inverted repeat 

TSD = target site duplication 

MBE Advance Access published June 29, 2005 

© The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology 

and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org 

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Abstract 

While transposons have traditionally been viewed as genomic parasites or “junk DNA”, the 

discovery of transposon-derived host genes has fuelled an ongoing debate over the evolutionary 

role of transposons. In particular, while mobility-related open reading frames have been known 

to acquire host functions, the contribution of these types of events to the evolution of genes is not 

well known. Here we report that genome-wide searches for Mutator transposase-derived host 

genes in Arabidopsis thaliana (Columbia-0) and Oryza sativa ssp. japonica (cv. Nipponbare) 

(domesticated rice) identified 121 sequences, including the taxonomically-conserved 

MUSTANG1. Syntenic MUSTANG1 orthologs in such varied plant species as rice, poplar, 

Arabidopsis, and Medicago truncatula appear to be under purifying selection. However, despite 

the evidence of this pathway of gene evolution, MUSTANG1 belongs to one of only two Mutator- 

like gene families with members in both monocotyledonous and dicotyledonous plants, 

suggesting that Mutator-like elements seldom evolve into taxonomically widespread host genes. 


http://mbe.oxfordjournals.org/ 

by guest on July 15, 2013

Introduction 

Mobile elements have traditionally been viewed as “junk DNA” or as genomic parasites 

(Doolittle and Sapienza 1980; Orgel and Crick 1980). However, reports of transposon-derived 

genes contributing to cellular processes (Agrawal, Eastman, and Schatz 1998; Mi et al. 2000; 

Hudson, Lisch, and Quail 2003) have indicated that such genetic elements may influence the 

evolution of their host by providing a source of novel coding capacity. Other lines of evidence 

such as sequence analysis and conservation studies, also support this view (International Human 

Genome Sequencing Constorium 2001; Hammer, Strehl, and Hagemann 2005; Zdobnov et al. 

2005). This, in turn, has spurred controversy concerning the extent to which transposable 

elements contribute to host evolution (Hurst and Werren 2001). 

In plants, the only transposon-like genes with known host functions, far-red impaired 

response protein 1 (FAR1), far-red elongated hypocotyl 3 (FHY3) and the FAR1 related 

sequences (FRS), are related to the family of DNA transposons called Mutator-like elements 

(MULEs) (Hudson, Lisch, and Quail 2003; Lin and Wang 2004). MULEs are DNA transposons, 

which move via a “cut-and-paste” mechanism (Lisch 2002). The mudrA gene of autonomous 

elements encodes the transposase MURA that is required for MULE mobility (Lisch et al. 1999). 

Though most MULEs are bounded by terminal-inverted repeats (TIRs) of ~200 bp in length, 

some MULEs lack terminal symmetry and are termed non-TIR MULEs (Yu, Wright, and Bureau 

2000). In maize, MURA binds specifically to the terminal sequences of MULEs (Benito and 

Walbot 1997). This association between the transposase and the terminal sequences appears 

necessary for excision of the element to occur, as it is in other DNA transposons (Steiniger- 

White, Rayment, and Reznikoff 2004). Upon insertion of the MULE into the genome, 9-11 bp 

target site duplications (TSDs) are created (Bennetzen 1996). 




Transposon-derived genes with known host functions, such as FAR1 and FHY3, lack 

transposon-specific terminal sequences (Hudson, Lisch, and Quail 2003). This is likely because 

the transposition of a domesticated mobile element, one that has gained a host function, could be 

detrimental to its host. The removal of such a gene from the vicinity of cis-regulatory factors, or 

its insertion into a heterochromatic region, could dramatically change its expression (van 

Leeuwen et al. 2001). Thus, selection should act to remove mobility-enabling features such as 

TIRs. 

We took advantage of this expected historical selection pressure in our search for 

potentially domesticated genes, by mining the genomes of Oryza sativa ssp. japonica (cv. 

Nipponbare) (domesticated rice) and Arabidopsis thaliana (Columbia-0) for sequences that 

shared similarity to mudrA, but lacked transposon features such as TIRs. We then examined the 

evolutionary histories of these putative transposon-derived genes by performing phylogenetic 

reconstructions and clustering analyses on all mudrA-related sequences. The results of our 

analysis are discussed with respect to the importance that transposons play in the evolution of 

plant genes. 

Materials and Methods 

Identification of transposon-associated and transposon-dissociated mudrA genes 

In order to determine the abundance of domesticated mudrA genes in plants, we searched 

the Oryza sativa ssp. japonica (cv. Nipponbare) (domesticated rice) and Arabidopsis thaliana 

(Columbia-0) genomes for sequences with similarity to mudrA that were not associated with 

transposon structures such as terminal sequences and TSDs. mudrA-like sequences were 

identified by using Zea mays mudrA (GI:540581) as a query for a BLASTP (Altschul et al. 1997) 

search (cutoff = E < 1e-10) of predicted genes from the Arabidopsis TIGR 5.0 release 




(www.tigr.org) and from rice build 2 pseudomolecules (DDBJ accessions numbers AP006867 

through AP006877 and NCBI accession number AE016959), predicted using FGENESH 

(http://www.softberry.com/berry.phtml?topic=fgenesh) in the monocot trained settings. 

Similarly, Zea mays mudrA (GI:540581) was used as a query for a PSI-BLAST (Altschul et al. 

1997) search against the rice predicted gene set and The Arabidopsis Information Resource 

(TAIR) UNIPROT dataset (www.arabidopsis.org) (cutoff = E < e-15, second PSI-BLAST 

iteration) in order to identify more divergent mudrA-like sequences. Mined mudrA genes are 

listed in supplementary table 1. 

Whether the mined mudrA genes were associated with a transposon structure was 

determined by (a) identifying correctly oriented MULE termini separated by less than 30 kbp via 

homology searches with models and queries based on the TIR and non-TIR termini of previously 

identified families using HMMER (Eddy 1998) and BLASTN (Altschul et al. 1997); (b) using 

BL2SEQ (Tatusova and Madden 1999) with an expect value of 100 and a mismatch penalty of –1 

to identify TIRs in the 10 kbp flanking regions; (c) using the genes and flanking sequences as 

BLASTN (Altschul et al. 1997) queries to identify repetitiveness extending beyond the putative 

coding region; and (d) scanning extreme terminal sequences of TIRs and repetitive regions for 

TSDs. Genes with TSDs were considered transposon-associated mudrA genes (TAMs), those 

lacking TSDs but with other transposon features were considered potentially transposon- 

associated mudrA genes (pTAMs), while those lacking all transposon features were considered 

transposon-dissociated mudrA genes (TDMs). 

Arabidopsis genes were considered to have open reading frames (ORFs) if they were so 

annotated (www.arabidopsis.org), or if they corresponded to a gene in the TAIR UNIPROT 

dataset. Rice genes were considered to have ORFs if they corresponded to a predicted gene from 

the rice build 2 pseudomolecules. Sequences for predicted rice mudrA genes are listed in 




supplementary table 2. The TAIR sequence viewer tool was used to determine if Arabidopsis 

genes were expressed. To determine which rice genes were expressed, rice cDNAs were mapped 

to the build 2 rice pseudomolecules using BLAT (Kent 2002) with default parameters and the 

“fine” option. Similarly, rice expressed sequence tags (ESTs) were mapped using BLAT (Kent 

2002) with trimmed trailing poly-A and leading poly-T tails, 3 kbp maximum intron size, and 

90% overall identity (matches/length), rejecting ESTs with alignments to more than one location 

with less than 3% identity difference. cDNAs and ESTs that overlapped predicted mudrA genes 

by > 100 bp were considered to be associated with them. 

Clustering and phylogenetic analyses 

Inter-relationships among TDMs and TAMs were examined through clustering and 

phylogenetic analyses based on the conserved MULE-specific domain, MUDR (Hershberger et 

al. 1995; Marchler-Bauer et al. 2003). To identify MUDR domains in the mined mudrA genes, 

the mudrA genes were used as queries for TBLASTN (Altschul et al. 1997) and BLASTP 

(Altschul et al. 1997) searches against the 21 diverse MUDR domains listed in the NCBI Entrez 

(http://www.ncbi.nlm.nih.gov/entrez) entry for pfam03108. Where both protein and nucleotide 

sequences were available, the BLASTP-identified MUDR domain was retained, though it was 

manually edited if a predicted intron eliminated a large portion of the domain. More divergent 

mined mudrA genes were used as PSI-BLAST (Altschul et al. 1997) queries in order to identify 

regions within them that aligned with the MUDR domain of conserved mudrA genes. See 

supplementary table 1 for a list of detected MUDR domain regions. 

For the clustering analysis, MUDR regions were iteratively clustered at every 5% change 

in identity using BLASTCLUST (Altschul et al. 1997) with default parameters (fig. 1 and 

supplementary table 3). 




For the phylogenetic analysis, CLUSTALW (Higgins, Thompson, and Gibson 1994) was 

used to generate a multiple alignment of the MUDR domain regions, omitting truncated 

sequences less than 100 amino acid residues in length. In addition, the sequences were edited so 

that gaps in the alignment caused by less than 1% of the sequences were eliminated. A neighbor- 

joining tree with 100 bootstrap repetitions was generated from this alignment using PHYLIP 

(Retief 2000; Felsenstein 2004) with default parameters (fig. 2 and supplementary fig. 1) 

Phylogenetic and syntenic relationships within the MUSTANG (MUG) gene family 

MUG1 genes in other plant species were identified by using rice and Arabidopsis MUG1 

as queries for TBLASTN (Altschul et al. 1997) searches against the nonredundant NCBI 

(http://www.ncbi.nlm.nih.gov/BLAST), JGI (www.jgi.doe.gov/poplar), and Plant Genome 

Database (http://www.plantgdb.org) databases. A multiple alignment of the amino acid 

sequences of fungal MULE hop78 (Chalvet et al. 2003), TAMs At2g07100 and At1g12720, and 

the MUG family proteins was generated using CLUSTALW (Higgins, Thompson, and Gibson 

1994). The alignment was edited so that the ends corresponded to those of MUG1. A maximum 

parsimony tree with 100 bootstrap replications was constructed from this alignment using 

PHYLIP (Retief 2000; Felsenstein 2004) with default parameters (fig. 3). 

Predicted proteins encoded by the 50 kbp regions flanking the MUG1 genes of 

Arabidopsis, rice, and poplar were identified from the TAIR (www.arabidopsis.org), TIGR 

(www.tigr.org), and JGI (www.jgi.doe.gov/poplar) datasets, respectively. Predicted proteins 

encoded by the Medicago contig (GI:50284582) were determined using the GENSCAN 

webserver with Arabidopsis trained settings (Burge and Karlin 1997). These predicted genes 

were used for an unfiltered all-against-all BLASTP (Altschul et al. 1997) search with an effective 




database length of 740,307,180 in order to uncover microsynteny (fig. 4, supplementary fig. 2, 

and supplementary table 4). 

dN/dS estimates 

A multiple alignment of MUG1 orthologs was generated using CLUSTALW (Higgins, 

Thompson, and Gibson 1994). Corresponding nucleotide sequences were manually aligned using 

the amino acid alignment as a guide. After eliminating ambiguity sites, the CODEML program 

in the PAML package (Yang 1997) was used to estimate the dN/dS ratio for the provided 

nucleotide sequence alignment and maximum parsimony tree (constructed using the PHYLIP 

(Retief 2000; Felsenstein 2004) package with default parameters). dN/dS values for the N- 

terminal, MUDR domain, central, zinc finger domain and C-terminal subsections of MUG1 were 

similarly estimated. 

Results and Discussion 

Transposon-dissociated mudrA-like genes in rice and Arabidopsis 

Our search for sequences with similarity to mudrA that were not associated with 

transposon structures revealed 45 such transposon-dissociated mudrA genes (TDMs) in 

Arabidopsis and 76 in rice (table 1). Approximately nine-tenths of these have predicted ORFs 

(table 1). Strikingly, 40% (18/45) and 37% (28/76) of TDMs in Arabidopsis and rice, 

respectively, match ESTs and/or cDNAs as compared to only 1% (2/143) and 6% (32/498) of 

Arabidopsis and rice transposon-associated mudrA genes (TAMs) (table 1), hinting that the 

expression profile of TDMs is more similar to that of host genes than that of mobile elements. 

Taxonomic distribution of TDMs and TAMs 




Clustering analysis, based on the MUDR domains of the mined TAMs and TDMs, found 

that most of these sequences clustered exclusively with other MUDR domains from the same 

species until the identity threshold was < 40% (fig.1). A neighbor-joining tree also indicated that 

the MUDR domains form mainly species-specific clades (fig. 2). This suggests that many TDMs 

have evolved from MULEs since the monocot-dicot divergence 140-150 million years ago (Chaw 

et al. 2004). Nonetheless, two exceptional groups with both monocot and dicot members were 

identified, and both of these involve TDMs (fig. 1, fig. 2, and supplementary fig. 1). 

The first of these exceptional groups includes members of the FAR1 gene family, including 

two TDMs, FAR1 and FHY3, that have proven host functions in far-red light response (Hudson, 

Lisch, and Quail 2003). The second group, a previously unreported family henceforth referred to 

as the MUSTANG (MUG) gene family, is composed, with one exception, entirely of TDMs 

(supplementary table 3, supplementary fig. 1). However, while the neighbor-joining tree 

indicates that a TAM gene, At2g07100, is a part of the MUG gene family, the clustering analysis 

places it with other Arabidopsis TAMs. To resolve this discrepancy, as well as to gain greater 

resolution of the internal branches of the MUG gene family, a maximum parsimony tree was 

constructed using the entire protein sequences (fig. 3). 

The maximum parsimony tree places At2g07100 with other TAMs, rather than inside the 

MUG gene family, with high bootstrap support of 97% (fig. 3). The shorter sequences used to 

construct the original neighbor-joining tree, as well as the fact that the predicted ORF of 

At2g07100 has accumulated several stop codons, may account for the differing placement of 

At2g07100 and lower associated bootstrap values in this tree (supplementary fig. 1). The 

maximum parsimony tree divides the remaining TDMs into two primary clades, both of which 

include monocot and dicot members. This indicates that there were several members of the 

MUG gene family before the divergence of the monocot and dicot lineages. 




MUSTANG1 is a novel, taxonomically widespread gene 

Interestingly, a pair of potential rice/Arabidopsis orthologs called MUG1 are not only 

highly similar to each other at the amino acid level (BL2SEQ E = 0.0, identity = 71%), but are 

also similar to sequences encoded by the indica subspecies of O. sativa, Medicago truncatula, 

Populus trichocarpa (poplar), and Zea mays (maize) (BL2SEQ E = 0.0, identity > 66%). The 

maximum parsimony tree supports the hypothesis that these genes are MUG1 orthologs (fig. 3). 

The tree also indicates that the two poplar MUG1 genes probably arose through a duplication 

event sometime after the Medicago and poplar lineages diverged. The duplication is unlikely to 

be the result of transposition as the ~20 kbp regions flanking the poplar genes share significant 

nucleotide level similarity (BL2SEQ E < e-120, identity > 78%). The predicted genes in the 50 

kbp flanking regions of the rice and Arabidopsis MUG1 genes are similar to the predicted genes 

in the poplar flanking regions (fig. 4 and supplementary fig. 2). Even the flanking regions of the 

Medicago MUG1 gene, which is located on an unordered contig, display microsynteny with the 

flanking regions of the other plant species (fig. 4 and supplementary fig. 2). It is impossible to 

determine whether the maize MUG1-like gene is located in a syntenic region as the available 

sequence is only 3547 bp in length. However, the syntenic and phylogenetic relationships 

between the other MUG1 genes support their orthology and eliminate the possibility that the 

distribution of MUG1 genes arose through horizontal transfer. 

MUG1 functionality 

The rice, Arabidopsis, maize, and one of the poplar MUG1 orthologs are associated with 

cDNAs, ESTs or both (fig. 3). This expression information comes from such varied tissues as 

rice calli, corn endosperm and ears, and Arabidopsis calli, developing seeds, inflorescence 




meristem, and seedling leaves. As an indication of whether this expressed gene is performing a 

host function, we calculated the non-synonymous to synonymous substitution rate ratio (dN/dS) 

for the six identified orthologs. A dN/dS ratio of significantly less than 1 suggests that a coding 

sequence is being, or has until recently been, maintained by purifying selection, and is thus a 

good indication of whether a gene has protein coding functionality (Hurst 2002). The dN/dS 

ratio of 0.0683 for the MUG1 genes is significantly smaller than 1 (P < 0.001; log likelihood ratio 

test). 

Like the MUG1 genes, the transcription factors FAR1 and FHY3, which function as 

regulators of far-red light response (Hudson, Lisch, and Quail 2003), have also evolved from a 

MULE transposase to perform a host function. Given how dissimilar MUG1 is to these proteins 

(BL2SEQ E > 0.17, identity < 24%), it seems unlikely that MUG1 also functions in far-red light 

response. However, like FAR1 and FHY3, MUG1 does have a highly conserved zinc finger 

domain. In fact, with a dN/dS ratio of 0.0072, the zinc finger domain appears to be the most 

highly conserved region of the MUG1 gene. Therefore, as with FAR1, MUG1 may bind DNA in 

a manner that evolved directly from the way in which MURA transposase binds to TIRs 

(Hudson, Lisch, and Quail 2003). This would be consistent with other transposon-derived host 

genes whose mode of action is reminiscent of that of the transposon gene they evolved from. For 

instance, the vertebrate immune system proteins RAG1 and RAG2 assist V(D)J recombination of 

antibody genes by binding to and nicking DNA in a manner highly similar to DNA transposases, 

from which they are likely derived (Agrawal, Eastman, and Schatz 1998). In addition, the co- 

opted human endogenous retroviral envelope (env) protein syncytin mediates trophoblast cell 

fusion during placental development (Chang et al. 2004), while env proteins in general are 

thought to promote membrane fusion for viral entry into cells (Colman and Lawrence 2003). 




Conclusions 

The rarity of taxonomically widespread MULE-dissociated mudrA genes is such that they 

appear to represent an unusual detour rather than a common mechanism for generating novel 

proteins. These results, however, do not exclude the possibility that more recently domesticated 

mudrA genes may be common among individual monocot and dicot lineages not investigated 

here. The six rice and four Arabidopsis expressed TDMs that do not belong to either the FAR1 

or MUG gene families may be representative of such cases. Moreover, TDMs may frequently be 

“born”, but “die out” soon after. One reason for this could be that many domesticated genes may 

benefit their host under specific environmental conditions. Another possible explanation is that 

newly domesticated genes, which have not yet lost their mobility enabling structures, may 

transpose, thus losing the cis regulatory factors required for their host function, and thereby 

reverting to selfish DNA. In fact, in evolutionary terms, it is likely that such sequences have 

been more successful as transposons than as host genes. 

Acknowledgements 

We would like to thank Nikoleta Juretic and Ken Hastings for their helpful comments on the 

manuscript. This study was supported by Discovery grants from the National Science and 

Engineering Research Council (NSERC) of Canada to T.E.B. and D.J.S. and a grant from the 

McGill University William Dawson Scholar Chair to T.E.B. 




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Figure 1 Relationships between monocot and dicot mudrA genes as determined by clustering at 

40% identity. The number of clusters of various sizes is shown for monocot (diagonal fill), dicot 

(light grey fill) and ‘other’ (dark grey fill). The number of MUG (M) and FAR1 (F) clusters of a 

given size is indicated by a number or is equal to one where labeled with no number. The 

‘Number of Clusters’ axis is discontiguous. The ‘other’ category includes MUG and FAR1 

clusters containing both monocots and dicots as well as one cluster containing as its single 

member the fungal outgroup hop78 (Chalvet et al. 2003). Note that FAR1 family is still in 

several clusters at this at this level of identity. 

Figure 2 Relationships between monocot and dicot mudrA genes as determined by phylogenetic 

analysis, depicted as a majority rule neighbor-joining tree rooted at MULE hop78 (Chalvet et al. 

2003). Monocot leaves are labelled in grey, dicot in black. 

Figure 3 Maximum parsimony tree of the MUG gene family. This majority rule consensus tree 

is rooted with fungal MULE hop78 (Chalvet et al. 2003). Bootstrap values are shown at nodes. 

All genes shown are transposon-dissociated mudrA genes (TDMs) and belong to the MUG gene 

family except for hop78 and At2g07100 and At1g12720 which are transposon-associated mudrA 

genes (TAMs). Genes indicated by one or two asterisks are associated with ESTs and cDNAs, 

respectively. Oryza sativa subspecies japonica gene names are shown beginning with “R”, 

Oryza sativa subspecies indica with “Ri”, Arabidopsis thaliana with “A”, Zea mays with “Z”, 

Medicago truncatula with “M”, and Populus trichocarpa with “P”. 

Figure 4 Syntenic 100 kbp region surrounding MUG1 genes. The MUG1 genes are depicted in 

red. Genes shown with the same shading/pattern share protein-level similarity according to an 

all-against-all BLASTP search. A color version of this figure is available as supplementary fig. 

2. See supplementary table 4 for E values. Vertical lines indicate gaps in the sequence. The 




Medicago sequence represents an unordered contig. Poplar I and II refer, respectively, to the 

molecules scaffold_201 containing the MUG1 gene eugene3.02010019, and LG_XIII containing 

the MUG1 gene grail3.0016035401. Note that diagram is not to scale. 




Supplementary Figure 1 Neighbor-joining tree of the mudrA superfamily. The tree depicted is a 

majority rule consensus tree constructed from an alignment of the MUDR domain regions of 

mudrA-like genes. The tree is rooted with fungal MULE hop78 (Chalvet et al. 2003). Bootstrap 

values are shown at nodes. Oryza sativa subspecies japonica gene names are shown beginning 

with “R”, Oryza sativa subspecies indica with “Ri”, Arabidopsis thaliana with “A”, Zea mays 

with “Z”, Medicago truncatula with “M”, and Populus trichocarpa with “P”. 

Supplementary Figure 2 Color version of Figure 4. Genes shown in the same color share 

protein-level similarity according to an all-against-all BLASTP search. See supplementary table 

4 for E values. 




Table 1 The number and distribution of mudrA genes in rice and Arabidopsis. 

Arabidopsis 

Rice 

TAMs TDMs pTAMs 

total 143 45 18 

ORF 77 36 9 

expressed 2 18 0 

cDNA 1 11 0 

EST 2 18 0 

total 498 76 60 

ORF 493 76 60 

expressed 32 28 6 

cDNA 26 25 3 

EST 12 20 4 

TAMs, TDMs, and pTAMs refer to transposon-associated mudrA genes, transposon-dissociated 

mudrA genes, and potentially transposon-associated mudrA genes, respectively. The latter class 

includes elements that lack well-defined TSDs but have other transposon features such as TIRs 

and repetitiveness extending beyond the coding region. 







a 

MUG 

FAR1 

FAR1 

MUG 




97 

97 

97 

94 

97 

89 

90 

hop78 

At2g07100 

97 

At1g12720 

R2_001_0826** 

97 

R6_003_1496** 

R10_001_0051** 

97 

At5g34853* 

At3g05850** 

97 

At5g48965** 

97 

At3g06940** 

R1_005_0969** 

97 

At5g16505** 

At1g06740* 

94 

At2g30640* 

ZmGSStuc04-27- 13378.1* 

97 

R12_011_1237** 

97 

RiCtg035198 

At3g04605** 

95 

Mth2-35g8* 

87 

Pgrail3.0016035401* 

97 

Peugene3.02010019 




ice 

Arabidopsis 

poplar I 

poplar II 

Medicago

MUSTANG is a novel family of domesticated transposase genes ...

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