Non-autonomous Transposable Elements Research Articles

Sequences enhancing cassava mosaic disease symptoms occur in the cassava genome and are associated with South African cassava mosaic virus infection.

Cassava is an important food security crop in Sub-Saharan Africa. Two episomal begomovirus-associated sequences, named Sequences Enhancing Geminivirus Symptoms (SEGS1 and SEGS2), were identified in field cassava affected by the devastating cassava mosaic disease (CMD). The sequences reportedly exacerbated CMD symptoms in the tolerant cassava landrace TME3, and the model plants Arabidopsis thaliana and Nicotiana benthamiana, when biolistically co-inoculated with African cassava mosaic virus-Cameroon (ACMV-CM) or East African cassava mosaic virus-UG2 (EACMV-UG2). Following the identification of small SEGS fragments in the cassava EST database, the intention of this study was to confirm their presence in the genome, and investigate a possible role for these sequences in CMD. We report that multiple copies of varying lengths of both SEGS1 and SEGS2 are widely distributed in the sequenced cassava genome and are present in several other cassava accessions screened by PCR. The endogenous SEGS1 and SEGS2 are in close proximity or overlapping with cassava genes, suggesting a possible role in regulation of specific biological processes. We confirm the expression of SEGS in planta using EST data and RT-PCR. The sequence features of endogenous SEGS (iSEGS) are unique but resemble non-autonomous transposable elements (TEs) such as MITEs and helitrons. Furthermore, many SEGS-associated genes, some involved in virus-host interactions, are differentially expressed in susceptible (T200) and tolerant TME3) cassava landraces infected by South African cassava mosaic virus (SACMV) of susceptible (T200) and tolerant (TME3) cassava landraces. Abundant SEGS-derived small RNAs were also present in mock-inoculated and SACMV-infected T200 and TME3 leaves. Given the known role of TEs and associated genes in gene regulation and plant immune responses, our observations are consistent with a role of these DNA elements in the host's regulatory response to geminiviruses.

A non-autonomous insect piggyBac transposable element is mobile in tobacco.

The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid only requires expression of piggyBac transposase. To determine if piggyBac could function in dicotyledonous plants, a two-element system was developed in tobacco (Nicotiana tabacum) to test for transposable element excision and insertion. The first transgenic line constitutively expressed piggyBac transposase, while the second transgenic line contained at least two non-autonomous piggyBac transposable elements. Progeny from crosses of the two transgenic lines was analyzed for piggyBac excision and transposition. Several progeny displayed excision events, and all the sequenced excision sites exhibited evidence of the precise excision mechanism characteristic of piggyBac transposase. Two unique transposition insertion events were identified that each included diagnostic duplication of the target site. These data indicate that piggyBac transposase is active in a dicotyledonous plant, although at a low frequency.

FB-NOF is a non-autonomous transposable element, expressed in Drosophila melanogaster and present only in the melanogaster group

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this.In this work we test the hypothesis that FB-NOF is an active transposon nowadays. We search for its expression product, obtaining its cDNA, and propose the ORF and the sequence of its potential protein. We found that the NOF protein is not a transposase as it lacks any of the motifs of known transposases and also shows structural homology with hydrolases, therefore FB-NOF cannot belong to the superfamily MuDR/foldback, as up to now it has been classified, and can be considered as a non-autonomous transposable element. The alignment with the published genomes of 12 Drosophila species shows that NOF presence is restricted only to the 6 Drosophila species belonging to the melanogaster group.

NcRNAclassifier: a tool for detection and classification of transposable element sequences in RNA hairpins

BackgroundInverted repeat genes encode precursor RNAs characterized by hairpin structures. These RNA hairpins are then metabolized by biosynthetic pathways to produce functional small RNAs. In eukaryotic genomes, short non-autonomous transposable elements can have similar size and hairpin structures as non-coding precursor RNAs. This resemblance leads to problems annotating small RNAs.ResultsWe mapped all microRNA precursors from miRBASE to several genomes and studied the repetition and dispersion of the corresponding loci. We then searched for repetitive elements overlapping these loci. We developed an automatic method called ncRNAclassifier to classify pre-ncRNAs according to their relationship with transposable elements (TEs). We showed that there is a correlation between the number of scattered occurrences of ncRNA precursor candidates and the presence of TEs. We applied ncRNAclassifier on six chordate genomes and report our findings. Among the 1,426 human and 721 mouse pre-miRNAs of miRBase, we identified 235 and 68 mis-annotated pre-miRNAs respectively corresponding completely to TEs.ConclusionsWe provide a tool enabling the identification of repetitive elements in precursor ncRNA sequences. ncRNAclassifier is available at http://EvryRNA.ibisc.univ-evry.fr.

A transposon-mediate inactivation of a CYCLOIDEA-like gene originates polysymmetric and androgynous ray flowers in Helianthus annuus

In several eudicots, including members of the Asteraceae family, the CYCLOIDEA (CYC) genes, which belong to the TCP class of transcription factors, are key players for floral symmetry. The sunflower inflorescence is heterogamous (radiate capitulum) with sterile monosymmetric ray flowers located in the outermost whorl of the inflorescence and hermaphrodite polysymmetric disk flowers. In inflorescence of Heliantheae tribe, flower primordia development initiates from the marginal ray flowers while disk flowers develop later in an acropetal fashion in organized parastichies along a number found to be one of Fibonacci patterns. Mutants for inflorescence morphology can provide information on the role of CYC-like genes in radiate capitulum evolution. The tubular ray flower (turf) mutant of sunflower shows hermaphrodite ray flowers with a nearly polysymmetric tubular-like corolla. Here, we demonstrate that this mutation is caused by the insertion in the TCP motif of a sunflower CYC-like gene (HaCYC2c) of non-autonomous transposable element (TE), belonging to the CACTA superfamily of transposons. We named this element Transposable element of turf1 (Tetu1). The Tetu1 insertion changes the reading frame of turf-HaCYC2c for the encoded protein and leads to a premature stop codon. Although in Tetu1 a transposase gene is lacking, our results clearly suggest that it is an active TE. The excision of Tetu1 restores the wild type phenotype or generates stable mutants. Co-segregation and sequence analysis in progenies of F(2) and self-fertilized plants derived from reversion of turf to wild type clearly identify HaCYC2c as a key regulator of ray flowers symmetry. Also, HaCYC2c loss-of-function promotes the developmental switch from sterile to hermaphrodite flowers, revealing a novel and unexpected role for a CYC-like gene in the repression of female organs.

Transposition behavior of nonautonomous a hAT superfamily transposon nDart in rice (Oryza sativa L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0-13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.

MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences

Miniature inverted-repeat transposable elements (MITEs) are a special type of Class 2 non-autonomous transposable element (TE) that are abundant in the non-coding regions of the genes of many plant and animal species. The accurate identification of MITEs has been a challenge for existing programs because they lack coding sequences and, as such, evolve very rapidly. Because of their importance to gene and genome evolution, we developed MITE-Hunter, a program pipeline that can identify MITEs as well as other small Class 2 non-autonomous TEs from genomic DNA data sets. The output of MITE-Hunter is composed of consensus TE sequences grouped into families that can be used as a library file for homology-based TE detection programs such as RepeatMasker. MITE-Hunter was evaluated by searching the rice genomic database and comparing the output with known rice TEs. It discovered most of the previously reported rice MITEs (97.6%), and found sixteen new elements. MITE-Hunter was also compared with two other MITE discovery programs, FINDMITE and MUST. Unlike MITE-Hunter, neither of these programs can search large genomic data sets including whole genome sequences. More importantly, MITE-Hunter is significantly more accurate than either FINDMITE or MUST as the vast majority of their outputs are false-positives.

Novel non-autonomous transposable elements on W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm Bombyx mori are designated ZW (XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in theWGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.

Remobilization of Tol2 transposons in Xenopus tropicalis

BackgroundThe Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.ResultsTo test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis.ConclusionsIn this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.

Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes

BackgroundBacterial repetitive extragenic palindromes (REPs) compose a distinct group of genomic repeats. They usually occur in high abundance (>100 copies/genome) and are often arranged in composite repetitive structures - bacterial interspersed mosaic elements (BIMEs). In BIMEs, regularly spaced REPs are present in alternating orientations. BIMEs and REPs have been shown to serve as binding sites for several proteins and suggested to play role in chromosome organization and transcription termination. Their origins are, at present, unknown.ResultsIn this report, we describe a novel class of putative transposases related to IS200/IS605 transposase family and we demonstrate that they are obligately associated with bacterial REPs. Open reading frames coding for these REP-associated tyrosine transposases (RAYTs) are always flanked by two REPs in inverted orientation and thus constitute a unit reminiscent of typical transposable elements. Besides conserved residues involved in catalysis of DNA cleavage, RAYTs carry characteristic structural motifs that are absent in typical IS200/IS605 transposases. DNA sequences flanking rayt genes are in one third of examined cases arranged in modular BIMEs. RAYTs and their flanking REPs apparently coevolve with each other. The rayt genes themselves are subject to rapid evolution, substantially exceeding the substitution rate of neighboring genes. Strong correlation was found between the presence of a particular rayt in a genome and the abundance of its cognate REPs.ConclusionsIn light of our findings, we propose that RAYTs are responsible for establishment of REPs and BIMEs in bacterial genomes, as well as for their exceptional dynamics and species-specifity. Conversely, we suggest that BIMEs are in fact a special type of nonautonomous transposable elements, mobilizable by RAYTs.

Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata

Insect transgenesis is mainly based on the random genomic integration of DNA fragments embedded into non-autonomous transposable elements. Once a random insertion into a specific location of the genome has been identified as particularly useful with respect to transgene expression, the ability to make the insertion homozygous, and lack of fitness costs, it may be advantageous to use that location for further modification. Here we describe an efficient method for the modification of previously inserted transgenes by the use of the site-specific integration system from phage phiC31 in a tephritid pest species, the Mediterranean fruit fly Ceratitis capitata. First, suitable transgenic strains with randomly integrated attP landing sites within transposon-based vectors were identified by molecular and functional characterization. Second, donor plasmids containing an attB site, with additional markers, and transposon ends were integrated into attP sites by phiC31 integrase-mediated recombination. Third, transposase-encoding 'jumpstarter' strains were created and mated to transgenic strains resulting in the postintegrational excision of transposon ends, which left stably integrated transgene insertions that could not be remobilized. This three-step integration and stabilization system will allow the combination of several transgene-encoded advantageous traits at evaluated genomic positions to generate optimized strains for pest control that minimize environmental concerns.

Site specific cytosine methylation in rice nonautonomous transposable element nDart

The mobile nonautonomous element nDart, which is active in intact rice plants, exhibits locus specific transposition. Due to the high homogeneity of nDart elements, the locus specificity of nDart transposition might be controlled by factors other than genetic differences. In this study, we elucidated the regulation of the locus specificity of nDart transposition. The difference of transpositional activities in 10 nDart elements among rice varieties exhibiting nDart transposition was clearly correlated with the methylation state of nDart elements. Both hyper- and hypo-methylated nDart elements were inactive, while site specific methylation in both subterminal regions was identified in active nDart loci. The specific methylation sites contain the pentamer motif GCC/ACG. The repeated motifs in the subterminal region of nDart elements may contribute to the stable secondary structure of nDart elements with low free energy. Our results suggested that site specific cytosine methylation may loosen the stable secondary structure of the nDart element to allow it to bind TPase, which then perform the excision of nDart elements from genomic loci.

Spip and Squiq, two novel rice non-autonomous LTR retro-element families related to RIRE3 and RIRE8

Transposable elements are the main components of complex genomes. Their impact on the genomes in terms of structural changes during evolution is thus one of the main focuses of today's structural genomics. Although non-autonomous transposable elements have been known for a long time, their contribution to genome evolution is poorly understood. Our present study describes two new non-autonomous LTR retrotransposons in the rice ( Oryza sativa L) genome, Spip and Squiq, the LTR of which are closely related to those of the gypsy-like RIRE3 and RIRE8 LTR retrotransposon families, respectively, but the internal region of which is completely different and harbours none of the characteristic coding domains of LTR retrotransposons. Spip and Squiq thus belong to the class of LArge Retrotransposon Derivatives (LARDs). A phylogenetic study based on the sequence alignment of the LTRs of Spip/ RIRE3, and of Squiq/ RIRE8 show that both Spip and Squiq elements are of monophyletic origin. In addition, the estimation of the date of insertion of the copies suggests that Spip and Squiq families are of recent origin (that is, they amplified mainly within the last 2 My). Spip and Squiq are the fourth and fifth LARD families to be described in the rice genome, suggesting that this type of sequences is not rare. Moreover, these two families appear to be as numerous as their autonomous counterparts, suggesting that they have played an equivalent role in the recent history of rice.

Transposon-free insertions for insect genetic engineering

Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.

Identification of Novel Non-autonomous CemaT Transposable Elements and Evidence of their Mobility within the C. elegans Genome

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.

Molecular mapping of k2 Mdh1-n y20, an unstable chromosomal region in soybean [Glycine max (L.) Merr.

In the soybean genome, a chromosomal region covering three tightly linked genes, k2, Mdh1-n, and y20, was found very unstable. It was suspected that the instability of the k2 Mdh1-n y20 chromosomal region was caused by a non-autonomous transposable element residing adjacent to or in this region. In this study, we located and mapped this region with simple sequence repeat (SSR) markers on the soybean integrated map using five mapping populations. The k2 Mdh1-n y20 chromosomal region was located on molecular linkage group H. The integrated map from five mapping populations consisted of 13 loci in the order Satt541, Satt469, Sat_122, Satt279, Satt253, Satt314, Mdh1-n,y20, k2, Satt302, Satt142, Satt181, and Satt434. The k2 Mdh1-n y20 chromosomal region was very close to Satt314, Satt253, and Satt279. The genetic distance between the Mdh1-n gene and Satt314 was less than 1 cM. The results of the mapping study were consistent with the results from previous studies that the Mdh1-n mutation in T261 (k2 Mdh1-n) and the Mdh1-n y20 mutation in T317 (Mdh1-n y20) were caused by deletions. In addition, another putative deletion was found in the genome of T261 which covered three SSR markers (Satt314, Satt253, and Satt279).

Fusion of the Transposase with a Classical Nuclear Localization Signal to Increase the Transposition Efficiency of Ac Transposon

A new strategy was applied to improve the transposition efficiency of the maize transposon Activator (Ac) in heterologous plants. The Ac transposase was fused with a classical nuclear localization signal (NLS) of SV40 to promote the transport of transposase into a nucleus. Base on this, two NLS-TPase constructs were yielded, one containing the full length transposase gene (termed as SV40TPase), the other containing the truncated transposase gene (lacking its first NLS-like signal, termed as SV39TPase). These two NIS-TPase genes were expressed in transgenic tobacco plants under the control of PR-1a promoter. Excision of non-autonomous transposable element (Ds) from luciferase (LUC) reporter gene constructs was employed to analyze the induction of Ac transposase containing NLS. Applying the LUC assay and PCR analysis, these new NIS-TPase sources triggered higher Ds excision efficiencies then the native transposase. Furthermore, the SV40TPase showed more ability then the SV39TPase to trigger the Ds element. The usage of this new inducible transposon for plant functional genomics is discussed.

Isolation and characterization of the first non-autonomous transposable element in amphioxus, ATE-1

We report the first class II transposon in cephalochordates, which we have named Amphioxus Transposable Element 1 (ATE-1). ATE-1 members have been identified in the genome of Branchiostoma lanceolatum ( BlATE-1) and B. floridae ( BfATE-1). Structural analysis revealed that ATE-1 elements consist of a central region (CR) with no homology with any coding sequence, nor any detectable open reading frame (ORF), flanked by internal direct repeats (DR) of about 100 nt, each adjacent to a terminal inverted repeat (TIR) of 16 nt. Although the estimated copy number in the amphioxus genome is low, there is evidence of mobility. Sequence and hybridization analyses pointed to TIR and DR inter-species conservation, whereas no structural similarities among the CR were detected. Moreover, this element is found in two amphioxus species that diverged long ago, which argues in favor of its ancient origin. However, the structural hallmarks needed for transposition are still preserved.

Pearl, a novel family of putative transposable elements in bivalve mollusks.

While genome sequencing projects have discovered numerous types of transposable elements in diverse eukaryotes, there are many taxa of ecological and evolutionary significance that have received little attention, such as the molluscan class Bivalvia. Examination of a 0.7-MB genomic sequence database from the cupped oyster Crassostrea virginica revealed the presence of a common interspersed element, CvA. CvA possesses subterminal inverted repeats, a tandemly repeated core element, a tetranucleotide microsatellite region, and the ability to form stable secondary structures. Three other less abundant repetitive elements with a similar structure but little sequence similarity were also found in C. virginica. Ana-1, a repetitive element with similar features, was discovered in the blood ark Anadara trapezia by probing a genomic library with a dimeric repeat element contained in intron 2 of a minor globin gene in that species. All of these elements are flanked by the dinucleotide AA, a putative target-site duplication. They exhibit structural similarity to the sea urchin Tsp family and Drosophila SGM insertion sequences; in addition, they possess regions of sequence similarity to satellite DNA from several bivalve species. We suggest that the Crassostrea repetitive elements and Ana-1 are members of a new MITE-like family of nonautonomous transposable elements, named pearl. Pearl is the first putative nonautonomous DNA transposon to be identified in the phylum Mollusca.

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants.

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.