Autonomous Transposon Research Articles

A concerted increase in readthrough and intron retention drives transposon expression during aging and senescence.

Aging and senescence are characterized by pervasive transcriptional dysfunction, including increased expression of transposons and introns. Our aim was to elucidate mechanisms behind this increased expression. Most transposons are found within genes and introns, with a large minority being close to genes. This raises the possibility that transcriptional readthrough and intron retention are responsible for age-related changes in transposon expression rather than expression of autonomous transposons. To test this, we compiled public RNA-seq datasets from aged human fibroblasts, replicative and drug-induced senescence in human cells, and RNA-seq from aging mice and senescent mouse cells. Indeed, our reanalysis revealed a correlation between transposons expression, intron retention, and transcriptional readthrough across samples and within samples. Both intron retention and readthrough increased with aging or cellular senescence and these transcriptional defects were more pronounced in human samples as compared to those of mice. In support of a causal connection between readthrough and transposon expression, analysis of models showing induced transcriptional readthrough confirmed that they also show elevated transposon expression. Taken together, our data suggest that elevated transposon reads during aging seen in various RNA-seq dataset are concomitant with multiple transcriptional defects. Intron retention and transcriptional readthrough are the most likely explanation for the expression of transposable elements that lack a functional promoter.

A concerted increase in readthrough and intron retention drives transposon expression during aging and senescence

Aging and senescence are characterized by pervasive transcriptional dysfunction, including increased expression of transposons and introns. Our aim was to elucidate mechanisms behind this increased expression. Most transposons are found within genes and introns, with a large minority being close to genes. This raises the possibility that transcriptional readthrough and intron retention are responsible for age-related changes in transposon expression rather than expression of autonomous transposons. To test this, we compiled public RNA-seq datasets from aged human fibroblasts, replicative and drug-induced senescence in human cells, and RNA-seq from aging mice and senescent mouse cells. Indeed, our reanalysis revealed a correlation between transposons expression, intron retention, and transcriptional readthrough across samples and within samples. Both intron retention and readthrough increased with aging or cellular senescence and these transcriptional defects were more pronounced in human samples as compared to those of mice. In support of a causal connection between readthrough and transposon expression, analysis of models showing induced transcriptional readthrough confirmed that they also show elevated transposon expression. Taken together, our data suggest that elevated transposon reads during aging seen in various RNA-seq dataset are concomitant with multiple transcriptional defects. Intron retention and transcriptional readthrough are the most likely explanation for the expression of transposable elements that lack a functional promoter.

Autonomous transposons tune their sequences to ensure somatic suppression

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns1, and also functional2 or non-functional chimeric transcripts3. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1’s conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline.

Schlafen-5 inhibits LINE-1 retrotransposition

Long interspersed element 1 (LINE-1) is the only currently known active autonomous transposon in humans, and its retrotransposition may cause deleterious effects on the structure and function of host cell genomes and result in sporadic genetic diseases. Host cells therefore developed defense strategies to restrict LINE-1 mobilization. In this study, we demonstrated that IFN-inducible Schlafen5 (SLFN5) inhibits LINE-1 retrotransposition. Mechanistic studies revealed that SLFN5 interrupts LINE-1 ribonucleoprotein particle (RNP) formation, thus diminishing nuclear entry of the LINE-1 RNA template and subsequent LINE-1 cDNA production. The ability of SLFN5 to bind to LINE-1 RNA and the involvement of the helicase domain of SLFN5 in its inhibitory activity suggest a mechanism that SLFN5 binds to LINE-1 RNA followed by dissociation of ORF1p through its helicase activity, resulting in impaired RNP formation. These data highlight a new mechanism of host cells to restrict LINE-1 mobilization.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

Comprehensive analysis of both long and short read transcriptomes of a clonal and a seed-propagated model species reveal the prerequisites for transcriptional activation of autonomous and non-autonomous transposons in plants

BackgroundTransposable element (TE) transcription is a precursor to its mobilisation in host genomes. However, the characteristics of expressed TE loci, the identification of self-competent transposon loci contributing to new insertions, and the genomic conditions permitting their mobilisation remain largely unknown.ResultsUsing Vitis vinifera embryogenic callus, we explored the impact of biotic stressors on transposon transcription through the exposure of the callus to live cultures of an endemic grapevine yeast, Hanseniaspora uvarum. We found that only 1.7–2.5% of total annotated TE loci were transcribed, of which 5–10% of these were full-length, and the expressed TE loci exhibited a strong location bias towards expressed genes. These trends in transposon transcription were also observed in RNA-seq data from Arabidopsis thaliana wild-type plants but not in epigenetically compromised Arabidopsis ddm1 mutants. Moreover, differentially expressed TE loci in the grapevine tended to share expression patterns with co-localised differentially expressed genes. Utilising nanopore cDNA sequencing, we found a strong correlation between the inclusion of intronic TEs in gene transcripts and the presence of premature termination codons in these transcripts. Finally, we identified low levels of full-length transcripts deriving from structurally intact TE loci in the grapevine model.ConclusionOur observations in two disparate plant models representing clonally and seed propagated plant species reveal a closely connected transcriptional relationship between TEs and co-localised genes, particularly when epigenetic silencing is not compromised. We found that the stress treatment alone was insufficient to induce large-scale full-length transcription from structurally intact TE loci, a necessity for non-autonomous and autonomous mobilisation.

Characterization and expression pattern of ZB and PS transposons in zebrafish

Despite comprising much of the genome, transposons were once thought of as junk. However, transposons play many roles in the eukaryotic genome, such as providing new proteins as domesticated genes, expressing during germline-soma differentiation, function in DNA rearrangement in the offspring, and so on. We sought to describe the distribution and structural organization of the two autonomous transposons (ZB and PS) in the zebrafish genome and examine their expression patterns in embryos and adult tissues. The intact copy of ZB and PS was queried by BLAST on NCBI and ENSEMBL using default parameters. Of the copies with coverage and identity, more than 90 % were downloaded to do structural analysis. Spatial and temporal expression patterns were detected by qRT-PCR and Whole-mount in situ hybridization (WISH). There are 19 intact copies of ZB, encoding 341 amino acid residues with DD34E catalytic domain and flanked by 201bp TIRs, and seven intact PS copies, containing 425 amino acid residues with DD35D catalytic domain flanked by 28bp TIRs, were detected in the genome of zebrafish respectively. Analysis of genomic insertions indicated that both ZB and PS transposons are prone to be retained in the intron and intergenic regions of the zebrafish genome. The sense and antisense transcripts of ZB and PS were detected during embryonic development stages and exhibited similar expression patterns. The difference is that the sense strand transcript of ZB was explicitly expressed in midbrain-hindbrain boundary (MHB) and otic vesicle (OV), and pharyngeal arches and pharyngeal pouches (PA&PP) at 48 hpf. In adult zebrafish, the expressions of ZB and PS in muscle and brain are much higher than in other tissues. Our study results indicate that ZB and PS transposons may be involved in the embryonic development and regulation of somatic cells of certain adult tissues, such as the brain and muscle.

Cloning and Detection of Genomic Retrozymes and Their circRNA Intermediates.

Retrozymes are a novel family of non-autonomous retrotransposable elements that contain hammerhead ribozyme motifs. These retroelements are found widespread in eukaryotic genomes, with active copies present in many species, which rely on other autonomous transposons for mobilization. Contrary to other retrotransposons, transcription of retrozymes in vivo leads to the formation and accumulation of circular RNAs, which can be readily detected by RNA blotting. In this chapter, we describe the procedures needed to carry out the cloning of genomic retrozymes, and to detect by northern blot their circular RNA retrotransposition intermediates.

SLFN14 inhibits LINE-1 transposition activity

Long interspersed nuclear element-1 (LINE-1) is the only active autonomous transposon in the human genome. Its transposition frequently induces host genome instability, leading to a variety of genetic diseases, including cancers. The host factors play important roles in inhibiting LINE-1 retrotransposition. As an important component of the immune system, the host factor SLFN14 has antiviral activity. Our laboratory shows that SLFN14 possesses potent inhibitory activity against LINE-1 retrotransposition. To explore the potential mechanism of SLFN14 inhibition, we analyzed its effects on transcription, translation, reverse transcription and insertion in the LINE-1 replication cycle. We confirmed that SLFN14 could suppress the LINE-1 mRNA level by affecting its transcription and degradation, thereby diminishing the protein and cDNA levels of LINE-1, which eventually block the LINE-1 retrotransposition. Further, by mapping the active domains of SLFN14, we found its inhibitory activity on LINE-1 being closely related to its endoribonuclease and ribosome binding domains. These results demonstrate the mechanism of SLFN14 in regulating LINE-1 replication, which further provide new insights for improving the regulation network of host factors for controlling genomic instability caused by LINE-1 replication.

Evolution and domestication of Tc1/mariner transposons in the genome of African coelacanth (Latimeria chalumnae).

Here, we comprehensively analysed the abundance, diversity, and activity of Tc1/mariner transposons in African coelacanth (Latimeria chalumnae). Fifteen Tc1/mariner autonomous transposons were identified and grouped into six clades: DD34E/Tc1, DD34D/mariner, DD35D/Fot, DD31D/pogo, DD30-31D/pogo-like, and DD32-36D/Tigger, belonging to three known families: DD34E/Tc1, DD34D/mariner, and DD×D/pogo (DD35D/Fot, DD31D/pogo, DD30-31D/pogo-like, and DD32-36D/Tigger). Thirty-one miniature inverted-repeat transposable element (MITE) transposons of Tc1/mariner were also identified, and 20 of them display similarity to the identified autonomous transposons. The structural organization of these full Tc1/mariner elements includes a transposase gene flanked by terminal inverted repeats (TIRs) with TA dinucleotides. The transposases contain N-terminal DNA binding domain and a C-terminal catalytic domain characterized by the presence of a conservative D(Asp)DE(Glu)/D triad that is essential for transposase activity. The Tc1/mariner superfamily in coelacanth exhibited very low genome coverage (0.3%), but it experienced an extraordinary difference of proliferation dynamics among the six clades identified; moreover, most of them exhibited a very recent and current proliferation, suggesting that some copies of these transposons are putatively active. Additionally, at least four functional genes derived from Tc1/mariner transposons were found. We provide an up-to-date overview of Tc1/mariner in coelacanth, which may be helpful in determining genome and gene evolution in this living fossil.

Transgenerational activation of an autonomous DNA transposon, Dart1-24, by 5-azaC treatment in rice.

Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

Abstract 3096: LINE-1 retrotransposition L1-FGGY promotes the occurrence and progression of lung squamous cell carcinoma by inhibiting the tumor-suppressor gene FGGY

Abstract Retrotransposition is a kind of chromatin rearrangement containing the non-coding region, which makes up about half of the human genome. Long interspersed element-1 (LINE-1) retrotransposition is the only currently known active autonomous transposon in humans and might cause genetic instability, which was reported to occur with high frequency in a variety of tumor tissues, including colon cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, etc. However, the relationship between LINE-1 and lung squamous cell carcinoma (LUSC) is unclear. In this study, we analyzed 504 cases of LUSC samples based on the RNA-seq database in The Cancer Genome Atlas (TCGA), and found that 1/3 of tumor samples possessed LINE-1 inserted retrotransposons. We screened out 14 LINE-1 retrotransposons with the highest occurrence frequency and detected the expression of LINE-1 retrotransposons in 110 clinical LUSC samples and 52 matched adjacent normal tissue samples. The results showed that among 14 screened retrotransposon genes, the expression of 13 retrotransposon genes in LUSC was significantly higher than that in the corresponding para-carcinoma tissues, indicating that the presence of LINE-1 retrotransposon was related to the occurrence of LUSC. Furthermore, by analyzing the correlationship between the expression levels of LINE-1 retrotransposons and overall survival time of patients, we also found that the survival time of patients with low retrotransposon gene expression was long, while the survival time of patients with high retrotransposon gene expression was short. Among them, L1-FGGY and L1-ATP8B1 showed significant differences. In this study, we focused on one LINE-1 retrotransposon, i.e. L1-FGGY. Then we further investigated the mechanism of the LINE-1 retrotransposition in regulating the occurrence and progression of LUSC and discovered that the expression of L1-FGGY and FGGY was negatively correlated in LUSC patients (coefficient of association=-0.232, p<0.05). In order to further explore the function of FGGY in cancer development, we constructed 3 different siRNAs which could specifically interfere with the expression of FGGY and discovered that knockdown of FGGY could promote cell proliferation, inhibit cell apoptosis, as well as facilitate cell migration and invasion in vitro. The further study showed that inhibition of FGGY could promote tumorigenesis and tumor metastasis in vivo, which collectively indicated that FGGY might function as a tumor-suppressor gene. In conclusion, we not only uncovered that the LINE-1 retrotransposition L1-FGGY promotes the development of LUSC by inhibiting the expression and function of the tumor-suppressor gene FGGY, but also revealed that LINE-1 retrotransposon might be a new biomarker for early diagnosis, prognosis evaluation, and targeted therapy in the future clinical translation. Citation Format: Rui Zhang, Jinpu Yu. LINE-1 retrotransposition L1-FGGY promotes the occurrence and progression of lung squamous cell carcinoma by inhibiting the tumor-suppressor gene FGGY [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3096.

Border collies of the genome: domestication of an autonomous retrovirus-like transposon.

Retrotransposons often spread rapidly through eukaryotic genomes until they are neutralized by host-mediated silencing mechanisms, reduced by recombination and mutation, and lost or transformed into benevolent entities. But the Ty1 retrotransposon appears to have been domesticated to guard the genome of Saccharomyces cerevisiae.

Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators.

SummaryTransposable elements (TEs) are now recognized not only as parasitic DNA, whose spread in the genome must be controlled by the host, but also as major players in genome evolution and regulation1,2,3,4,5,6. Long INterspersed Element-1 (LINE-1 or L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and continues to generate inter- and intra-individual genetic variation, in some cases resulting in disease1,2,3,4,5,6,7. Nonetheless, how L1 activity is controlled and what function L1s play in host gene regulation remain incompletely understood. Here, we use CRISPR/Cas9 screening strategies in two distinct human cell lines to provide the first genome-wide survey of genes involved in L1 retrotransposition control. We identified functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, often associated with human diseases, control the L1 lifecycle at transcriptional or post-transcriptional levels and in a manner that can depend on the endogenous L1 sequence, underscoring the complexity of L1 regulation. We further investigated L1 restriction by MORC2 and human silencing hub (HUSH) complex subunits MPP8 and TASOR8. HUSH/MORC2 selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environment, and promote H3K9me3 deposition for transcriptional silencing. Interestingly, these silencing events often occur within introns of transcriptionally active genes and lead to down-regulation of host gene expression in a HUSH/MORC2-dependent manner. Together, we provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway, and illustrate how epigenetic silencing of TEs rewires host gene expression programs.

Hsp90 and Physiological Stress Are Linked to Autonomous Transposon Mobility and Heritable Genetic Change in Nematodes.

Transposable elements (TEs) have been recognized as potentially powerful drivers of genomic evolutionary change, but factors affecting their mobility and regulation remain poorly understood. Chaperones such as Hsp90 buffer environmental perturbations by regulating protein conformation, but are also part of the PIWI-interacting RNA pathway, which regulates genomic instability arising from mobile TEs in the germline. Stress-induced mutagenesis from TE movement could thus arise from functional trade-offs in the dual roles of Hsp90. We examined the functional constraints of Hsp90 and its role as a regulator of TE mobility by exposing nematodes (Caenorhabditis elegans and Caenorhabditis briggsae) to environmental stress, with and without RNAi-induced silencing of Hsp90. TE excision frequency increased with environmental stress intensity at multiple loci in several strains of each species. These effects were compounded by RNAi-induced knockdown of Hsp90. Mutation frequencies at the unc-22 marker gene in the offspring of animals exposed to environmental stress and Hsp90 RNAi mirrored excision frequency in response to these treatments. Our results support a role for Hsp90 in the suppression of TE mobility, and demonstrate that that the Hsp90 regulatory pathway can be overwhelmed with moderate environmental stress. By compromising genomic stability in germline cells, environmentally induced mutations arising from TE mobility and insertion can have permanent and heritable effects on both the phenotype and the genotype of subsequent generations.

The characteristics and functions of a miniature inverted-repeat transposable element TaMITE81 in the 5' UTR of TaCHS7BL from Triticum aestivum.

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in eukaryotic and prokaryotic genomes. In this article, a MITE, TaMITE81, was isolated from the 5' untranslated region (UTR) of TaCHS7BL, chalcone synthase (CHS) catalyzing the first committed step of anthocyanin biosynthesis, in the wheat cultivar 'Opata' with white grain. TaMITE81 was only 81 nucleotides, including a terminal inverted repeat with 39 nucleotides and was flanked by two nucleotides, "TA", target site duplications that were typical features of stowaway-like MITEs. Compared with the wheat cultivar 'Gy115' with purple grain, which is without the insertion, the expression of TaCHS7BL was lower in several organs of 'Opata'. The insertion of TaMITE81 into the 5' UTR of the GUS gene also reduced the transient expression of GUS on the coleoptiles of 'Opata', which means the insertion of TaMITE81 was the reason for the low expression of TaCHS7BL in 'Opata'. But the genotype of TaCHS7BL was not linked to phenotype of grain color in the RILs derived from a cross 'Gy115' and 'Opata'. The TaMITE81 density of the hexaploid variety of T. aestivum was more than 10 times that of diploid relatives, which implies that polyploidization caused the amplification of TaMITE81 homologous sequences. Further research should be conducted on decoding the relationship between TaCHS7BL and other traits relative to anthocyanin biosynthesis in wheat, and discovering the mechanism of TaMITE81 transposon action.

The connection between LINE-1 retrotransposition and human tumorigenesis

LINE-1 is the only currently known active autonomous transposon in humans with an estimated 500 000 copies representing 17% of the human genome. LINE-1 propagates itself through a process called retrotransposition, which includes transcription and reverstranscription, to produce new DNA copies. The new produced DNA copies can integrate into new genomic loci. The insertion of LINE-1 is able to cause genetic instability by affecting the expression or regulation of the nearby genes, thus causing dozens of genetic diseases or tumors. In this review, we summarize the recent research progress on the connection between LINE-1 retrotransposition and human tumorigenesis, which might shed light on the biology mechanisms and treatment of the tumorigenesis.

ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs.

ABSTRACTBacterial genomes encode numerous homologs of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. Here, we show that Cas9 homologs represent a distinct group of nonautonomous transposons, which we denote ISC (insertion sequences Cas9-like). We identify many diverse families of full-length ISC transposons and demonstrate that their terminal sequences (particularly 3′ termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene and often also encode the TnpB protein containing the RuvC-like endonuclease domain. The terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication. We also identify autonomous ISC transposons that encode TnpA-like Y1 transposases. Thus, the nonautonomous ISC transposons could be mobilized in trans either by Y1 transposases of other, autonomous ISC transposons or by Y1 transposases of the more abundant IS605 transposons. These findings imply an evolutionary scenario in which the ISC transposons evolved from IS605 family transposons, possibly via insertion of a mobile group II intron encoding the HNH domain, and Cas9 subsequently evolved via immobilization of an ISC transposon.IMPORTANCE Cas9 endonucleases, the effectors of type II CRISPR-Cas systems, represent the new generation of genome-engineering tools. Here, we describe in detail a novel family of transposable elements that encode the likely ancestors of Cas9 and outline the evolutionary scenario connecting different varieties of these transposons and Cas9.

Evolutionary genomics of miniature inverted-repeat transposable elements (MITEs) in Brassica.

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in most eukaryotic genomes. We aimed to characterize various MITEs families in Brassica in terms of their presence, sequence characteristics and evolutionary activity. Dot plot analyses involving comparison of homoeologous bacterial artificial chromosome (BAC) sequences allowed identification of 15 novel families of mobile MITEs. Of which, 5 were Stowaway-like with TA Target Site Duplications (TSDs), 4 Tourist-like with TAA/TTA TSDs, 5 Mutator-like with 9-10bp TSDs and 1 novel MITE (BoXMITE1) flanked by 3bp TSDs. Our data suggested that there are about 30,000 MITE-related sequences in Brassica rapa and B. oleracea genomes. In situ hybridization showed one abundant family was dispersed in the A-genome, while another was located near 45S rDNA sites. PCR analysis using primers flanking sequences of MITE elements detected MITE insertion polymorphisms between and within the three Brassica (AA, BB, CC) genomes, with many insertions being specific to single genomes and others showing evidence of more recent evolutionary insertions. Our BAC sequence comparison strategy enables identification of evolutionarily active MITEs with no prior knowledge of MITE sequences. The details of MITE families reported in Brassica enable their identification, characterization and annotation. Insertion polymorphisms of MITEs and their transposition activity indicated important mechanism of genome evolution and diversification. MITE families derived from known Mariner, Harbinger and Mutator DNA transposons were discovered, as well as some novel structures. The identification of Brassica MITEs will have broad applications in Brassica genomics, breeding, hybridization and phylogeny through their use as DNA markers.

The diversification and activity of hAT transposons in Musa genomes.

Sequencing of plant genomes often identified the hAT superfamily as the largest group of DNA transposons. Nevertheless, detailed information on the diversity, abundance and chromosomal localization of plant hAT families are rare. By in silico analyses of the reference genome assembly and bacterial artificial chromosome (BAC) sequences, respectively, we performed the classification and molecular characterization of hAT transposon families in Musa acuminata. Musa hAT transposons are organized in three families designatedMuhAT I, MuhAT II and MuhAT III. In total, 70 complete autonomous elements of the MuhAT I and MuhAT II families were detected, while no autonomous MuhAT III transposons were found. Based on the terminal inverted repeat (TIR)-specific sequence information of the autonomous transposons, 1722 MuhAT I- and MuhAT II-specific miniature inverted-repeat transposable elements (MuhMITEs) were identified. Autonomous MuhAT I and MuhAT II elements areonly moderately abundant in the sections of the genus Musa, while the corresponding MITEs exhibit an amplification in Musa genomes. By fluorescent in situ hybridization(FISH), autonomous MuhAT transposons as well as MuhMITEs were localized in subtelomeric, most likely gene-rich regions of M. acuminata chromosomes. A comparison of homoeologous regions of M. acuminata and Musa balbisiana BACs revealed the species-specific mobility of MuhMITEs. In particular, the activity of MuhMITEs II showing transduplications of genomic sequences might indicate the presence of active MuhAT transposons, thus suggesting a potential role of MuhMITEs as modulators of genome evolution of Musa.