Topi, an IS630/Tc1/mariner-type transposable element in the African malaria mosquito, Anopheles gambiae

IS630/Tc1/mariner elements are diverse and widespread within insects. The African malaria mosquito, Anopheles gambiae, contains over 30 families of IS630/Tc1/mariner elements although few have been studied in any detail. To examine the history of Topi elements in An. gambiae populations, Topi elements (n=73) were sampled from five distinct populations of An. gambiae from eastern and western Africa and evaluated with respect to copy number, nucleotide diversity and insertion site-occupancy frequency. Topi 1 and 2 elements were abundant (10-34 per diploid genome) and highly diverse (pi=0.051). Elements from mosquitoes collected in Nigeria were Topi 2 elements and those from mosquitoes collected in Mozambique were Topi 1 elements. Of the 49 Topi transposase open reading frames sequenced none were found to be identical. Intact elements with complete transposase open reading frames were common, although based on insertion site-occupancy frequency data it appeared that genetic drift was the major force acting on these IS630/Tc1/mariner-type elements. Topi 3 elements were not recovered from any of the populations sampled in this study and appear to be rare elements in An. gambiae, possibly due to a recent introduction.     

Transposition of cyanobacterium insertion element ISY100 in Escherichia coli

The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

Expanding the diversity of the IS630-Tc1-mariner superfamily: discovery of a unique DD37E transposon and reclassification of the DD37D and DD39D transposons.

A novel transposon named ITmD37E was discovered in a wide range of mosquito species. Sequence analysis of multiple copies in three Aedes species showed similar terminal inverted repeats and common putative TA target site duplications. The ITmD37E transposases contain a conserved DD37E catalytic motif, which is unique among reported transposons of the IS630-Tc1-mariner superfamily. Sequence comparisons and phylogenetic analyses suggest that ITmD37E forms a novel family distinct from the widely distributed Tc1 (DD34E), mariner (DD34D), and pogo (DDxD) families in the IS630-Tc1-mariner superfamily. The inclusion in the phylogenetic analysis of recently reported transposons and transposons uncovered in our database survey provided revisions to previous classifications and identified two additional families, ITmD37D and ITmD39D, which contain DD37D and DD39D motifs, respectively. The above expansion and reorganization may open the doors to the discovery of related transposons in a broad range of organisms and help illustrate the evolution and structure-function relationships among these distinct transposases in the IS630-Tc1-mariner superfamily. The presence of intact open reading frames and highly similar copies in some of the newly characterized transposons suggests recent transposition. Studies of these novel families may add to the limited repertoire of transgenesis and mutagenesis tools for a wide range of organisms, including the medically important mosquitoes.     

Transposon Tc1 of the nematode Caenorhabditis elegans jumps in human cells.

The transposon Tc1 of the nematode Caenorhabditis elegans is a member of the widespread family of Tc1/mariner transposons. The distribution pattern of virtually identical transposons among insect species that diverged 200 million years ago suggested horizontal transfer of the elements between species. Thishypothesis gained experimental support when it was shown that Tc1 and later also mariner transposons could be made to jump in vitro , with their transposase as the only protein required. Later it was shown that mariner transposons from one fruit fly species can jump in other fruit fly species and in a protozoan and, recently, that a Tc1-like transposon from the nematode jumps in fish cells and that a fish Tc1-like transposon jumps in human cells. Here we show that the Tc1 element from the nematode jumps in human cells. This provides further support for the horizontal spread hypothesis. Furthermore, it suggests that Tc1 can be used as vehicle for DNA integration in human gene therapy.     

Tc1 transposase of Caenorhabditis elegans is an endonuclease with a bipartite DNA binding domain.

The Tc1 transposon of Caenorhabditis elegans is a member of the Tc1/mariner family of mobile elements. These elements have inverted terminal repeats that flank a single transposase gene. Here we show that Tc1 transposase, Tc1A, has a bipartite DNA binding domain related to the paired domain of mammalian and Drosophila genes. Both the DNA binding domain of Tc1A and the DNA binding site in the inverted repeat of Tc1 can be divided into two subdomains. Methylation interference studies demonstrate adjacent minor and major groove contacts at the inner part of the binding site by the N-terminal 68 amino acids of the DNA binding domain. In addition, Tc1A amino acids 69-142 are essential for major groove contacts at the outer part of the binding site. Recombinant Tc1A is found to be able to introduce a single strand nick at the 5' end of the transposon in vitro. Furthermore, Tc1A can mediate a phosphoryl transfer reaction. A mutation in a DDE motif abolishes both endonucleolytic and phosphoryl transfer activities, suggesting that Tc1A carries a catalytic core common to retroviral integrases and IS transposases.     

Network‐based visualisation reveals new insights into transposable element diversity

Transposable elements (TEs) are widespread across eukaryotic genomes, yet their content varies widely between different species. Factors shaping the diversity of TEs are poorly understood. Understanding the evolution of TEs is difficult because their sequences diversify rapidly and TEs are often transferred through non‐conventional means such as horizontal gene transfer. We developed a method to track TE evolution using network analysis to visualise TE sequence and TE content across different genomes. We illustrate our method by first using a monopartite network to study the sequence evolution of Tc1/mariner elements across focal species. We identify a connection between two subfamilies associated with convergent acquisition of a domain from a protein‐coding gene. Second, we use a bipartite network to study how TE content across species is shaped by epigenetic silencing mechanisms. We show that the presence of Piwi‐interacting RNAs is associated with differences in network topology after controlling for phylogenetic effects. Together, our method demonstrates how a network‐based approach can identify hitherto unknown properties of TE evolution across species.     

A New Basal Subfamily of mariner Elements in Ceratitis rosa and Other Tephritid Flies

Several copies of highly related transposable elements, Crmar2, Almar1, and Asmar1, are described from the genomes of Ceratitis rosa, Anastrepha ludens, and A. suspensa, respectively. One copy from C. rosa, Crmar2.5, contains a full-length, uninterrupted ORF. All the other copies, from the three species contain a long deletion within the putative ORF. The consensus Crmar2 element has features typical of the mariner/Tc1 superfamily of transposable elements. In particular, the Crmar2 consensus encodes a D,D41D motif, a variant of the D,D34D catalytic domain of mariner elements. Phylogenetic analysis of the relationships of these three elements and other members of the mariner/Tc1 superfamily, based on their encoded amino acid sequences, suggests that they form a new basal subfamily of mariner elements, the rosa subfamily. BLAST analyses identified sequences from other diptera, including Drosophila melanogaster, which appear to be members of the rosa subfamily of mariner elements. Analyses of their molecular evolution suggests that Crmar2 entered the genome of C. rosa in the recent past, a consequence of horizontal transfer.     

Regulation of the transposable element mariner

The mariner/ Tc1 superfamily of transposable elements is widely distributed in animal genomes and is especially prevalent in insects. Their wide distribution results from their ability to be disseminated among hosts by horizontal transmission and also by their ability to persist in genomes through multiple speciation events. Although a great deal is known about the molecular mechanisms of transposition and excision, very little is known about the mechanisms by which transposition is controlled within genomes. The issue of mariner/Tc1 regulation is critical in view of the great interest in these elements as vectors for germline transformation of insect pests and vectors of human disease. Several potentially important regulatory mechanisms have been identified in studies of genetically engineered mariner elements. One mechanism is overproduction inhibition, in which excessive wild-type transposase reduces the rate of excision of a target element. A second mechanism is mediated by certain mutant transposase proteins, which antagonize the activity of the wild-type transposase. The latter process may help explain why the vast majority of MLEs in nature undergo ‘vertical inactivation’ by multiple mutations and, eventually, stochastic loss. Another potential mechanism of regulation may result from transposase titration by defective elements that retain their DNA binding sites and ability to transpose. There is also evidence that some mariner/Tc1 elements can be mobilized in a type of hybrid dysgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Target sequences of Tc1, Tc3 and Tc5 transposons of Caenorhabditis elegans

We report here the consensus target sequence of transposons Tc1, Tc3 and Tc5 of Caenorhabditis elegans. These sequences were obtained by molecular analysis of 1008 random new insertions which have not been exposed to natural selection. This analysis reveals consensus target sites slightly different from those previously reported, and confirms that the mariner elements Tc1 and Tc3 insert in sites which are not preferentially palindromic.     

A rice Tc1/mariner-like element transposes in yeast

The Tc1/mariner transposable element superfamily is widely distributed in animal and plant genomes. However, no active plant element has been previously identified. Nearly identical copies of a rice (Oryza sativa) Tc1/mariner element called Osmar5 in the genome suggested potential activity. Previous studies revealed that Osmar5 encoded a protein that bound specifically to its own ends. In this report, we show that Osmar5 is an active transposable element by demonstrating that expression of its coding sequence in yeast promotes the excision of a nonautonomous Osmar5 element located in a reporter construct. Element excision produces transposon footprints, whereas element reinsertion occurs at TA dinucleotides that were either tightly linked or unlinked to the excision site. Several site-directed mutations in the transposase abolished activity, whereas mutations in the transposase binding site prevented transposition of the nonautonomous element from the reporter construct. This report of an active plant Tc1/mariner in yeast will provide a foundation for future comparative analyses of animal and plant elements in addition to making a new wide host range transposable element available for plant gene tagging.     

DNA binding activities of the Caenorhabditis elegans Tc3 transposase.

Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves.     

Transposon mediated transgenesis in a marine invertebrate chordate: <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis.     

“睡美人”转座系统研究进展

“睡美人 (SleepingBeauty ,SB)”转座系统是Tc1/mariner转座因子超家族中的一员 ,是目前唯一取材于脊椎动物的具有活性的转座系统 .对近年来有关“睡美人”的研究进展作一个综述 ,并针对存在的问题提出相应的解决方案 .     

Detection of de novo insertion of the medaka fish transposable element Tol2

Tol2 is a terminal-inverted-repeat transposable element of the medaka fish Oryzias latipes. It is a member of the hAT (hobo/Activator/Tam3) transposable element family that is distributed in a wide range of organisms. We here document direct evidence for de novo insertion of this element. A Tol2 clone marked with the bacterial tetracycline-resistance gene was microinjected into fertilized eggs together with a target plasmid, and the plasmid was recovered from embryos. The screening of plasmid molecules after transformation into Escherichia coli demonstrated transposition of tet into the plasmid and, by inference, precise insertion of Tol2 in medaka fish cells. De novo excision of Tol2 has previously been demonstrated. The present study provides direct evidence that the Tol2 element has the entire activity necessary for cut-and-paste transposition. Some elements of the mariner/Tc1 family, another widespread group, have already been applied to development of gene tagging systems in vertebrates. The Tol2 element of the hAT family, having different features from mariner/Tc1 family elements, also has potential as an alternative gene tagging tool in vertebrates.     

Structural role of the flanking DNA in mariner transposon excision

During cut-and-paste mariner/Tc1 transposition, transposon DNA is cut precisely at its junction with flanking DNA, ensuring the transposon is neither shortened nor lengthened with each transposition event. Each transposon end is flanked by a TpA dinucleotide: the signature target site duplication of mariner/Tc1 transposition. To establish the role of this sequence in accurate DNA cleavage, we have determined the crystal structure of a pre-second strand cleavage mariner Mos1 transpososome. The structure reveals the route of an intact DNA strand through the transposase active site before second strand cleavage. The crossed architecture of this pre-second strand cleavage paired-end complex supports our proposal that second strand cleavage occurs in trans. The conserved mariner transposase WVPHEL and YSPDL motifs position the strand for accurate DNA cleavage. Base-specific recognition of the flanking DNA by conserved amino acids is revealed, defining a new role for the WVPHEL motif in mariner transposition and providing a molecular explanation for in vitro mutagenesis data. Comparison of the pre-TS cleavage and post-cleavage Mos1 transpososomes with structures of Prototype Foamy Virus intasomes suggests a binding mode for target DNA prior to Mos1 transposon integration.     

Shotgun cloning of transposon insertions in the genome of Caenorhabditis elegans

We present a strategy to identify and map large numbers of transposon insertions in the genome of Caenorhabditis elegans. Our approach makes use of the mutator strain mut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence of C. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predicted C. elegans genes. The strategy presented provides an approach to isolate insertions of natural transposable elements in many C. elegans genes and to create a large-scale collection of C. elegans mutants.     

The DNA transposon <Emphasis Type="Italic">Minos</Emphasis>as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates

Transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific, economic, or medical interest. Minos, a member of the Tc1/mariner family of DNA transposons, exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates. Its use as a tool for transgenesis and genome analysis of rather different animal species is described.