Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.     

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.     

Target choice determinants of the Tc1 transposon of Caenorhabditis elegans.

The Tc1 transposon of Caenorhabditis elegans always integrates into the sequence TA, but some TA sites are preferred to others. We investigated a TA target site from the gpa-2 gene of C.elegans that was previously found to be preferred (hot) for Tc1 integration in vivo . This site with its immediate flanks was cloned into a plasmid, and remained hot in vitro , showing that sequences immediately adjacent to the TA dinucleotide determine this target choice. Further deletion mapping and mutagenesis showed that a 4 bp sequence on one side of the TA is sufficient to make a site hot; this sequence nicely fits the previously identified Tc1 consensus sequence for integration. In addition, we found a second type of hot site: this site is only preferred for integration when the target DNA is supercoiled, not when it is relaxed. Excision frequencies were relatively independent of the flanking sequences. The distribution of Tc1 insertions into a plasmid was similar when we used nuclear extracts or purified Tc1 transposase in vitro , showing that the Tc1 transposase is the protein responsible for the target choice.     

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.     

Target-site preferences of Sleeping Beauty transposons

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has applications in vertebrate animals for gene transfer, gene-tagging, and human gene therapy. In this study, we analyzed the target-site preferences of the SB transposon. At the genomic level, integration of SB transposons with respect to genes (exons and introns) and intergenic regions appears fairly random but not on a micro-scale. Although there appears to be a consensus sequence around the vicinity of the target sites, the primary sequence is not the determining factor for target selection. When integrations were examined over a limited topography, the sites used most often for integration did not match the consensus sequence. Rather, a unique deformation inherent in the sequence may be a recognition signal for target selection. The deformation is characterized by an angling of the target site such that the axis around the insertion site is off-center, the rotation of the helix (twisting) is non-uniform and there is an increase in the distance between the central base-pairs. Our observations offer several hypothetical insights into the transposition process. Our observations suggest that particular deformations of the double helix predicted by the V(step) algorithm can distinguish TA sites that vary by about 16-fold in their preferences for accommodating insertions of SB transposons.     

The Tc2 transposon of Caenorhabditis elegans has the structure of a self-regulated element.

We have analyzed the sequence of the Tc2 transposon of the nematode Caenorhabditis elegans. The Tc2 element is 2,074 bp in length and has perfect inverted terminal repeats of 24 bp. The structure of this element suggests that it may have the capacity to code for a transposase protein and/or for regulatory functions. Three large reading frames on one strand exhibit nonrandom codon usage and may represent exons. The first open coding region is preceded by a potential CAAT box, TATA box, and consensus heat shock sequence. In addition to its inverted terminal repeats, Tc2 has an unusual structural feature: subterminal degenerate direct repeats that are arranged in an irregular overlapping pattern. We have also examined the insertion sites of two Tc2 elements previously identified as the cause of restriction fragment length polymorphisms. Both insertions generated a target site duplication of 2 bp. One element had inserted inside the inverted terminal repeat of another transposon, splitting it into two unequal parts.     

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.     

Tc7, a Tc1-hitch hiking transposon in Caenorhabditis elegans.

We have found a novel transposon in the genome of Caenorhabditis elegans. Tc7 is a 921 bp element, made up of two 345 bp inverted repeats separated by a unique, internal sequence. Tc7 does not contain an open reading frame. The outer 38 bp of the inverted repeat show 36 matches with the outer 38 bp of Tc1. This region of Tc1 contains the Tc1-transposase binding site. Furthermore, Tc7 is flanked by TA dinucleotides, just like Tc1, which presumably correspond to the target duplication generated upon integration. Since Tc7 does not encode its own transposase but contains the Tc1-transposase binding site at its extremities, we tested the ability of Tc7 to jump upon forced expression of Tc1 transposase in somatic cells. Under these conditions Tc7 jumps at a frequency similar to Tc1. The target site choice of Tc7 is identical to that of Tc1. These data suggest that Tc7 shares with Tc1 all the sequences minimally required to parasitize upon the Tc1 transposition machinery. The genomic distribution of Tc7 shows a striking clustering on the X chromosome where two thirds of the elements (20 out of 33) are located. Related transposons in C. elegans do not show this asymmetric distribution.     

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.     

The <Emphasis Type="Italic">piggyBac</Emphasis> Transposon as a Tool in Genetic Engineering

Transposons are mobile genetic elements that are part of the genomic DNA of numerous organisms and belong to two classes. Unlike class I transposons, class II DNA transposons do not use the stage of RNA synthesis in their transition; they perform it by the cut-and-paste mechanism or with a replicative transposition. The integration of a DNA transposon in a new site results in the duplication of a target sequence on either side of a transposon, and its excision is, as a rule, associated with insertions and deletions. The piggyBac transposon isolated from the Trichoplusia ni moth differs from other mobile elements of its class. Due to its unique ability to leave no traces after excision from an insertion site and to perform successful transposition and transference of large DNA fragments, piggyBac is a convenient tool for the development of gene engineering approaches. The TTAA sequence serves as a target site for transposon integration: insertion in the AT-rich DNA regions is more frequent. The ability of piggyBac to be transferred to a new area independently of the cell apparatus and to restore a DNA site without error after excision lies in the mechanism of its transposition, which is discussed in detail in the present review. Along with other transposons and viruses, the piggyBac transposon is widely used in the transgenesis of various organisms; it also finds application in insertion mutagenesis and gene therapy.     

Patterns of selection against transposons inferred from the distribution of Tc1, Tc3 and Tc5 insertions in the mut-7 line of the nematode Caenorhabditis elegans

To identify the factors (selective or mutational) that affect the distribution of transposable elements (TEs) within a genome, it is necessary to compare the pattern of newly arising element insertions to the pattern of element insertions that have been fixed in a population. To do this, we analyzed the distribution of recent mutant insertions of the Tc1, Tc3, and Tc5 elements in a mut-7 background of the nematode Caenorhabditis elegans and compared it to the distribution of element insertions (presumably fixed) within the sequenced genome. Tc1 elements preferentially insert in regions with high recombination rates, whereas Tc3 and Tc5 do not. Although Tc1 and Tc3 both insert in TA dinucleotides, there is no clear relationship between the frequency of insertions and the TA dinucleotide density. There is a strong selection against TE insertions within coding regions: the probability that a TE will be fixed is at least 31 times lower in coding regions than in noncoding regions. Contrary to the prediction of theoretical models, we found that the selective pressure against TE insertions does not increase with the recombination rate. These findings indicate that the distribution of these three transposon families in the genome of C. elegans is determined essentially by just two factors: the pattern of insertions, which is a characteristic of each family, and the selection against insertions within coding regions.     

Continuous exchange of sequence information between dispersed Tc1 transposons in the Caenorhabditis elegans genome

In a genome-wide analysis of the active transposons in Caenorhabditis elegans we determined the localization and sequence of all copies of each of the six active transposon families. Most copies of the most active transposons, Tc1 and Tc3, are intact but individually have a unique sequence, because of unique patterns of single-nucleotide polymorphisms. The sequence of each of the 32 Tc1 elements is invariant in the C. elegans strain N2, which has no germline transposition. However, at the same 32 Tc1 loci in strains with germline transposition, Tc1 elements can acquire the sequence of Tc1 elements elsewhere in the N2 genome or a chimeric sequence derived from two dispersed Tc1 elements. We hypothesize that during double-strand-break repair after Tc1 excision, the template for repair can switch from the Tc1 element on the sister chromatid or homologous chromosome to a Tc1 copy elsewhere in the genome. Thus, the population of active transposable elements in C. elegans is highly dynamic because of a continuous exchange of sequence information between individual copies, potentially allowing a higher evolution rate than that found in endogenous genes.     

Cis requirements for transposition of Tc1-like transposons in C. elegans

The Caenorhabditis elegans transposons Tc1 and Tc3 are able to transpose in heterologous systems such as human cell lines and zebrafish. Because these transposons might be useful vectors for transgenesis and mutagenesis of diverse species, we determined the minimal cis requirements for transposition. Deletion mapping of the transposon ends shows that fewer than 100?bp are sufficient for transposition of Tc3. Unlike Tc1, Tc3 has a second, internal transposase binding site at each transposon end. We found that these binding sites play no major role in the transposition reaction, since they can be deleted without reduction of the transposition frequency. Site-directed mutagenesis was performed on the conserved terminal base pairs at the Tc3 ends. The four terminal base pairs at the ends of the Tc3 inverted repeats were shown to be required for efficient transposition. Finally, increasing the length of the transposon from 1.9?kb to 12.5 kb reduced the transposition frequency by 20-fold, both in vivo and in vitro.     

Testing the palindromic target site model for DNA transposon insertion using the Drosophila melanogaster P-element

Understanding the molecular mechanisms that influence transposable element target site preferences is a fundamental challenge in functional and evolutionary genomics. Large-scale transposon insertion projects provide excellent material to study target site preferences in the absence of confounding effects of post-insertion evolutionary change. Growing evidence from a wide variety of prokaryotes and eukaryotes indicates that DNA transposons recognize staggered-cut palindromic target site motifs (TSMs). Here, we use over 10 000 accurately mapped P-element insertions in the Drosophila melanogaster genome to test predictions of the staggered-cut palindromic target site model for DNA transposon insertion. We provide evidence that the P-element targets a 14-bp palindromic motif that can be identified at the primary sequence level, which predicts the local spacing, hotspots and strand orientation of P-element insertions. Intriguingly, we find that the although P-element destroys the complete 14-bp target site upon insertion, the terminal three nucleotides of the P-element inverted repeats complement and restore the original TSM, suggesting a mechanistic link between transposon target sites and their terminal inverted repeats. Finally, we discuss how the staggered-cut palindromic target site model can be used to assess the accuracy of genome mappings for annotated P-element insertions.     

Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons

The Sleeping Beauty and PiggyBac DNA transposon systems have recently been developed as tools for insertional mutagenesis. We have compared the chromosomal mobilization efficiency and insertion site preference of the two transposons mobilized from the same donor site in mouse embryonic stem (ES) cells under conditions in which there were no selective constraints on the transposons' insertion sites. Compared with Sleeping Beauty, PiggyBac exhibits higher transposition efficiencies, no evidence for local hopping and a significant bias toward reintegration in intragenic regions, which demonstrate its utility for insertional mutagenesis. Although Sleeping Beauty had no detectable genomic bias with respect to insertions in genes or intergenic regions, both Sleeping Beauty and PiggyBac transposons displayed preferential integration into actively transcribed loci. genesis 47:404–408, 2009. © 2009 Wiley‐Liss, Inc.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

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.     

MMTS, a new subfamily of Tc1-like transposons

A novel Tc1-like transposable element has been identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. The M. mizolepis Tc1-like transposon (MMTS) is comprised of inverted terminal repeats and a single gene that codes Tc1-like transposase. The deduced amino acid sequence of the transposase-encoding region of MMTS transposon contains motifs including DDE motif, which was previously recognized in other Tc1-like transposons. However, putative MMTS transposase has only 34-37% identity with well-known Tc1, PPTN, and S elements at the amino acid level. In dot-hybridization analysis used to measure the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that the MMTS transposon is present at about 3.36 x 104 copies per 2 x 109 bp, and accounts for approximately 0.027% of the mud loach genome. Here, we also describe novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fishes, which bear conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches and phylogenetic analysis indicated that MMTS-like transposons evolved uniquely in fishes, and comprise a new subfamily of Tc1-like transposons, with only modest similarity to Drosophila melanogaster (foldback element FB4, HB2, HB1), Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae (Frisky).