Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.     

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.     

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.     

Use of the piggyBac transposon for germ-line transformation of insects

Germ-line transformation of insects is now possible with four independent transposable element vector systems. Among these, the TTAA-insertion site specific transposon, piggyBac, discovered in Trichoplusia ni, is one of the most widely used. Transformations have been achieved in a wide variety of dipterans, lepidopterans, and a coleopteran, and for many species, piggyBac transposition was first tested by plasmid-based mobility assays in cell lines and embryos. All plasmid and genomic insertions are consistent with the duplication of a TTAA insertion site, and most germ-line integrations appear to be stable, though this is largely based on stable marker phenotypes. Of the vector systems presently in use for non-drosophilids, piggyBac is the only one not currently associated with a superfamily of transposable elements, though other elements exist that share its TTAA insertion site specificity. While functional piggyBac elements have only been isolated from T. ni, nearly identical elements have been discovered in a dipteran species, Bactrocera dorsalis, and closely related elements exist in another moth species, Spodoptera frugiperda. It appears that piggyBac has recently traversed insect orders by horizontal transmission, possibly mediated by a baculovirus or other viral system. This interspecies movement has important implications for the practical use of piggyBac to create transgenic insect strains for field release.     

The C-terminus of the Hermes transposase contains a protein multimerization domain

Transposase activity that mediates the mobility of class II transposable elements, is most commonly initiated by the assembly of higher order synaptic complexes, called transpososomes. The formation of these complexes, that contain the transposable element's DNA as well as two or more molecules of the transposase, is dependent on interactions between transposase molecules. Using the yeast Two-Hybrid system, we were able to identify three regions mediating multimerization of the Hermes transposase, an element used for germline transformation of insects belonging to the hAT family of transposable elements. One region facilitating protein binding of Hermes transposase molecules was found within the first 252 amino acids of the transposase. The second region was located at the C-terminus of the transposase, and was found to be specific for Hermes transposase multimerization. Amino acids 551-569 were not only required for multimerization but were also necessary for transposition of the element. The third region was located between amino acids 253 and 380 and was found to eliminate the non-specific protein binding ability of the N-terminal protein interaction region but was required for the specific protein binding ability of the C-terminal region of the transposase. Five point mutations affecting the structural integrity of the C-terminal multimerization region abolished or significantly reduced transpositional activity. The same region had been previously identified to mediate dimerization in Activator (Ac), another hAT element, indicating that hAT transposase multimerization is likely to be a prerequisite for mobility of their elements.     

Temperature sensitivity of transposition of class II transposons

It has been reported that transposition of Tn3 is temperature-sensitive. The effect of temperature on the transposition of other class II bacterial transposable elements is reported here: Tn21, Tn501, Tn1721, Tn2501 and Tn3926 all also display temperature-sensitivity of transposition. The temperature at which the highest transposition frequency was observed varied between room temperature and 30 degrees C.     

Post-integration stability of piggyBac in Aedes aegypti

The post-integration activity of piggyBac transposable element gene vectors in Aedes aegypti mosquitoes was tested under a variety of conditions. The embryos from five independent transgenic lines of Ae. aegypti, each with a single integrated non-autonomous piggyBac transposable element gene vector, were injected with plasmids containing the piggyBac transposase open-reading frame under the regulatory control of the Drosophila melanogaster hsp70 promoter. No evidence for somatic remobilization was detected in the subsequent adults whereas somatic remobilization was readily detected when similar lines of transgenic D. melanogaster were injected with the same piggyBac transposase-expressing plasmid. Ae. aegypti heterozygotes of piggyBac reporter-containing transgenes and piggyBac transposase-expressing transgenes showed no evidence of somatic and germ-line remobilization based on phenotypic and molecular detection methods. The post-integration mobility properties of piggyBac in Ae. aegypti enhance the utility of this gene vector for certain applications, particularly those where any level of vector remobilization is unacceptable.     

Mobility of the piggyBac transposon in embryos of the vectors of Dengue fever (Aedes albopictus) and La Crosse encephalitis (Ae. triseriatus)

The re-emergence of arboviral diseases such as Dengue Fever and La Crosse encephalitis is primarily due to the failure of insect vector control strategies. The development of a procedure capable of producing stable germ-line transformants in the insect vectors of these diseases would bridge the gap between gene expression systems being developed to curb vector transmission and the identification of important genes and regulatory sequences and their reintroduction back into the insect genome in the form of vector control strategies. The transposable element piggyBac is capable of transposition in a variety of insect species, and could serve as a versatile insect transformation vector. Using plasmid-based excision and transposition assays, we report that this short-ITR transposon undergoes precise, transposase-dependent excision and transposition in embryos of Aedes albopictus and Aedes triseriatus, the vectors of Dengue fever and LaCrosse encephalitis, respectively. These assays allow us easily and rapidly to confirm and assess the potential utility of piggyBac as a gene transfer tool in a given species. piggyBac is an exceptionally mobile and versatile genetic transformation vector, comparable to other transposons currently in use for the transformation of insects. The mobility of the piggyBac element seen in both Ae. albopictus and Ae. triseriatus is further evidence that it can be employed as a germ-line vector in important insect disease vectors.     

Copy Number Control of Tn5 Transposition

Transposition of Tn5 in Escherichia coli strains containing one or multiple copies of the transposable element was investigated. It was found that the overall frequency of transposition within a cell remained constant regardless of the number of copies of Tn5 present in that cell. Experiments measuring the transposition frequency of differentially marked Tn5s confirmed that the frequency of transposition of an individual Tn5 decreased proportionally with the total number of copies of the element present in a cell. The IS50R -encoded function, protein 2, which has previously been shown to be an inhibitor of transposition, is sufficient to mediate this inhibitory effect. The concentration of protein 2 in a cell appears to modulate the transposition of individual Tn5 elements in such a way that the overall transposition of Tn5 in a cell remains constant.     

A derivative of Tn5 with direct terminal repeats can transpose

The 5.7 kb⁴ transposable kanamycin resistance determinant Tn5 contains 1.5 kb terminal inverted repeats which we here call arms. Tn5's arms contain the genes and sites necessary for Tn5 transposition, and are not homologous to previously described transposable elements. To determine whether one or both arms is a transposable (IS) element, we transposed Tn5 to pBR322 and used restriction endonuclease digestion and ligation in vitro to generate plasmid derivatives designated pTn5-DR1 and pTn5-DR2 in which Tn5's arms were present in direct rather than in inverted orientation. Analysis of transposition products from dimeric forms of the pTn5-DR1 plasmid to phage λ showed that the outside and inside termini of right and of left arms could function in transposition. We conclude that both of Tn5's arms are transposable elements and name them IS50L (left) and IS50R (right). IS50R, which encodes transposase, was used several-fold more frequently than IS50L, which contain an ochre mutant allele of transposase: this implies that Tn5's transposase acts preferentially on the DNA segment which encodes it. Analysis of transpositions of the amp^rkan^r element Tn5-DR2 to the lac operon showed that Tn5-DR2, like Tn5 wild-type, exhibits regional preference without strict site specificity in the choice of insertion sites.     

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida

The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.     

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.     

The Tn21 subgroup of bacterial transposable elements

The Tn3 family of transposable elements is probably the most successful group of mobile DNA elements in bacteria: there are many different but related members and they are widely distributed in gram-negative and gram-positive bacteria. The Tn21 subgroup of the Tn3 family contains closely related elements that provide most of the currently known variation in Tn3-like elements in gram-negative bacteria and that are largely responsible for the problem of multiple resistance to antibiotics in these organisms. This paper reviews the structure, the mechanism of transposition, the mode of acquisition of accessory genes, and the evolution of these elements.     

Tn7: a target site-specific transposon

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.     

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.     

Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.

The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.