Factors affecting transposition of the Himar1 mariner transposon in vitro.

Mariner family transposable elements are widespread in animals, but their regulation is poorly understood, partly because only two are known to be functional. These are particular copies of the Dmmar1 element from Drosophila mauritiana, for example, Mos1, and the consensus sequence of the Himar1 element from the horn fly, Haematobia irritans. An in vitro transposition system was refined to investigate several parameters that influence the transposition of Himar1. Transposition products accumulated linearly over a period of 6 hr. Transposition frequency increased with temperature and was dependent on Mg2+ concentration. Transposition frequency peaked over a narrow range of transposase concentration. The decline at higher concentrations, a phenomenon observed in vivo with Mos1, supports the suggestion that mariners may be regulated in part by "overproduction inhibition." Transposition frequency decreased exponentially with increasing transposon size and was affected by the sequence of the flanking DNA of the donor site. A noticeable bias in target site usage suggests a preference for insertion into bent or bendable DNA sequences rather than any specific nucleotide sequences beyond the TA target site.     

Developmental profile of P element transposition inDrosophila somatic cells

Transposition of the P element duringDrosophila ontogenesis was monitored. A modified P element was transposed by the PΔ2-3 transposase source. P elements inserted into the genome were cloned by the plasmid rescue at various developmental stages of the G1 hybrid to trace events in somatic cells. The transposed elements were directly counted by analyzing RFLP of genomic DNA fragments flanking the P elements. Transposition began from the late embryonic stage, but occurred rarely. Frequent transposition was observed from the late third instar to early pupal stage. From these results, transposition of the P element would appear to be affected by the developmental state of somatic host cells.     

Analysis of P element transposase protein-DNA interactions during the early stages of transposition

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.     

Reprogramming the purine nucleotide cofactor requirement of Drosophila P element transposase in vivo.

Guanosine triphosphate (GTP)-binding proteins are involved in controlling a wide range of fundamental cellular processes. In vitro studies have indicated a role for GTP during Drosophila P element transposition. Here we show that P element transposase contains a non-canonical GTP-binding domain that is critical for its ability to mediate transposition in Drosophila cells. Moreover, a single amino acid substitution could switch the nucleotide binding-specificity of transposase from GTP to xanthosine triphosphate (XTP). Importantly, this mutant protein could no longer function effectively in transposition in vivo but required addition of exogenous xanthine or xanthosine for reactivation. These results suggest that transposition may be controlled by physiological GTP levels and demonstrate that a single mutation can switch the nucleotide specificity for a complex cellular process in vivo.     

P Element Transposition Contributes Substantial New Variation for a Quantitative Trait in Drosophila Melanogaster

The P-M system of transposition in Drosophila melanogaster is a powerful mutator for many visible and lethal loci. Experiments using crosses between unrelated P and M stocks to assess the importance of transposition-mediated mutations affecting quantitative loci and response to selection have yielded unrepeatable or ambiguous results. In a different approach, we have used a P stock produced by microinjection of the ry506 M stock. Selection responses were compared between transposition lines that were initiated by crossing M strain females with males from the "co-isogenic" P strain, and ry506 M control lines. Unlike previous attempts to quantify the effects of P element transposition, there is no possibility of P transposition in the controls. During 10 generations of selection for the quantitative trait abdominal bristle number, none of the four control lines showed any response to selection, indicative of isogenicity for those loci affecting abdominal bristle number. In contrast, three of the four transposition lines showed substantial response, with regression of cumulative response on cumulative selection differential ranging from 15% to 25%. Transposition of P elements has produced new additive genetic variance at a rate which is more than 30 times greater than the rate expected from spontaneous mutation.     

Sequence and positional requirements for DNA sites in a mu transpososome.

Transposition of bacteriophage Mu uses two DNA cleavage sites and six transposase recognition sites, with each recognition site divided into two half-sites. The recognition sites can activate transposition of non-Mu DNA sequences if a complete set of Mu sequences is not available. We have analyzed 18 sequences from a non-Mu DNA molecule, selected in a functional assay for the ability to be transposed by MuA transposase. These sequences are remarkably diverse. Nonetheless, when viewed as a group they resemble a Mu DNA end, with a cleavage site and a single recognition site. Analysis of these "pseudo-Mu ends" indicates that most positions in the cleavage and recognition sites contribute sequence-specific information that helps drive transposition, though only the strongest contributors are apparent from mutagenesis data. The sequence analysis also suggests variability in the alignment of recognition half-sites. Transposition assays of specifically designed DNA substrates support the conclusion that the transposition machinery is flexible enough to permit variability in half-site spacing and also perhaps variability in the placement of the recognition site with respect to the cleavage site. This variability causes only local perturbations in the protein-DNA complex, as indicated by experiments in which altered and unaltered DNA substrates are paired.     

In vitro transposition of transposon Tn3

Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract. In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules. Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase. The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3. The reaction also required DNA synthesis but not RNA synthesis by E. coli RNA polymerase.     

Mobilization of RAG-generated signal ends by transposition and insertion in vivo

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.     

Rec dependence of mu transposition from P22-transduced fragments.

Derivatives of bacteriophage Mu carrying a lac operon and a selectable drug resistance element (Mu d phages) are frequently used tools of bacterial genetics. Mu d prophages used in this way can be treated as transposons, in that the inserted material can be transduced from one strain to another by general transducing phages, such as P1 and P22. When a Mu d prophage is transduced into a new recipient by P1 or P22, the Mu d element can transpose from the transduced fragment into the bacterial chromosome. Transposition of the Mu d element from a P22-transduced fragment shows several striking differences from transposition of a Mu d genome injected by a Mu virion. First, the frequency of transposition from a transduced fragment is greatly enhanced by a P22 helper genome. Second, transposition requires the host recA, B, and C functions. Transposition of Mu following injection by a Mu virion is rec independent. While the basis of these observations is not understood, we suggest that the Mu X protein, a 65-kilodalton protein injected by a Mu virion and required for Mu transposition, may not be packaged by P22. We suggest that the effects seen reflect the behavior of a Mu genome in the absence of the X protein.     

Molecular genetic analysis of the Tn5041 transposition system

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).     

Regulation of RAG1/RAG2-mediated transposition by GTP and the C-terminal region of RAG2

The RAG1 and RAG2 proteins perform critical DNA recognition and cleavage functions in V(D)J recombination, and also catalyze efficient DNA transposition in vitro. No transposition in vivo by the RAG proteins has been reported, suggesting regulation of the reaction by as yet unknown mechanisms. Here we report that RAG-mediated transposition is suppressed by physiological concentrations of the guanine nucleotide GTP, and by the full-length RAG2 protein. Both GTP and full-length RAG2 inhibit transposition by blocking the non-covalent 'capture' of target DNA, and both are capable of inhibiting RAG-mediated hybrid joint formation in vitro. We also observe that another intracellular signaling molecule, Ca(2+), stimulates RAG-mediated transposition and is capable of activating transposition even in reactions containing full-length RAG2 and GTP. RAG-mediated transposition has been proposed to contribute to the chromosomal translocations that underlie the development of lymphoid malignancies, and our findings highlight regulatory mechanisms that might prevent such occurrences, and circumstances in which these regulatory mechanisms could be overcome.     

Artificial transposable elements in the study of the ends of IS1

We have constructed artificial IS1-based transposons by attaching synthetic oligodeoxynucleotides, corresponding to the sequence of the ends of IS1, to a selectable DNA segment ['omega' fragment; Prentki and Krisch, Gene 29 (1984) 303-313]. These transposons were used to examine the sequence requirements at the ends for IS1 transposition. We show here that a 24- to 28-bp sequence from the left or right ends of IS1 is capable of transposition when present at both ends of the omega fragment in the correct orientation. Transposition activity requires the presence of an intact IS1 in cis on the same plasmid molecule. In trans, however, neither resident genomic copies of IS1, nor copies carried by a compatible, high-copy-number plasmid present in the same cell, complement the artificial transposons efficiently. Transposition frequencies in the presence of a cis-complementing IS1 are, however, similar to those of the naturally occurring IS1-based transposon, Tn9. In addition, transposition results in a 9-bp duplication in the target DNA molecule as is usually the case for insertion of the intact IS1. Using this system, we have obtained evidence indicating that the activity of a synthetic IS1 end is not determined exclusively by its sequence, but can be strongly enhanced by a second, wild-type end used in the transposition event. The data also show that single base pair mutations can exhibit a cumulative effect in reducing transposition activity.     

Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition.

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.     

Developing a transposon tagging system to isolate rust-resistance genes from flax

Summary A line of flax, homozygous for four genes controlling resistance to flax rust, was transformed with T-DNA vectors carrying the maize transposable elements Ac and Ds to assess whether transposition frequency would be high enough to allow transposon tagging of the resistance genes. Transposition was much less frequent in flax than in Solanaceous hosts such as tobacco, tomato and potato. Transposition frequency in callus tissue, but not in plants, was increased by modifications to the transposase gene of Ac. Transactivation of the excision of a Ds element was achieved by expressing a cDNA copy of the Ac transposase gene from the Agrobacterium T-DNA 2 promoter. Progeny of three plants transformed with Ac and 15 plants transformed with Ds and the transposase gene, were examined for transposition occurring in the absence of selection. Transposition was observed in the descendants of only one plant which contained at least nine copies of Ac. Newly transposed Ac elements were observed in 25–30% of the progeny of some members of this family and one active Ac element was located 28.8 (SE=6.3) map units from the L ⁶ rust-resistance gene. This family will be potentially useful in our resistance gene tagging program.     

Mechanism and regulation of P element transposition

P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans. This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.     

Transposition of reversed Ac element ends generates chromosome rearrangements in maize

In classical "cut-and-paste" transposition, transposons are excised from donor sites and inserted at new locations. We have identified an alternative pathway in which transposition involves the 5' end of an intact Ac element and the 3' end of a nearby terminally deleted fAc (fractured Ac). The Ac and fAc elements are inserted at the maize p1 locus on chromosome 1s in the same orientation; the adjacent ends of the separate elements are thus in reversed orientation with respect to each other and are separated by a distance of approximately 13 kb. Transposition involving the two ends in reversed orientation generates inversions, deletions, and a novel type of local rearrangement. The rearrangement breakpoints are bounded by the characteristic footprint or target site duplications typical of Ac transposition reactions. These results demonstrate a new intramolecular transposition mechanism by which transposons can greatly impact genome evolution.