In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria

mariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.     

Transposase is the only nematode protein required for in vitro transposition of Tc1

The Tc1 element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature. Here, we describe efficient and precise transposition of Tc1 in a cell-free system. Tc1 appears to jump by a cut-and-paste mechanism of transposition. The terminal 26 bp of the Tc1 terminal repeats together with the flanking TA sequence are sufficient for transposition. The target site choice in vitro is similar to that in vivo. Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tc1 transposase but also by recombinant transposase purified from Escherichia coli. The simple reaction requirements explain why horizontal spread of Tc1/mariner transposons can occur. They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.     

Covalent labeling of muscarinic acetylcholine receptors by tritiated aryldiazonium photoprobes

Members of the Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.     

Tiggers and DNA transposon fossils in the human genome

We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.     

Assembly of a strong promoter following IS911 circularization and the role of circles in transposition

When supplied with high levels of the IS911-encoded transposase, IS911-based transposons can excise as circles in which the right and left terminal inverted repeats are abutted. Formation of the circle junction is shown here to create a promoter, p(junc), which is significantly stronger than the indigenous promoter, pIRL, and is also capable of driving expression of the IS911 transposition proteins. High transposase expression from the circular transposon may promote use of the circle as an integration substrate. The results demonstrate that IS911 circles are highly efficient substrates for insertion into a target molecule in vivo. Insertion leads to the disassembly of p(junc) and thus to a lower level of synthesis of the transposition proteins. The observation that normal levels of IS911 transposition proteins supplied by wild-type copies of IS911 are also capable of generating transposon circles, albeit at a low level, reinforces the idea that the transposon circles might form part of the natural transposition cycle of IS911. These observations form the elements of a feedback control mechanism and have been incorporated into a model describing one possible pathway of IS911 transposition.     

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata. A piggyBac vector marked with the medfly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3-5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G1 transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a piggyBac-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic piggyBac TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the piggyBac system will be functional in a broad range of insects.     

Human cortical-evoked fields during detection, localisation, and identification of ''pop-out'' targets

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons.     

Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Infrequent transposition of Ac in lettuce, Lactuca sativa

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R1 plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2' promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.     

Conjugating plasmids are preferred targets for Tn7

Most transposons display target site selectivity, inserting preferentially into sites that contain particular features. The bacterial transposon Tn7 possesses the unusual ability to recognize two different classes of target sites. Tn7 inserts into these classes of target sites through two transposition pathways mediated by different combinations of the five Tn7-encoded transposition proteins. In one transposition pathway, Tn7 inserts into a unique site in the bacterial chromosome, attTn7, through specific recognition of sequences in attTn7; the other transposition pathway ignores the attTn7 target. Here we examine targets of the non-attTn7 pathway and find that Tn7 preferentially inserts into bacterial plasmids that can conjugate between cells. Furthermore, Tn7 appears to recognize preferred targets through the conjugation process, as we show that Tn7 inserts poorly into plasmids containing mutations that block plasmid transfer. We propose that Tn7 recognizes preferred targets through features of the conjugation process, a distinctive target specificity that offers Tn7 the ability to spread efficiently through bacterial populations.     

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.     

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.     

Transposition of group II intron aI1 in yeast and invasion of mitochondrial genes at new locations

Intron mobility at the RNA level by splicing reversal at allelic (homing) and non-allelic locations (transposition) has been reported in vitro. In the living cell, however, only intron homing by unidirectional gene conversion has been described. Supposing that intron insertions at non-allelic sites might occur in vivo, we speculated that group II splice-site-associated macro-deletions in fungal mitochondrial DNA might result from group II intron transposition to new locations followed by recombination. We used polymerase chain reaction techniques to detect this critical, infrequent intermediate in mtDNA populations. Here we report on group II intron aI1 transposition to non-allelic, splicing-compatible locations within the cox1 gene of yeast mtDNA. The identified integration sites are preceded by motifs similar to the upstream exon A1. Sequences flanking intron aI1 are not co-converted to the insertion sites and cis- and trans-acting mutations within aI1 reduce intron mobility below detection levels. These findings suggest the involvement of an RNA intermediate in group II intron transposition.     

Transposition without transposase: a spontaneous mutation in bacteria

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.