The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells

Mariner transposons belong to the mariner /Tc1 superfamily of class II, DNA-mediated elements. One of these transposons, Himar1 , isolated from the horn fly, is independent of host-specific factors that would limit transfer between different species, making it an ideal candidate for gene transfer technology development. To determine the activity of Himar1 transposase in mammalian cells, we introduced the Himar1 transposase gene into an adenovirus (Ad) vector under control of the phage T7 RNA polymerase promoter. Mammalian cells infected with the Ad vector carrying the Himar1 gene efficiently expressed the Himar1 transposase in the presence of T7 polymerase. In in vitro inter-plasmid transposition reactions, Himar1 transposase expressed by the Ad vector mediated precise cut-and-paste transposition and resulted in a characteristic duplication of TA at the integration site of the target plasmid. Further studies showed that this transposase was capable of catalyzing transposition between twoplasmids co-transfected into 293T7pol cells, which express T7 RNA polymerase. Combining the integration capability of mariner transposons with the transduction efficiency of Ad vectors is expected to provide a powerful tool for introducing transgenes into the host chromosome.     

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.     

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.     

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.     

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.     

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.     

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.     

Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

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.     

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.     

Two classes of Tn10 transposase mutants that suppress mutations in the Tn10 terminal inverted repeat

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named "SEM" for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

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.     

An ATP-ADP switch in MuB controls progression of the Mu transposition pathway

MuB protein, an ATP-dependent DNA-binding protein, collaborates with Mu transposase to promote efficient transposition. MuB binds target DNA, delivers this target DNA segment to transposase and activates transposase's catalytic functions. Using ATP-bound, ADP-bound and ATPase-defective MuB proteins we investigated how nucleotide binding and hydrolysis control the activities of MuB protein, important for transposition. We found that both MuB-ADP and MuB-ATP stimulate transposase, whereas only MuB-ATP binds with high affinity to DNA. Four different ATPase-defective MuB mutants fail to activate the normal transposition pathway, further indicating that ATP plays critical regulatory roles during transposition. These mutant proteins fall into two classes: class I mutants are defective in target DNA binding, whereas class II mutants bind target DNA, deliver it to transposase, but fail to promote recombination with this DNA. Based on these studies, we propose that the switch from the ATP- to ADP-bound form allows MuB to release the target DNA while maintaining its stimulatory interaction with transposase. Thus, ATP-hydrolysis by MuB appears to function as a molecular switch controlling how target DNA is delivered to the core transposition machinery.     

Effects of phorbol myristate acetate (PMA) on functions of macrophages and microglia in vitro

Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac). Here, we describe experiments designed to determine the basis for the high activity of Tag1. The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity. Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed. The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines. This result was maintained through at least six generations after transformation. These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active. Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1. Tag1 elements were found in only a few A. thaliana ecotypes but were present in four other Arabidopsis species.     

A modified autonomous En transposon in maize (Zea mays L.) elicits a differential response of reporter alleles

Transposable elements in maize are composed of a defined molecular structure that includes coding sequences, determiners of functionality and ordered terminal motifs that provide binding sites for transposase proteins. Alterations in these components change the phenotypic expression of unstable genes with transposon inserts. The molecular basis for the altered timing and frequency of transposition as determined by the size and number of spots on kernels or stripes on leaves has generally been described for defective inserts in genes. Most differential patterns can be ascribed to alterations in the terminal motifs of the reporter allele structure that supplies a substrate (terminal inverted repeat motifs) for transposase activity. For autonomously functioning alleles, the explanations for changes in phenotype are not so clear. In this report, an En-related element identified as F-En is described that shares with En the recognition of a specific defective element cl(mr)888104 but differs from En in that this F-En element does not recognize the canonical cl(mr) elements that are recognized by En. Evidence is provided suggesting that F-En does not recognize other En/Spm-related defective elements, some of whose sequences are known. This modified En arose from a cl-m autonomously mutating En allele.     

Tn5 in vitro transposition

This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition. The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3' and 5' strands at the ends of the specific end sequences.     

Defining functional regions of the IS903 transposase

The insertion sequence IS903 encodes a 307 amino acid residue protein, transposase, that is essential for transposition. It is a multi-functional DNA-binding protein that specifically recognizes the 18 bp inverted repeats at the ends of the element and also recognizes DNa non-specifically when it captures a target site. In addition, transposase performs catalytic functions when it mediates the cleavage and religation steps of transposition. We have carried out deletion and mutational analyses to define functional domains of the transposase protein. The deletion studies delineate a 99 residue region of the protein (residues 31 to 129) that specifies binding to the inverted repeat. A slightly larger maltose-binding protein-transposase fusion that includes residues 22 to 139 (Tnp 22-139) binds as efficiently and with the same specificity as the full-length transposase protein. Tnp 22-139 also induces a DNA bend similar to that of the wild-type protein, and so we conclude that all binding and bending specificity is contained within the N-terminal domain of the protein. Unlike full-length transposase, Tnp 22-139 forms additional higher-order complexes in band-shift gels suggesting that the deletion has exposed a surface(s) capable of participating in protein-protein interactions. Six highly conserved residues in the C-terminal portion of the protein were mutated to alanine. Each mutant protein was binding-proficient but defective in transposition. The phenotype of these substitutions, and their alignment with residues shown to abolish catalysis of other transposases and integrases, suggest that these are residues responsible for catalytic steps in transposition of IS903; we believe three of these residues comprise the DDE motif, conserved in transposases and integrases. Our data are consistent with IS903 transposase being composed of two domains: an N-terminal domain primarily involved in DNA binding and a C-terminal domain that is involved in catalysis.     

Structure and process: the relationship between practice management and actual clinical performance in general practice

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F. oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events.