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PubMed1998

Evidence for circular transposition derivatives from the fungal hAT-transposon Restless.

The structure and function of eukaryotic hAT-transposons has already been elucidated; however, their transposition mechanism is barely understood. We recently have discovered Restless, a fungal member of the hAT-transposon family, which shows transposition in its host Tolypocladium inflatum. Investigations of two strains from T. inflatum, carrying either about 15 copies or only a single copy of the Restless element, indicate the presence of circular transposition intermediates. Using PCR technology, amplicons were identified which carry the joined end of the Restless transposon fused at its inverted repeats. All of eight sequenced PCR fragments contained the intact transposon ends with a short insertion of 1-93 bp of genomic DNA. Remarkably, one of the discovered genomic sequences matches a previously described integration site. Our data are discussed with respect to the transposition mechanism and the horizontal transfer of hAT-transposons.

DNA Transposable Elements↗
PubMed2005

Identification of an active transposon in intact rice plants.

A transposable element that is active in intact plants has been identified in rice (Oryza sativa L.). The 607-bp element itself, termed nonautonomous DNA-based active rice transposon (nDart), has no coding capacity. It was found inserted in the gene encoding Mg-protoporphyrin IX methyltransferase in a chlorophyll-deficient albino mutant isolated from backcross progeny derived from a cross between wild-type japonica varieties. The nDart has 19-bp terminal inverted repeats (TIRs) and, when mobilized, generates an 8-bp target-site duplication (TSD). At least 13 nDart elements were identified in the genome sequence of the japonica cultivar Nipponbare. Database searches identified larger elements, termed DNA-based active rice transposon (Dart) that contained one ORF for a protein that contains a region with high similarity to the hAT dimerization motif. Dart shares several features with nDart, including identical TIRs, similar subterminal sequences and the generation of an 8-bp TSD. These shared features indicate that the nonautonomous element nDart is an internal deletion derivative of the autonomous element Dart. We conclude that these active transposon systems belong to the hAT superfamily of class II transposons. Because the transposons are active in intact rice plants, they should be useful tools for tagging genes in studies of functional genomics.

Base Sequence↗
PubMed1998

Tfo1: an Ac-like transposon from the plant pathogenic fungus Fusarium oxysporum.

A transposable element from a plant pathogenic fungus, Fusarium oxrysporum, was isolated and characterized. Four clones carrying moderately repetitive DNA were selected from a genomic library of the strain MAFF305118 of F. oxysporum f. sp. lagenariae, which causes wilt of bottle gourd. One the four clones was found to include a transposable element, which we have named Tfo1. This element is 2763 bp in size and appears to contain a long ORF that can encode a polypeptide of 777 amino acids. The amino acid sequence shows significant similarity to transposases from the hAT family of transposons, such as the maize transposon Activator (Ac). The element has 15-bp terminal inverted repeats and causes an 8-bp target site duplication upon insertion, as expected for an hAT-family transposon. Northern analysis detected a transcript, which hybridized to the putative transposase-encoding region of Tfo1. The size of this transcript (about 2.3 kb) corresponds to that of the ORF. A Southern analysis using pulsed-field gel electrophoresis showed that multiple chromosomal bands carry Tfo1 elements. PCR amplification of the Tfo1 elements with a 15-base inverted repeat primer produced a single DNA fragment of about 2.8 kb in all bottle gourd-infecting strains used. The element was found in multiple copies in the genome of all these strains and also in strains from other formae speciales tested. The sequence similarity of the Tfo1 element to other transposons, together with its transcriptional expression and genomic distribution, strongly suggests that Tfo1 is a member of the hAT transposon family.

Amino Acid Sequence↗
PubMed2026

Genetic interactions between PIWI subfamily genes and hobo transposons modulate Drosophila melanogaster lifespan under chronic low-intensity irradiation.

In recent decades, there has been active research into how ionizing radiation at low doses, an inevitable factor in human activity, affects aging processes and which molecular genetic mechanisms underlie this influence. This study investigates the effects of mutations in PIWI subfamily genes (piwi and aub), which regulate transposable elements, on the lifespan of Drosophila melanogaster under conditions of genome instability induced by hobo transposons and chronic low-intensity irradiation (20 cGy). It is shown that dysfunction of piwi and aub modulates the activity of hobo transposons, increasing the frequency of their excisions/transpositions and recombinogenic activity, as confirmed by phenotypic and PCR analyses. The presence of hobo transposons in the genome elevates the spontaneous level of DNA fragmentation in ovarian cells, and chronic irradiation enhances this effect, leading to increased DNA damage in somatic and germline cells of most studied strains. Despite increased genetic instability and reduced fertility in some genotypes, the combined presence of mutations and hobo transposons paradoxically increases lifespan both under control conditions and after irradiation. Analysis of the interaction between genetic factors reveals a predominantly antagonistic, and in one case synergistic, effect on lifespan, depending on the type of mutation, the structure of the hobo transposons (full-size or defective copies), sex, and irradiation conditions. These results demonstrate the complex interplay between systems controlling transpositional activity and stress-induced processes that affect key viability parameters.

Animals↗
PubMed1989

A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment.

We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition. These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends. Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site. SOS induction presumably results from the degradation of the donor site. Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends. SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function. A single additional mutation maps within a region that is highly conserved among IS element transposases. The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition.

Alleles↗
PubMed1984

A physical and functional analysis of Tn917, a Streptococcus transposon in the Tn3 family that functions in Bacillus.

The erythromycin-resistance (Emr)-conferring transposon Tn917, first isolated in the genus Streptococcus, has in previous work been shown to function efficiently in the spore-forming species Bacillus subtilis, where it has been developed as a tool for identifying and studying sporulation genes. In the present work, a physical analysis of Tn917 was undertaken, including detailed restriction mapping, chemical DNA sequencing, heteroduplex studies, and Southern hybridization analysis, as a first step in understanding the genetic organization of this useful insertion element. The location and transcriptional orientation of the transposon-borne erm gene (the gene responsible for the Emr phenotype) have been determined, and a partial sequence of DNA 5' to the coding sequence of this gene indicates that its inducibility is probably the result of "translational attenuation," a mechanism known to be responsible for the regulation of at least two other gram-positive erm genes. Restriction mapping and heteroduplex analysis have revealed extensive homology between Tn917 and the Staphylococcus transposon Tn551, throughout virtually their entire lengths, and DNA sequencing studies have revealed a remarkably high degree of sequence correspondence within the terminal inverted repeats of Tn917, Tn551 and the gram-negative transposon Tn3. Tn917 was also shown to generate a 5-bp duplication upon insertion, as do Tn3 and Tn551 (and all of the other Tn3-related elements studied thus far), strengthening the conclusion that these three transposons are members of a highly dispersed family of related insertion elements which populate both gram-positive and gram-negative genera.

Bacillus↗
PubMed1984

Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene.

A 1.3-kb restriction fragment carrying a cat gene derived from Staphylococcus aureus was inserted by ligation in both possible orientations into a HpaI restriction site located less than 300 bp from one end of Tn917. The resulting transposon derivatives were unimpaired in their ability to make and resolve transpositions into the chromosome of Bacillus subtilis and they displayed no detectable defect in expression of the inducible erm gene carried by the transposon. This demonstrates that the HpaI site itself, and perhaps the entire 250- to 300-bp region between the HpaI site and the nearest transposon terminal inverted repeat consists of nonessential DNA, and is there fore available to be modified or used as a cloning site with the expectation that the resulting transposon derivatives should be capable of normal transposition activity. To facilitate such manipulations, the HpaI site was "replaced" by a 24-bp DNA segment which contains a BamHI site flanked on either side by SmaI sites; these BamHI and SmaI sites are unique to the transposon. Several of the plasmid constructions undertaken in the course of this work illustrate ways in which homologous recombination may be used in conjunction with ligation in B. subtilis (and other bacteria, such as Streptococcus pneumoniae, which have similar mechanisms for DNA uptake during competence) to facilitate significantly the recovery of certain kinds of recombinant molecules.

Bacillus subtilis↗
PubMed1990

Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages.

Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.

Animals↗
PubMed2004

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.

AT Rich Sequence↗
PubMed2006

The implications of DNA transposons in the evolution of P elements in zebrafish (Danio rerio).

We have carried out detailed analyses of Drosophila P-transposon-homologous Pdre elements in zebrafish (Danio rerio) and present a model to explain the evolutionary origin of the complete and terminally truncated copies. Analysis of the most complex terminally truncated element led to the identification of novel DNA transposons, referred to as demio, hexi, and lady elements. We also show that the existence of a P-homologous stable gene can be explained in two ways: it is a P-transposon source gene, and insertion into or recombination with another transposon can lead to mobilization by recruitment of structures necessary for transposition (known as the source gene hypothesis), or it is the result of a domestication event involving a P transposon (domestication hypothesis). Our results indicate that both scenarios are possible and that they reflect different stages of the P-element life cycle.

Animals↗
PubMed2004

Development of hyperactive sleeping beauty transposon vectors by mutational analysis.

The Sleeping Beauty (SB) transposable element is a promising vector for transgenesis in vertebrates and is being developed as a novel, nonviral system for gene therapeutic purposes. A mutagenesis approach was undertaken to improve various aspects of the transposon, including safety and overall efficiency of gene transfer in human cells. Deletional analysis of transposon sequences within first-generation SB vectors showed that the inverted repeats of the element are necessary and sufficient to mediate high-efficiency transposition. We constructed a "sandwich" transposon, in which the DNA to be mobilized is flanked by two complete SB elements arranged in an inverted orientation. The sandwich element has superior ability to transpose >10-kb transgenes, thereby extending the cloning capacity of SB-based vectors. We derived hyperactive versions of the SB transposase by single-amino-acid substitutions. These mutations act synergistically and result in an almost fourfold enhancement of activity compared to the wild-type transposase. When combined with hyperactive transposons and transiently overexpressed HMGB1, a cellular cofactor of SB transposition, hyperactive transposases elevate transposition by almost an order of magnitude compared to the first-generation transposon system. The improved vector system should prove useful for efficient gene transfer in vertebrates.

Amino Acid Sequence↗
PubMed2004

Endothelial targeting of the Sleeping Beauty transposon within lung.

Endothelial cells have complex roles in the pathophysiology of vascular and heart disease and are increasingly being recognized as targets for gene therapy. The intravenous administration of plasmid DNA complexed to lipid tends to target transfection of endothelial cells within the lung; however, expression from the transgene remains transient. Here we utilize the integrating capability of the Sleeping Beauty (SB) transposon for durable gene transfer within lung endothelia. To restrict expression of the transgene, an endothelial cell-specific promoter, endothelin-1, was placed within the transposon. Further refinements to the transposon increased in vitro transposition efficiency by 3.6-fold. Utilizing this optimized transposon we evaluated the expression of two reporter molecules, secreted alkaline phosphatase (SEAP) and intracellular GFP, following administration of DNA-polyethylenimine complexes to mice. Long-term expression (>2 months) of SEAP occurred only with cotransfection of adequate amounts of transposase. Localization studies using the GFP reporter, at 3 days and 6 weeks postinjection, demonstrated that the majority of transgene-expressing cells were of endothelial origin, while the second most abundant cell type was type II pneumocyte. These results suggest that the SB transposon can be adapted to target particular cell types, in this case, endothelial cells. Such an approach may be useful for gene therapy paradigms involving the long-term modulation of vascular and endothelial cell biology.

Alkaline Phosphatase↗
PubMed2003

Transposon mutagenesis of Mycoplasma gallisepticum.

There are few systems available for studying the genetics of the important avian respiratory pathogen, Mycoplasma gallisepticum. These techniques are needed to develop a mechanism to study the molecular pathogenesis of M. gallisepticum. Tn916 has the ability to transpose into the M. gallisepticum genome by both transformation and conjugation. In this study, PEG-mediated transformation was employed for the transfer of Tn916 into M. gallisepticum and create a transposon mutant library. Transformants were obtained at a frequency of approximately 5 x 10(-8) per recipient CFU. A total of 424 MG/Tn916 mutants were constructed and sequence data from the transposon junctions of 71 mutants was obtained and used to identify transposon insertion sites. Insertions were found throughout the genome in nearly all of the major gene categories, making this the first extensive characterization of a transposon mutant library of M. gallisepticum. Transposon stability was also examined, and it was determined that for two mutants the element was stably maintained in vivo in the absence of selective pressure.

Animals↗
PubMed2001

New Mos1 mariner transposons suitable for the recovery of gene fusions in vivo and in vitro.

The Drosophila Mos1 element can be mobilized in species ranging from prokaryotes to protozoans and vertebrates, and the purified transposase can be used for in vitro transposition assays. In this report we developed a 'mini-Mos1' element and describe a number of useful derivatives suitable for transposon mutagenesis in vivo or in vitro. Several of these allow the creation and/or selection of tripartite protein fusions to a green fluorescent protein-phleomycin resistance (GFP-PHLEO) reporter/selectable marker. Such X-GFP-PHLEO-X fusions have the advantage of retaining 5' and 3' regulatory information and N- and C-terminal protein targeting domains. A Mos1 derivative suitable for use in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is described, and transposons bearing selectable markers suitable for use in the protozoan parasite Leishmania were made and tested. A novel 'negative selection' approach was developed which permits in vitro assays of transposons lacking bacterial selectable markers. Application of this assay to several Mos1 elements developed for use in insects suggests that the large mariner pM[cn] element used previously in vivo is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly active.

Animals↗
PubMed2003

Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis.

Methylation of cytosine residues in eukaryotic genomes is often associated with repeated sequences including transposons and their derivatives. Methylation has been implicated in control of two potential deleterious effects of these repeats: (1) uncontrolled transcription, which often disturbs proper expression of nearby host genes, and (2) changes in genome structure by transposition and ectopic recombination. Arabidopsis thaliana provides a genetically tractable system to examine these possibilities, since viable mutants in DNA methyltransferases are available. Arabidopsis MET1 (METHYLTRANSFERASE1, ortholog of mammalian DNA methyltransferase Dnmt1) is necessary for maintaining genomic cytosine methylation at 5'-CG-3' sites. Arabidopsis additionally methylates non-CG sites using CHROMOMETHYLASE3 (CMT3). We examined the mobility of endogenous CACTA transposons in met1, cmt3, and cmt3-met1 mutants. High-frequency transposition of CACTA elements was detected in cmt3-met1 double mutants. Single mutants in either met1 or cmt3 were much less effective in mobilization, despite significant induction of CACTA transcript accumulation. These results lead us to conclude that CG and non-CG methylation systems redundantly function for immobilization of transposons. Non-CG methylation in plants may have evolved as an additional epigenetic tag dedicated to transposon control. This view is consistent with the recent finding that CMT3 preferentially methylates transposon-related sequences.

Arabidopsis↗
PubMed1997

Ds elements on all five Arabidopsis chromosomes and assessment of their utility for transposon tagging.

The maize transposons Activator (Ac) and Dissociation (Ds) tend to transpose to sites close to their original position and can be efficiently used to transposon-tag genetically linked genes. To facilitate this approach, we describe the locations of seven T-DNAs carrying Ds elements, including at least one on each of the five chromosomes. For five of these T-DNAs, we have confirmed that the Ds element transposes preferentially to genetically linked sites. A large-scale transposon-tagging experiment was performed by activating Ds from eight chromosomal locations that included at least one on each of the five chromosomes. These experiments produced a total of 1132 F3 families that were predicted to carry around 870 independent Ds insertions. In these populations, 33 independently isolated mutants that were visibly different from wild-type were identified. Twenty-nine of these mutants were studied genetically, and 14 were not tagged with Ds because the element could be separated from the mutation by recombination. The remaining 15 mutations were possibly tagged because the transposon and the mutation were not separated by recombination. These experiments provide tools for transposon-tagging on each chromosome, and indicate that approximately 50% of identified mutations are likely to be tagged, thereby enabling cloning of the affected genes.

Arabidopsis↗
PubMed2000

Technical advance: display and isolation of transposon-flanking sequences starting from genomic DNA or RNA.

Insertion mutagenesis using transposons has become a powerful tool for the isolation of genes involved in any given biochemical or developmental pathway. We describe here ligation-mediated PCR techniques for the isolation of sequences flanking the transposable elements En/Spm, Mu1 and Cin4 in Zea mays and Arabidopsis thaliana. Two versions of this transposon insertion display (TID) method use biotinylated linkers or biotinylated primers to rapidly isolate transposon-flanking sequences starting from digested genomic DNA. TID protocols have been employed to clone several genes from En/Spm insertion mutants of Arabidopsis. A novel procedure, expression TID (ETID), is also introduced, which provides a direct approach for the isolation of transposon insertions that tag transcribed portions of genes. ETID uses RNA as a starting material and exploits 5' RACE PCR to identify transposon copies that form parts of gene transcripts. The detection of several En/Spm insertion mutations in Arabidopsis illustrates the power of this method. ETID offers important advantages for the isolation of mutant alleles of novel genes that are expressed in specific tissues in plants and animals.

Alleles↗
PubMed2005

Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse.

The Sleeping Beauty (SB) transposon system can integrate foreign sequences of DNA in the genome of mouse somatic cells eliciting long-term expression in vivo. This technology holds great promise for human gene therapy as a nonviral technology to deliver therapeutic genes. SB also provides a means to study the effects of defined genetic elements, such as oncogenes, on somatic cells in mice. Here, we test the ability of the SB transposon system to facilitate somatic integration of a transposon containing an activated NRAS oncogene in mouse hepatocytes to elicit tumor formation. NRAS oncogene-driven tumors developed when such vectors were delivered to the livers of p19Arf-null or heterozygous mice. Delivery of the NRAS transposon cooperates with Arf loss to cause carcinomas of hepatocellular or biliary origin. These tumors allowed characterization of transposon integration and expression at the single-cell level, revealing robust NRAS expression and both transposase-mediated and random insertion of delivered vectors. Random integration and expression of the SB transposase plasmid was also observed in one instance. In addition, studies using effector loop mutants of activated NRAS provide evidence that mitogen-activated protein kinase activation alone cannot efficiently induce liver carcinomas. This system can be used to rapidly model tumors caused by defined genetic changes.

Animals↗