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PubMed2002

High frequencies of Minos transposon mobilization are obtained in insects by using in vitro synthesized mRNA as a source of transposase.

One of the most frequently encountered problems in transposon-mediated transgenesis is low transformation frequency, often resulting from difficulty in expressing from injected plasmid DNA constructs adequate levels of transposase in embryos. Capped RNA corresponding to the spliced transcript of the Minos transposable element has been synthesized in vitro and shown to be an effective source of transposase protein for Minos transposon mobilization. Transposase produced by this mRNA is shown to catalyze excision of a Minos transposon from plasmid DNA in Medfly embryos. When injected into Drosophila or Medfly embryos, transposase mRNA leads to a several-fold increase in transformation efficiencies compared with injected plasmids expressing transposase. Also, frequent mobilization of a Minos transposon from the X chromosome into autosomes was demonstrated after injections of Minos transposase mRNA into pre-blastoderm Drosophila embryos. The high rates of transposition achieved with transposase mRNA suggest that this is a powerful system for genetic applications in Drosophila and other insects.

Animals↗
PubMed1987

Overproduction and purification of the Tn3 transposase.

The Tn3 transposase accumulated to about 4% of total cell protein in a minicell-producing Escherichia coli strain harboring a transposase overproducer plasmid. This accumulation of the transposase seems to be due to four factors: derepression of transcription resulting from inactivation of the repressor gene (tnpR); efficient translation caused by a mutation within the Shine-Dalgarno (SD) sequence, the dosage effect of the increased plasmid copy number resulting from deletion of the rom gene of the plasmid; and use of a minicell-producing strain as the host. The Tn3 transposase was purified by a procedure involving five steps; sonication, precipitation of protein by adding polyethyleneimine to the sonic supernatant, followed by extraction of transposase fraction with a buffer containing ammonium sulfate, ammonium sulfate precipitation, gel filtration through Sephacryl S-300, and phosphocellulose and DNA-cellulose chromatography. Milligram quantities of pure transposase can be obtained from one gram of wet cells. A small fraction of the accumulated transposase had a blocked amino-terminus and was eluted separately from the normal protein in the chromatography.

Chromosome Deletion↗
PubMed2005

Putative transposases conserved in Exiguobacterium isolates from ancient Siberian permafrost and from contemporary surface habitats.

Gram-positive bacteria of the genus Exiguobacterium have been repeatedly isolated from Siberian permafrost ranging in age from 20,000 to 2 to 3 million years and have been sporadically recovered from markedly diverse habitats, including microbial mats in Lake Fryxell (Antarctic), surface water, and food-processing environments. However, there is currently no information on genomic diversity of this microorganism or on the physiological strategies that have allowed its survival under prolonged freezing in the permafrost. Analysis of the genome sequence of the most ancient available Exiguobacterium isolate (Exiguobacterium sp. strain 255-15, from 2 to 3 million-year-old Siberian permafrost) revealed numerous putative transposase sequences, primarily of the IS200/IS605, IS30, and IS3 families, with four transposase families identified. Several of the transposase genes appeared to be part of insertion sequences. Southern blots with different transposase probes yielded high-resolution genomic fingerprints which differentiated the different permafrost isolates from each other and from the Exiguobacterium spp. type strains which have been derived from diverse surface habitats. Each of the Exiguobacterium sp. strain 255-15 transposases that were used as probes had highly conserved homologs in the genome of other Exiguobacterium strains, both from permafrost and from modern sites. These findings suggest that, prior to their entrapment in permafrost, Exiguobacterium isolates had acquired transposases and that conserved transposases are present in Exiguobacterium spp., which now can be isolated from various modern surface habitats.

Bacillaceae↗
PubMed2004

Comparative sequence analysis of IS50/Tn5 transposase.

Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.

Amino Acid Sequence↗
PubMed1999

Variations in the maize Ac transposase transcript level and the Ds excision frequency in transgenic wheat callus lines

To investigate the excision of a maize transposable element in wheat cells, plasmid DNAs containing a Dissociation (Ds) element located between a rice actin 1 gene promoter and a beta-glucuronidase (GUS) gene (gus) were introduced into wheat callus lines by microprojectile bombardment, and transient GUS expression was assayed. The gus-expressing cells after Ds excision were detected only when the Activator (Ac) transposase gene was co-transformed. To further examine a relationship between the amount of Ac mRNA and the Ds excision frequency, the Ds-containing plasmids were introduced into 15 independent transgenic callus lines transformed with the Ac transposase gene. Ten lines expressed the Ac transposase gene under the control of either the cauliflower mosaic virus 35S promoter or the Ac native promoter. The gus gene expression that indicated the Ds excision was observed only in the transgenic callus lines stably expressing the Ac transposase gene. The number of blue spots reflecting the frequency of Ds excision was variable among them. Northern-blot analysis also showed a large variability in the amount of Ac transposase transcripts among the lines. It was however noted that the excision frequency was decreased at a high level of the Ac transposase transcripts, supporting the hypothesis that Ds excision is inhibited above a certain level of the Ac transposase as observed in maize and transgenic tobacco.

Journal Article↗
PubMed1989

A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein.

We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro. To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts. Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase. We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein. We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting. Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion. The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation.

Bacteriophage mu↗
PubMed1998

Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21.

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.

Base Sequence↗
PubMed1994

The presence of enhancers adjacent to the Ac promoter increases the abundance of transposase mRNA and alters the timing of Ds excision in Arabidopsis.

Two copies of domain B of the CaMV 35S promoter were inserted ca. 300 bp upstream of the transcriptional start site of the Ac transposase gene. Four independent Arabidopsis transformants containing this fusion (35SenhAc::TPase) were made and the abundance of transposase mRNA in each of them was determined. The presence of the enhancers increased the abundance of the transposase mRNA by about 12-fold compared to that found in plants containing an Ac promoter fusion to the transposase gene (Ac::TPase). Hybrid plants carrying 35SenhAc::TPase and a Ds element inserted in a streptomycin phosphotransferase (SPT) gene were constructed and the frequency with which Ds excision occurred in the developing cotyledons was measured. Moreover, the number of progeny of these hybrid plants which inherited an SPT gene activated by Ds excision was studied in individual F2 families. Those derived from 35SenhAc::TPase often contained higher proportions of streptomycin-resistant (strepR) F2 progeny than those derived from Ac::TPase. These high frequencies of strepR seedlings were comparable to those previously detected after activation of Ds by a CaMV 35S promoter fusion to transposase (35S::TPase), but occurred in fewer families. The higher frequency with which this occurred in families derived from 35SenhAc::TPase compared to Ac::TPase suggests that the presence of enhancers adjacent to the native Ac promoter can influence transposase gene expression, and in this case often results in earlier excision of Ds during plant development.

Arabidopsis↗
PubMed1993

DNA binding domains in Tn3 transposase.

Various segments of Tn3 transposase were fused individually to beta-galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay. Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1-242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243-632) had non-specific DNA binding ability. Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1-86 and 87-242), neither of which had specific DNA binding ability, but which both possessed non-specific DNA binding ability. The central segment included two subsegments (amino acid positions 243-289 and 439-505) with non-specific DNA binding ability. These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity. The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability. This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition.

Amino Acid Sequence↗
PubMed1992

Transactivation of Ds by Ac-transposase gene fusions in tobacco.

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Acyltransferases↗
PubMed2006

A hAT superfamily transposase recruited by the cereal grass genome.

Transposable elements are ubiquitous genomic parasites with an ancient history of coexistence with their hosts. A few cases have emerged recently where these genetic elements have been recruited for normal function in the host organism. We have identified an expressed hobo/Ac/Tam (hAT) family transposase-like gene in cereal grasses which appears to represent such a case. This gene, which we have called gary, is found in one or two copies in barley, two diverged copies in rice and two very similar copies in hexaploid wheat. No gary homologues are found in Arabidopsis. In all three cereal species, an apparently complete 2.5 kb transposase-like open reading frame is present and nucleotide substitution data show evidence for positive selection, yet the predicted gary protein is probably not an active transposase, as judged by the absence of key amino acids required for transposase function. Gary is expressed in wheat and barley spikes and gary cDNA sequences are also found in rice, oat, rye, maize, sorghum and sugarcane. The short inverted terminal repeats, flanked by an eight-nucleotide host sequence duplication, which are characteristic of a hAT transposon are absent. Genetic mapping in barley shows that gary is located on the distal end of the long arm of chromosome 2H. Wheat homologues of gary map to the same approximate location on the wheat group 2 chromosomes by physical bin-mapping and the more closely related of the two rice garys maps to the syntenic location near the bottom of rice chromosome 4. These data suggest that gary has resided in a single genomic location for at least 60 Myr and has lost the ability to transpose, yet expresses a transposase-related protein that is being conserved under host selection. We propose that the gary transposase-like gene has been recruited by the cereal grasses for an unknown function.

Amino Acid Sequence↗
PubMed2006

The N-terminus of Himar1 mariner transposase mediates multiple activities during transposition.

Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood. We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition, resulting in a population of "average" transposons.

Alanine↗
PubMed1986

Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression.

We have examined transcriptional start sites responsible for expression of the transposase and transposition inhibitor proteins encoded by IS50R, and determined the likely translational start site of transposase. Amino-terminal analysis of a transposase-beta-galactosidase fusion protein gave the sequence Met-Ile-Thr-Ser-Ala, which corresponds to the predicted amino acid sequence starting at position 93 of IS50. S1 nuclease mapping of IS50 RNA produced in vivo indicated that three transcripts, T1, T2 and T3, start near this position. Only T1 starts upstream from the transposase amino terminus. T2 corresponds to an in-vitro transcript described previously. Analysis of the transcripts and proteins produced from deletion derivatives of an IS50-lacZ construct suggested that the three transcripts initiate at independent but overlapping promoters clustered near the end of IS50. This analysis confirmed that only T1 can encode transposase, and that T2 is largely responsible for expression of the inhibitor protein. The coding capacity of T3 was not determined. Finally, transcripts that originate outside of IS50 are prevented from expressing transposase because of a secondary structure that is present in these transcripts only.

Amino Acid Sequence↗
PubMed1989

Drosophila P element transposase recognizes internal P element DNA sequences.

Drosophila P transposable elements encode an 87 kd trans-acting protein, transposase, that is required to catalyze P element transposition and excision. We show here that purified transposase is a site-specific DNA binding protein. P element transposase does not interact with the terminal 31 bp inverted repeats but instead interacts specifically with an internal 10 bp consensus sequence present at both the 5' and 3' ends of P element DNA. These binding sites lie within sequences known to be important for transposition in vivo. Transposase also displays an unusually high nonspecific affinity for DNA. The transposase binding site at the 5' and overlaps sequences we show to be essential for transcription from the P element promoter in vitro, which raises the possibility that either transposase or the related 66 kd P element protein may affect P element transcription. From these and other observations, we suggest that the P element transposition reaction probably requires the binding of additional Drosophila protein factors to the terminal DNA sequences.

Animals↗
PubMed2005

Functional zinc finger/sleeping beauty transposase chimeras exhibit attenuated overproduction inhibition.

The sleeping beauty (SB) transposon system has potential utility in gene transfer applications but lacks specificity for genomic integration and exhibits overproduction inhibition which limits in vivo activity. Targeting transposition may be possible by coupling a specific DNA binding domain to the SB transposase, but it is not known if this strategy will preserve or disrupt activity of the system. We engineered and tested chimeric SB transposases with two different human zinc finger DNA binding domain elements, Sp1 and zinc finger 202 (ZNF202). Addition of Sp1 to the C-terminus abolished transposase activity whereas N-terminal addition of either Sp1 or ZNF202 did not. Transposition activity exhibited by N-terminal chimeras was increased to levels similar to native SB through the use of a hyperactive transposase (SB12) and activating transposon mutations. Importantly, addition of DNA binding domains to the transposase N-terminus resulted in attenuation of overproduction inhibition, a major limitation of this system. These findings suggest that SB transposase chimeras may have specific advantages over the native enzyme.

Carrier Proteins↗
PubMed2005

RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues.

Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and reintegration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon. We conclude that in vitro-transcribed mRNA can be used as an effective source of transposase for SB-mediated transposition in mammalian cells and tissues.

Animals↗
PubMed1998

Functional characterization of the Tn5 transposase by limited proteolysis.

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.

Animals↗
PubMed1998

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination.

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.

Amino Acid Sequence↗