The Experts below are selected from a list of 11034 Experts worldwide ranked by ideXlab platform
Zoltan Ivics - One of the best experts on this subject based on the ideXlab platform.
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excision efficiency is not strongly coupled to transgenic rate cell type dependent transposition efficiency of sleeping beauty and piggybac dna transposons
Human Gene Therapy Methods, 2014Co-Authors: Orsolya Kolacsek, Zsuzsanna Izsvak, Zoltan Ivics, Zsuzsa Erdei, Agota Apati, Sara Sandor, Balazs Sarkadi, Tamas I OrbanAbstract:The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, "overproduction inhibition," a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of Transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in-house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive Transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the Transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.
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the frog prince a reconstructed transposon from rana pipiens with high transpositional activity in vertebrate cells
Nucleic Acids Research, 2003Co-Authors: Csaba Miskey, Zsuzsanna Izsvak, Ronald H A Plasterk, Zoltan IvicsAbstract:Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their Transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted Transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were approximately 90% identical to a predicted Transposase gene sequence from Xenopus laevis, but contained an unpredicted, approximately 180 bp region encoding the N-terminus of the putative Transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the Transposase gene was derived. This engineered Transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only approximately 50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows approximately 70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.
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the frog prince a reconstructed transposon from rana pipiens with high transpositional activity in vertebrate cells
Nucleic Acids Research, 2003Co-Authors: Csaba Miskey, Zsuzsanna Izsvak, Ronald H A Plasterk, Zoltan IvicsAbstract:Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their Transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted Transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were ~90% identical to a predicted Transposase gene sequence from Xenopus laevis, but contained an unpredicted, ~180 bp region encoding the N-terminus of the putative Transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the Transposase gene was derived. This engineered Transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only ~50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows ~70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.
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the dna bending protein hmgb1 is a cellular cofactor of sleeping beauty transposition
Nucleic Acids Research, 2003Co-Authors: Hatem Zayed, Zsuzsanna Izsvak, Dheeraj Khare, Udo Heinemann, Zoltan IvicsAbstract:Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two Transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB Transposase was found to interact with HMGB1 in vivo, suggesting that the Transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the Transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that Transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.
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involvement of a bifunctional paired like dna binding domain and a transpositional enhancer in sleeping beauty transposition
Journal of Biological Chemistry, 2002Co-Authors: Zsuzsanna Izsvak, Dheeraj Khare, Udo Heinemann, Ronald H A Plasterk, Joachim Behlke, Zoltan IvicsAbstract:Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two Transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB Transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between Transposase subunits. A tetrameric form of the Transposase was detected in solution, consistent with an interaction between the IR/DR structure and a Transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a Transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.
Zsuzsanna Izsvak - One of the best experts on this subject based on the ideXlab platform.
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excision efficiency is not strongly coupled to transgenic rate cell type dependent transposition efficiency of sleeping beauty and piggybac dna transposons
Human Gene Therapy Methods, 2014Co-Authors: Orsolya Kolacsek, Zsuzsanna Izsvak, Zoltan Ivics, Zsuzsa Erdei, Agota Apati, Sara Sandor, Balazs Sarkadi, Tamas I OrbanAbstract:The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, "overproduction inhibition," a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of Transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in-house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive Transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the Transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.
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the frog prince a reconstructed transposon from rana pipiens with high transpositional activity in vertebrate cells
Nucleic Acids Research, 2003Co-Authors: Csaba Miskey, Zsuzsanna Izsvak, Ronald H A Plasterk, Zoltan IvicsAbstract:Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their Transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted Transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were approximately 90% identical to a predicted Transposase gene sequence from Xenopus laevis, but contained an unpredicted, approximately 180 bp region encoding the N-terminus of the putative Transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the Transposase gene was derived. This engineered Transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only approximately 50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows approximately 70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.
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the frog prince a reconstructed transposon from rana pipiens with high transpositional activity in vertebrate cells
Nucleic Acids Research, 2003Co-Authors: Csaba Miskey, Zsuzsanna Izsvak, Ronald H A Plasterk, Zoltan IvicsAbstract:Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their Transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted Transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were ~90% identical to a predicted Transposase gene sequence from Xenopus laevis, but contained an unpredicted, ~180 bp region encoding the N-terminus of the putative Transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the Transposase gene was derived. This engineered Transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only ~50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows ~70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.
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the dna bending protein hmgb1 is a cellular cofactor of sleeping beauty transposition
Nucleic Acids Research, 2003Co-Authors: Hatem Zayed, Zsuzsanna Izsvak, Dheeraj Khare, Udo Heinemann, Zoltan IvicsAbstract:Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two Transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB Transposase was found to interact with HMGB1 in vivo, suggesting that the Transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the Transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that Transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.
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involvement of a bifunctional paired like dna binding domain and a transpositional enhancer in sleeping beauty transposition
Journal of Biological Chemistry, 2002Co-Authors: Zsuzsanna Izsvak, Dheeraj Khare, Udo Heinemann, Ronald H A Plasterk, Joachim Behlke, Zoltan IvicsAbstract:Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two Transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB Transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between Transposase subunits. A tetrameric form of the Transposase was detected in solution, consistent with an interaction between the IR/DR structure and a Transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a Transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.
Andrew Wilber - One of the best experts on this subject based on the ideXlab platform.
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unexpectedly high copy number of random integration but low frequency of persistent expression of the sleeping beauty Transposase after trans delivery in primary human t cells
Human Gene Therapy, 2010Co-Authors: Xin Huang, Andrew Wilber, Kari Haley, Marianna Wong, Hongfeng Guo, Xianzheng ZhouAbstract:Abstract We have shown that the Sleeping Beauty (SB) transposon system can mediate stable expression of both reporter and therapeutic genes in human primary T cells and that trans delivery (i.e., transposon and Transposase are on separate plasmids) is at least 3-fold more efficient than cis delivery. One concern about trans delivery is the potential for integration of the Transposase-encoding sequence into the cell genome with the possibility of continued expression, transposon remobilization, and insertional mutagenesis. To address this concern, human peripheral blood lymphocytes were nucleofected with Transposase plasmid and a DsRed transposon. Eighty-eight stable DsRed+ T cell clones were generated and found to be negative for the Transposase-encoding sequence by PCR analysis of genomic DNA. Genomic PCR was positive for Transposase in 5 of 15 bulk T cell populations that were similarly transfected and selected for transgene expression where copy numbers were unexpectedly high (0.007–0.047 per cell) by ...
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messenger rna as a source of Transposase for sleeping beauty transposon mediated correction of hereditary tyrosinemia type i
Molecular Therapy, 2007Co-Authors: Andrew Wilber, Joel L Frandsen, Scott R. Mcivor, Jason B Bell, Kirk J Wangensteen, Yixin Chen, Lijuan Zhuo, Zongyu J Chen, Stephen C Ekker, Xin WangAbstract:The Sleeping Beauty (SB) transposon system mediates chromosomal integration and stable gene expression when an engineered SB transposon is delivered along with Transposase. One concern in the therapeutic application of the SB system is that persistent expression of Transposase could result in transposon instability and genotoxicity. Here, we tested the use of Transposase-encoding RNA plus transposon DNA for correction of murine fumarylacetoacetate hydrolase (FAH) deficiency. A bi-functional transposon containing both mouse FAH and firefly luciferase sequences was used to track the growth of genetically corrected liver tissue by in vivo bioluminescence imaging after delivery of DNA or RNA as a source of Transposase. Supplying SB Transposase in the form of RNA resulted in selective repopulation of corrected hepatocytes with stable expression of FAH and luciferase. Plasma succinylacetone and amino acid levels were normalized, suggesting normal liver metabolism of catabolized protein products. Secondary FAH-deficient animals transplanted with hepatocytes (250,000) isolated from primary treated animals survived 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) withdrawal, gained weight consistently, and demonstrated stable expression of luciferase. We conclude that Transposase-encoding messenger RNA (mRNA) can be used to mediate stable non-viral gene therapy, resulting in complete phenotypic correction, and is thus an effective source of recombinase activity for use in human gene therapy.
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405 correction of the murine model of hereditary tyrosinemia type i using messenger rna as a source of Transposase for sleeping beauty mediated integration of the fah gene
Molecular Therapy, 2006Co-Authors: Andrew Wilber, Joel L Frandsen, Scott R. Mcivor, Jason B Bell, Kirk J Wangensteen, Yixin Chen, Stephen C Ekker, Lijuan Zhou, Zongyu Chen, Xin WangAbstract:Sleeping Beauty (SB) is a DNA transposon capable of mediating chromosomal integration and stable expression in vertebrate cells when co-delivered with a source of Transposase. In all pre-clinical reports where SB-mediated gene insertion in somatic cells has been used to correct mouse models of human disease, the Transposase component has been provided as 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 remobilization and reintegration of transposons. Such continued expression of Transposase is a key safety concern in development of the SB transposon system for clinical applications. As an alternate source of Transposase, we have previously shown that in vitro transcribed Transposase-encoding messenger RNA (mRNA) can effectively mediate transposon insertion both in vitro and in mouse liver (Wilber et al., Molecular Therapy, 2005, in press). Here, we test the use of Transposase- encoding mRNA plus transposon DNA for gene therapy of hereditary tyrosinemia type I by first evaluating several parameters for systemic delivery and expression of mRNA in mice. We also introduce a method to quantitatively track repopulating liver cells by in vivo bioluminescence imaging after co-delivery of a DNA or RNA source of Transposase with a bi-functional transposon encoding both mouse fumaryl acetoacetate hydrolase (FAH) and firefly luciferase (luc) genes in FAH deficient mice by rapid, high-volume injection into the tail vein. Liver repopulation was quantitatively monitored over time by increasing luc activity, measured as light emitted from the liver. Using this method, we determined that supplying SB Transposase in the form of mRNA results in selective repopulation of corrected hepatocytes with stable co-expression of both FAH and luc. Plasma taken from animals 5 months after co-infusion with Transposase mRNA contained levels of succinylacetone (the clinical determinant of tyrosinemia) that were nearly normalized. Amino acid levels were also normalized, suggesting normal liver metabolism of catabolized protein products (including urea and glucose). We further demonstrated the stability of integration by transplanting hepatocytes (250,000) into FAH deficient recipient mice. All transplanted animals survived NTBC withdrawal and gained weight consistently over a period of 90 days and demonstrated stable expression of luc. In summary, we demonstrate for the first time that Transposase-encoding mRNA can be used to mediate non-viral gene therapy resulting in complete phenotypic correction that is stable and not associated with any incidents of cellular transformation.
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rna as a source of Transposase for sleeping beauty mediated gene insertion and expression in somatic cells and tissues
Molecular Therapy, 2006Co-Authors: Andrew Wilber, Joel L Frandsen, Jennifer L Geurts, Perry B. Hackett, David A Largaespada, Scott R. McivorAbstract: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.
Deepak T. Nair - One of the best experts on this subject based on the ideXlab platform.
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Dimerization through the RING-Finger Domain Attenuates Excision Activity of the piggyBac Transposase
Biochemistry, 2018Co-Authors: Rahul Sharma, Shivlee Nirwal, Naveen Narayanan, Deepak T. NairAbstract:The movement of the piggyBac transposon is mediated through its cognate Transposase. The piggyBac Transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the Transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac Transposase. Surprisingly, the monomeric constructs exhibit >2-fold enhancement in the excision activity of the enzyme. Overall, our studies suggest that dimerization attenuates the excision activity of the piggyBac Transposase. This attribute of the piggyBac Transposase may serve to prevent excessive transposition of the piggyBac t...
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Dimerization through the RING-Finger Domain Attenuates Excision Activity of the piggyBac Transposase
2018Co-Authors: Rahul Sharma, Shivlee Nirwal, Naveen Narayanan, Deepak T. NairAbstract:The movement of the piggyBac transposon is mediated through its cognate Transposase. The piggyBac Transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the Transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac Transposase. Surprisingly, the monomeric constructs exhibit >2-fold enhancement in the excision activity of the enzyme. Overall, our studies suggest that dimerization attenuates the excision activity of the piggyBac Transposase. This attribute of the piggyBac Transposase may serve to prevent excessive transposition of the piggyBac transposon that might be catastrophic for the host cell
Nancy Kleckner - One of the best experts on this subject based on the ideXlab platform.
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two classes of tn10 Transposase mutants that suppress mutations in the tn10 terminal inverted repeat
Genetics, 1996Co-Authors: J Sakai, Nancy KlecknerAbstract:Tn 10 transposition requires IS 10 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.
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the three chemical steps of tn10 is10 transposition involve repeated utilization of a single active site
Cell, 1996Co-Authors: Silvia Bolland, Nancy KlecknerAbstract:Abstract Nonreplicative transposition by Tn10/IS10 involves three chemical steps at each transposon end: cleavage of the two strands plus joining of one strand to target DNA. These steps occur within a synaptic complex comprising two transposon ends and monomers of IS10 Transposase. We report four Transposase mutations that individually abolish each of the three chemical steps without affecting the synaptic complex. We conclude that a single constellation of residues, the "active site," directly catalyzes each of the three steps. Analyses of reactions containing mixtures of wild-type and catalysis-defective Transposases indicate that a single Transposase monomer at each end catalyzes the cleavage of two strands and that strand transfer is carried out by the same monomers that previously catalyzed cleavage. These and other data suggest that one active site unit carries out all three reactions in succession at one transposon end.
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tn10 is10 Transposase purification activation and in vitro reaction
Journal of Biological Chemistry, 1994Co-Authors: R M Chalmers, Nancy KlecknerAbstract:Abstract We describe a method for the purification of Tn10/IS10 Transposase that relies on the aggregation of the protein after overexpression in Escherichia coli. Aggregated Transposase was solubilized before the final purification step, a gel-filtration column, using a combination of salt and detergent. This procedure is the first reported for the preparation of concentrated and active Transposase from any IS element. The yield is 11 mg of purified protein at a concentration of 1 mg/ml from 2.5 g of cells. The procedure can be scaled up with ease. We also describe a treatment that activates Transposase in either a crude or purified state. This involves dilution into a solution of salt plus organic solvent. In transposition reactions using supercoiled substrate plasmid, the activity was directly proportional to the amount of Transposase added over a wide range of Transposase/DNA ratios (0.2-2.0 molecules/DNA substrate molecule). In this range 8 Transposase molecules were added per transposition event. Maximum conversion of substrate to product (40%) was with 18 Transposase molecules/transposition event. At higher levels of Transposase with a constant amount of substrate, activity was reduced but could be restored by addition of nonspecific DNA. Both the specific activity of Transposase and the type of products generated can be altered by changing in vitro assay conditions. The effects of salts, solvents, and pH value on the reaction are described.
